U.S. patent application number 13/640057 was filed with the patent office on 2013-03-28 for fibronectin type iii domain-based multimeric scaffolds.
This patent application is currently assigned to Medlmmune, LLC. The applicant listed for this patent is Manuel Baca, Jeffrey Swers, Thomas Thisted. Invention is credited to Manuel Baca, Jeffrey Swers, Thomas Thisted.
Application Number | 20130079280 13/640057 |
Document ID | / |
Family ID | 44799000 |
Filed Date | 2013-03-28 |
United States Patent
Application |
20130079280 |
Kind Code |
A1 |
Baca; Manuel ; et
al. |
March 28, 2013 |
FIBRONECTIN TYPE III DOMAIN-BASED MULTIMERIC SCAFFOLDS
Abstract
The present invention provides fibronectin type III (Fn3)-based
multimeric scaffolds that specifically bind to one or more specific
target antigen. The invention further provides bispecific
Fn3-derived binding molecules that bind to two or more target
antigens simultaneously, fusions, conjugates, and methods to
increase the stability of Fn3-based binding molecules. Furthermore,
the present invention is related to a prophylactic, therapeutic or
diagnostic agent, which contains Fn3-based multimeric
scaffolds.
Inventors: |
Baca; Manuel; (Gaithersburg,
MD) ; Thisted; Thomas; (Gaithersburg, MD) ;
Swers; Jeffrey; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baca; Manuel
Thisted; Thomas
Swers; Jeffrey |
Gaithersburg
Gaithersburg
Gaithersburg |
MD
MD
MD |
US
US
US |
|
|
Assignee: |
Medlmmune, LLC
Gaithersburg
MD
|
Family ID: |
44799000 |
Appl. No.: |
13/640057 |
Filed: |
April 12, 2011 |
PCT Filed: |
April 12, 2011 |
PCT NO: |
PCT/US11/32184 |
371 Date: |
December 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61323708 |
Apr 13, 2010 |
|
|
|
Current U.S.
Class: |
514/9.3 ;
530/395; 536/23.5 |
Current CPC
Class: |
C07K 2319/70 20130101;
C07K 2319/30 20130101; A61P 35/00 20180101; C07K 2317/90 20130101;
C07K 16/468 20130101; C07K 2319/00 20130101; A61P 31/00 20180101;
C07K 2318/20 20130101; A61K 2039/505 20130101; C07K 2317/73
20130101; C07K 14/78 20130101; C07K 16/2878 20130101; C07K 14/001
20130101; C07K 16/1027 20130101; A61P 43/00 20180101; C07K 2319/33
20130101; A61P 37/06 20180101; C07K 19/00 20130101; C07K 2319/74
20130101; A61P 29/00 20180101 |
Class at
Publication: |
514/9.3 ;
530/395; 536/23.5 |
International
Class: |
C07K 19/00 20060101
C07K019/00 |
Claims
1-76. (canceled)
77. A recombinant multimeric scaffold comprising two fibronectin
type III (FnIII) monomer scaffolds derived from one or more FnIII
domains of interest (FOI), wherein (a) each FnIII monomer scaffold
comprises seven beta strands designated A, B, C, D, E, F, and G
linked to six loop regions designated AB, BC, CD, DE, EF, and FG,
(b) the FnIII monomer scaffolds are connected in tandem, wherein at
least two of the monomers comprises a non-naturally occurring
intramolecular disulfide bond, (c) the recombinant multimeric
scaffold specifically binds to at least one target, and (d) the
action on the target is improved over that of a cognate FnIII
monomer scaffold.
78. The multimeric scaffold of claim 77, wherein the multimeric
scaffold comprises 3, 4, 5, 6, 7, or 8 FnIII monomer scaffolds.
79. The multimeric scaffold of claim 77, wherein at least two FnIII
monomer scaffolds are connected by a linker.
80. The multimeric scaffold of claim 77, wherein at least to FnIII
monomer scaffolds are directly connected without a linker
interposed between the FnIII monomer scaffolds.
81. The multimeric scaffold of claim 77, wherein at least one of
the FnIII monomer scaffolds is fused to a heterologous moiety.
82. The multimeric scaffold of claim 77, wherein at least two FnIII
monomer scaffolds are different.
83. The multimeric scaffold of claim 77, wherein each beta strand
has at least 50% homology to the cognate beta strand of a FnIII
domain of interest (FOI) and at least one loop is a non-naturally
occurring variant of the cognate loop in the FOI.
84. The multimeric scaffold of claim 77, wherein at the FOI of
comprises a sequence selected from the group consisting of any one
of SEQ ID NOs: 1-4 and 14.
85. The multimeric scaffold of claim 77, wherein the beta strands
of at least one of the FnIII monomer scaffolds have at least 90%
sequence identity to the cognate beta strands in SEQ ID NO: 1, 4 or
14.
86. The multimeric scaffold of claim 77, wherein for at least one
FnIII monomer scaffold, the A beta strand comprises SEQ ID NO: 41
or 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand
comprises SEQ ID NO: 45, or 131, the D beta strand comprises SEQ ID
NO: 46, the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO: 49, 50 or 51, and the G beta strand comprises
SEQ ID NO: 52 or 53.
87. An isolated nucleic acid molecule encoding the multimeric
scaffold of claim 77.
88. A composition comprising the recombinant multimeric scaffold of
claim 77 in a pharmaceutically acceptable excipient.
89. A multimeric scaffold comprising two fibronectin type III
(FnIII) monomer scaffolds, wherein each FnIII scaffold binds a
target and wherein at least one FnIII monomer scaffold comprises
the amino acid sequence: TABLE-US-00027
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTIDL-
(X.sub.DE).sub.nYSI(X.sub.EF).sub.n YEVSLIC(X.sub.FG).sub.nFETFTT,
or
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nIELX.sub.1YGI(X.sub.CD).sub.nTTIDL-
(X.sub.DE).sub.nYSI(X.sub.EF).sub.n YCVSLIS(X.sub.FG).sub.nKECFTT,
or
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTIDL-
(X.sub.DE).sub.nYSI(X.sub.EF).sub.n
YCVSLIC(X.sub.FG).sub.nKECFTT
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein the length of the loop
n=2-26
90. The multimeric scaffold of claim 89, wherein the multimeric
scaffold comprises 3, 4, 5, 6, 7, or 8 FnIII monomer scaffolds.
91. The multimeric scaffold of claim 89, wherein at least two FnIII
monomer scaffolds are connected by a linker.
92. The multimeric scaffold of claim 89, wherein at least to FnIII
monomer scaffolds are directly connected without a linker
interposed between the FnIII monomer scaffolds.
93. The multimeric scaffold of claim 89, wherein at least two FnIII
monomer scaffolds are different.
94. The multimeric scaffold of claim 89, wherein the AB loop
comprises SEQ ID NO: 35, the CD loop comprises SEQ ID NO: 37, and
the EF loop comprises SEQ ID NO: 39
95. The multimeric scaffold of claim 89, wherein the BC loop
comprises SEQ ID NO: 36, the DE loop comprises SEQ ID NO: 38, and
the FG loop comprises SEQ ID NO: 40.
96. An isolated nucleic acid molecule encoding the multimeric
scaffold of claim 89.
97. A composition comprising the recombinant multimeric scaffold of
claim 89 in a pharmaceutically acceptable excipient.
Description
REFERENCE TO THE SEQUENCE LISTING
[0001] This application incorporates by reference a Sequence
Listing submitted with this application via EFS-Web as text file
entitled "2943.011PC01_sequence_listing.txt" created on Apr. 12,
2011 and having a size of 221 kilobytes.
FIELD OF THE INVENTION
[0002] The present invention relates in general to the field of
antibody mimetics, specifically to multimeric scaffolds based on
the fibronectin type III (Fn3) domain useful, for example, for the
generation of products having novel binding characteristics.
BACKGROUND
[0003] Biomolecules capable of specific binding to a desired target
epitope are of great importance as therapeutics, research, and
medical diagnostic tools. A well known example of this class of
molecules is the antibody. Antibodies can be selected that bind
specifically and with affinity to almost any structural epitope.
However, classical antibodies are structurally complex
heterotetrameric molecules with are difficult to express in simple
eukaryotic systems. As a result, most antibodies are produced using
complex and expensive mammalian cell expression systems.
[0004] Proteins having relatively defined three-dimensional
structures, commonly referred to as protein scaffolds, may be used
as reagents for the design of engineered products. These scaffolds
typically contain one or more regions which are amenable to
specific or random sequence variation, and such sequence
randomization is often carried out to produce libraries of proteins
from which desired products may be selected.
[0005] One particular area in which such scaffolds are useful is
the field of antibody mimetic design. Antibody mimetics, i.e.,
small, non-antibody protein therapeutics, capitalize on the
advantages of antibodies and antibody fragments, such as high
affinity binding of targets and low immunogenicity and toxicity,
while avoiding some of the shortfalls, such as the tendency for
antibody fragments to aggregate and be less stable than full-length
IgGs.
[0006] These drawbacks can be addressed by using antibody fragments
created by the removal of parts of the antibody native fold.
However, this often causes aggregation when amino acid residues
which would normally be buried in a hydrophobic environment such as
an interface between variable and constant domain become exposed to
the solvent.
[0007] One example of an scaffold-based antibody mimetic is based
on the structure of a fibronectin module of type III (FnIII), a
domain found widely across phyla and protein classes, such as in
mammalian blood and structural proteins. The FnIII domain occurs
often in various proteins, including fibronectins, tenascin,
intracellular cytoskeletal proteins, cytokine receptors and
prokaryotic enzymes (Bork and Doolittle, Proc. Natl. Acad. Sci. USA
89:8990-8894, 1992; Bork et al., Nature Biotechnol. 15:553-557,
1997; Meinke et al., J. Bacteriol. 175:1910-1918, 1993; Watanabe et
al., J. Biol. Chem. 265:15659-15665, 1990). PCT Publication No: WO
2009/058379 describes scaffolds based on the FnIII domain, in
particular, the third FnIII domain of human tenascin C. FnIII
domains comprise seven beta strands, designated N-terminus to
C-terminus A, B, C, D, E, F, and G strands, each strand separated
by a loop region wherein the loop regions are designated N-terminus
to C-terminus, AB, BC, CD, DE, EF, and FG loops. Although the FnIII
domain is not an immunoglobulin, the overall fold of the third
FnIII domain of human tenascin C domain is closely related to that
of the smallest functional antibody fragment, the variable region
of the heavy chain, which comprises the entire antigen recognition
unit in camel and llama IgG. This makes it possible to display the
three fibronectin loops on each opposite side of a FnIII domain,
e.g., the third FnIII domain of human tenascin C in relative
orientations similar to those of CDRs in native antibodies.
[0008] There is a need to develop improved stable, artificial
antibody-like molecules, having increased specificity, affinity,
avidity, and stability for a variety of therapeutic and diagnostic
applications, as well as screening methods for identifying such
molecules.
[0009] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
SUMMARY OF THE INVENTION
[0010] The invention provides recombinant multimeric scaffold
comprising two fibronectin type III (FnIII) monomer scaffolds
derived from one or more FnIII domains of interest (FOI), wherein
(a) each FnIII monomer scaffold comprises a plurality of beta
strands linked to a plurality of loop regions, (b) the FnIII
monomer scaffolds are connected in tandem, wherein at least one of
the monomers comprises a non-naturally occurring intramolecular
disulfide bond, (c) the recombinant multimeric scaffold
specifically binds to at least one target, and (d) the action on
the target is improved over that of a cognate FnIII monomer
scaffold.
[0011] The invention also provides recombinant multimeric scaffold
comprising 3 fibronectin type III (FnIII) monomer scaffolds derived
from one or more FnIII domains of interest (FOI) wherein (a) each
FnIII monomer scaffold comprises a plurality of beta strands linked
to a plurality of loop regions, (b) the recombinant multimeric
FnIII scaffold specifically binds to at least one target, and (c)
the action on the target is improved over that of a cognate FnIII
monomer scaffold.
[0012] In some embodiments, the multimeric scaffolds of the
invention comprise 3, 4, 5, 6, 7, or 8 FnIII monomer scaffolds. In
some embodiments, all of the FnIII monomer scaffolds in the
multimeric scaffold are in tandem. In other embodiments, at least
two FnIII monomer scaffolds in a multimeric scaffold comprise a
non-naturally occurring intramolecular disulfide bond. In some
other embodiments, the multimeric scaffold of the invention binds
to at least 2 targets. In some embodiments, at least one FnIII
monomer scaffold in a multimeric scaffold is connected directly, by
a linker, or by a heterologous moiety to 2, 3, 4, 5, or 6 other
FnIII monomer scaffolds. In some embodiments, the multimeric
scaffold of the invention comprises 7, 8, 9, 10, 11 or 12 FnIII
monomer scaffolds, which in some embodiments can all be in
tandem.
[0013] In some embodiments, at least two FnIII monomer scaffolds in
a multimeric scaffold are connected by a linker. In other
embodiments, at least two FnIII monomer scaffolds in a multimeric
scaffold are directly connected without a linker interposed between
the FnIII monomer scaffolds. In some embodiments, the plurality of
beta strands in at least one FnIII monomer scaffold in the
multimeric scaffold comprises seven beta strands designated A, B,
C, D, E, F, and G. In other embodiments, the plurality of loop
regions in at least one FnIII monomer scaffold in the multimeric
scaffold comprises six loop regions designated AB, BC, CD, DE, EF,
and FG.
[0014] In some embodiments, for at least one FnIII monomer scaffold
in a multimeric scaffold of the invention there is an improvement
in binding over that of a cognate FnIII monomer scaffold wherein
the improvement is in binding affinity and/or avidity.
[0015] In some embodiments, binding affinity for the target and
protein stability are improved in the multimeric scaffold over
those of a cognate FnIII monomer scaffold. In other embodiments,
the binding avidity for the target and the protein stability of a
multimeric scaffold are improved over those of a cognate FnIII
monomer scaffold.
[0016] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold of the invention comprises at least two
non-naturally occurring intramolecular disulfide bonds. In some
embodiments, the multimeric scaffold comprises a peptide linker.
The peptide linker can be a flexible peptide linker. In some
embodiments, the linker comprises a functional moiety, which is
some cases can be an immunoglobulin or a fragment thereof.
[0017] In some embodiments, at least one of the FnIII monomer
scaffolds in a multimeric scaffold is fused to a heterologous
moiety, such as a protein, a peptide, a protein domain, a linker, a
drug, a toxin, a cytotoxic agent, an imaging agent, a radionuclide,
a radioactive compound, an organic polymer, an inorganic polymer, a
polyethylene glycol (PEG), biotin, a human serum albumin (HSA), a
HSA FcRn binding portion, an antibody, a domain of an antibody, an
antibody fragment, a single chain antibody, a domain antibody, an
albumin binding domain, an enzyme, a ligand, a receptor, a binding
peptide, a non-FnIII scaffold, an epitope tag, a recombinant
polypeptide polymer, a cytokine, and a combination of two or more
of said moieties.
[0018] In some embodiments, more than two of the FnIII monomer
scaffolds in a multimeric scaffold are connected by linkers, and at
least one linker is structurally and/or functionally different from
the other linkers. In other embodiments, the FnIII monomer
scaffolds in a multimeric FnIII scaffold are connected in a
branched format. In other embodiments, some FnIII monomer scaffolds
in the multimeric scaffold are connected in a linear tandem format
and some FnIII monomer scaffolds are connected in a branched
format.
[0019] In some embodiments, at least two FnIII monomer scaffolds in
the multimeric scaffold are identical, whereas is some other
embodiments at least two FnIII monomer scaffolds are different. In
some embodiments, the multimeric scaffold is a receptor agonist. In
other embodiments, the multimeric scaffold is a receptor
antagonist.
[0020] In some embodiments, at least two FnIII monomer scaffolds in
the multimeric scaffold bind the same target at the same epitope.
In other embodiments, at least two FnIII monomer scaffolds in a
multimeric scaffold bind the same target at different epitopes. In
some embodiments, the different epitopes are non-overlapping
epitopes, whereas in other embodiments the different epitopes are
overlapping epitopes.
[0021] In some embodiments, at least one FOI is selected from the
group consisting of: an animal FnIII domain, a bacterial FnIII
domain, an archaea FnIII domain, and a viral FnIII domain. This at
least one FOI can comprise a sequence selected from the group
consisting of any one of SEQ ID NOs: 1-34, 59, 69, and any of the
sequences presented in FIG. 16. In some embodiments, the at least
one FOI is an FnIII domain from a hyperthermophilic archaea.
[0022] In some embodiments, the FOI comprises the third FnIII
domain of human tenascin C (SEQ ID NO: 4) or a functional fragment
thereof. In some embodiments, the FOI comprises the 14th FnIII
domain of human fibronectin (SEQ ID NO: 69) or a functional
fragment thereof, or the 10th FnIII domain of human fibronectin
(SEQ ID NO: 54) or a functional fragment thereof.
[0023] In some embodiments, the FOI for each FnIII monomer in a
multimeric scaffold comprises the third FnIII domain of human
tenascin C (SEQ ID NO: 4) or a functional fragment thereof. In some
embodiments, the functional fragment of the third FnIII domain of
human tenascin C is an N-terminal truncated form (SEQ ID NO:
14).
[0024] In some embodiments, the beta strands of at least one of the
FnIII monomer scaffolds in a multimeric scaffold have at least 90%
sequence identity to the cognate beta strands in SEQ ID NO: 4. In
some embodiments, for at least one FnIII monomer scaffold in a
multimeric scaffold, the A beta strand domain comprises SEQ ID NOs:
41 or 42, the B beta strand comprises SEQ ID NO: 43, the C beta
strand comprises SEQ ID NO: 44 or 131, the D beta strand comprises
SEQ ID NO: 46, the E beta strand comprises SEQ ID NO: 47, the F
beta strand comprises SEQ ID NO: 48, and the G beta strand
comprises SEQ ID NO: 52.
[0025] In some embodiments, for at least one FnIII monomer scaffold
in a multimeric scaffold, the AB loop comprises SEQ ID NO: 35, the
CD loop comprises SEQ ID NO: 37, and the EF loop comprises SEQ ID
NO: 39. In other embodiments, for at least one FnIII monomer
scaffold in a multimeric scaffold, the BC loop comprises SEQ ID NO:
36, the DE loop comprises SEQ ID NO: 38 and the FG loop comprises
SEQ ID NO: 40.
[0026] In some embodiments, for at least one FnIII monomer scaffold
in a multimeric scaffold, the AB loop comprises SEQ ID NO: 35, the
BC loop comprises SEQ ID NO: 97, 98, 99, 100, or 101, the CD loop
comprises SEQ ID NO: 37, the DE loop comprises SEQ ID NO: 38, 102,
103, 104, or 105, the EF loop comprises SEQ ID NO: 39, and the FG
loop comprises SEQ ID NO:106, 107, 108, 109, 110, or 111.
[0027] In some embodiments, for at least one FnIII monomer scaffold
in a multimeric scaffold, the A beta strand comprises SEQ ID NO: 41
or 42, the B beta strand comprises SEQ ID NO: 43, the C beta strand
comprises SEQ ID NO: 44, 45, or 131, the D beta strand comprises
SEQ ID NO: 46, the E beta strand comprises SEQ ID NO:47, the F beta
strand comprises SEQ ID NO: 49, 50 or 51, and the G beta strand
comprises SEQ ID NO: 52 or 53.
[0028] In some embodiments, the FOI of at least one FnIII monomer
scaffold comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3.
[0029] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold comprises the amino acid sequence: [0030]
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKE TFTT
[0031] wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF,
and X.sub.FG represent the amino acid residues present in the AB,
BC, CD, DE, EF, and FG loops, respectively, wherein X.sub.1
represents amino acid residue A or T, and wherein the length of the
loop n is an integer between 2 and 26.
[0032] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold comprises the amino acid sequence: [0033]
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYCVSLIS(X.sub.FG).sub.nKE CFTT
[0034] wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF,
and X.sub.FG represent the amino acid residues present in the AB,
BC, CD, DE, EF, and FG loops, respectively, wherein X.sub.1
represents amino acid residue A or T, and wherein the length of the
loop n is an integer between 2 and 26.
[0035] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold comprises the amino acid sequence: [0036]
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYCVSLIC(X.sub.FG).sub.nKE CFTT
[0037] wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF,
and X.sub.FG represent the amino acid residues present in the AB,
BC, CD, DE, EF, and FG loops, respectively, wherein X.sub.1
represents amino acid residue A or T, and wherein the length of the
loop n is an integer between 2 and 26.
[0038] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold comprises an AB loop comprising SEQ ID NO:
35, a CD loop comprising SEQ ID NO: 37, and an EF loop comprising
SEQ ID NO: 39. In other embodiments, at least one FnIII monomer
scaffold in a multimeric scaffold comprises a BC loop comprising
SEQ ID NO: 36, a DE loop comprising SEQ ID NO: 38, and an FG loop
comprising SEQ ID NO: 40.
[0039] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold comprises at least one BC loop, DE, loop or
FG loop variant. In some embodiments, the BC loop variant comprises
SEQ ID NO: 97, 98, or 168. In other embodiments, the DE loop
variant comprises SEQ ID NO: 102, or 103. In some other
embodiments, the FG loop variant comprises SEQ ID NO: 106, 108,
109, 169, or 170. In some embodiments the BC loop, DE, loop or FG
loop variant comprises the amino acid sequence of the respective
BC, DE, or FG loop of SEQ ID NO: 178, 195, 196, 197, 198, 199, 200,
205, 206, 207, or 208.
[0040] In some embodiments, the increased protein stability of at
least one FnIII monomer scaffold is measured by differential
scanning calorimetry (DSC), circular dichroism (CD), polyacrylamide
gel electrophoresis (PAGE), protease resistance, isothermal
calorimetry (ITC), nuclear magnetic resonance (NMR), urea
denaturation, or guanidine denaturation.
[0041] In some embodiments, at least one FnIII monomer scaffold in
a multimeric scaffold is affinity matured.
[0042] The invention also provides a method for obtaining a
recombinant multimeric scaffold comprising: expressing, fusing or
conjugating 2 fibronectin type III (FnIII) monomer scaffolds
derived from one or more wild-type FnIII domains of interest (FOI),
wherein (a) each FnIII monomer scaffold comprises a plurality of
beta strands linked to a plurality of loop regions, (b) the FnIII
monomer scaffolds are connected in tandem, wherein at least one of
the FnIII monomer scaffolds comprises one non-naturally occurring
intramolecular disulfide bond, (c) the recombinant multimeric
scaffold specifically binds to at least one target, and (d) the
binding for the target is improved over that of a cognate FnIII
monomer scaffold.
[0043] The invention also provides a method for obtaining a
recombinant multimeric scaffold comprising: expressing, fusing or
conjugating at least 3 fibronectin type III (FnIII) monomer
scaffolds derived from one or more wild-type FnIII domains of
interest (FOI) wherein (a) each FnIII monomer scaffold comprises a
plurality of beta strands linked to a plurality of loop regions,
(b) the recombinant multimeric FnIII scaffold specifically binds to
at least one target, and (c) the binding for the target is improved
over that of a cognate FnIII monomer scaffold.
[0044] In some embodiments, at least one of the FOIs used in the
methods described above comprises a sequence selected from the
group consisting of any one of SEQ ID NOs:1-34, 54, 69, and any of
the sequences presented in FIG. 16.
[0045] The invention also provides a nucleic acid encoding any of
the multimeric scaffolds described above. In some embodiments, a
vector is operably linked to the nucleic acid. In other
embodiments, a host cell can comprise the vector.
[0046] The invention also provides a method of producing a
recombinant multimeric scaffold comprising culturing a host cell
under conditions in which the multimeric scaffold encoded by the
nucleic acid molecule is expressed.
[0047] In other embodiments, the scaffolds of the invention are
combined with a pharmaceutically acceptable excipient to yield a
pharmaceutical composition.
[0048] The invention also provides a method for treating a cancer,
an autoimmune disorder, an inflammatory disorder, or an infection
in a patient in need thereof comprising administering an effective
amount of the composition of a pharmaceutical composition
comprising a scaffold of the invention.
[0049] The invention also provides a method of detecting a protein
in a sample comprising labeling a multimeric FnIII scaffold of the
invention or a conjugate comprising a scaffold of the invention,
contacting the labeled multimeric FnIII scaffold or conjugate with
a sample, and detecting complex formation between the multimeric
FnIII scaffold or conjugate with the protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0051] FIG. 1 shows linear, antibody-like and fusion formats of
multivalent Tn3 scaffolds. Multivalent Tn3 scaffolds contain two or
more Tn3 modules attached by a spacer indicated by a black
octagonal block, where the spacer can be, e.g., a linker.
[0052] FIG. 2 shows TRAIL R2-specific multivalent Tn3 scaffolds,
designated as A2 to A9, which were generated according to the three
different molecular formats shown in FIG. 1 with valencies (number
of Tn3 modules) varying from 2 to 8.
[0053] FIG. 3 shows non reducing SDS-PAGE analysis of crude
bacterial media (right gel) and affinity purified samples (left
gel) corresponding to linear tandem constructs designated A1 to A5,
with valencies varying from 1 to 8, expressed in E. coli.
[0054] FIG. 4 shows a competition ELISA measuring binding of
monovalent (A1) and multivalent (A2, A3) Tn3 scaffolds to TRAIL
R2.
[0055] FIG. 5. shows a flow cytometry histogram of the TRAIL
R2-specific multivalent scaffold A9 binding to H2122 cells compared
to a cognate control scaffold (B9) that does not bind TRAIL R2.
[0056] FIG. 6A shows the effect of valency on the specific killing
of the TRAIL R2-expressing cell line H2122 by multivalent
scaffolds.
[0057] FIG. 6B shows the specificity of H2122 tumor cell killing by
TRAIL R2-specific multivalent scaffolds.
[0058] FIG. 7A shows the effect of molecular format on killing of
H2122 cells by TRAIL R2-specific multivalent scaffolds comprising 4
Tn3 modules.
[0059] FIG. 7B shows the effect of molecular format on killing of
H2122 cells by TRAIL R2-specific multivalent scaffolds comprising 8
Tn3 modules.
[0060] FIG. 8A shows the specific killing of colorectal
adenocarcinoma cell line Colo205 cells expressing TRAIL R2 by
linearly fused tetra-(A3) and octavalent (A5) TRAIL R2-specific Tn3
scaffolds.
[0061] FIG. 8B shows the specific killing of leukemic line Jurkat
cells expressing TRAIL R2 by linearly fused tetra-(A3) and
octavalent (A5) TRAIL R2-specific Tn3 scaffolds.
[0062] FIG. 9A shows the design of murine CD40L-specific tandem
bivalent Tn3 scaffolds (M13 constructs).
[0063] FIG. 9B shows the SDS-PAGE analysis of a purified monovalent
M13 construct (CD40L-specific Tn3 construct), or tandem bivalent
scaffolds with linkers containing 1, 3, 5 or 7 Gly.sub.4Ser units
(denoted as GS) joining two M13 modules. Monovalent M13 construct
was run in lane 2, Construct C1 in lanes 3 and 7, Construct C2 in
lanes 4 and 8, construct C3 in lanes 5 and 9, and construct C4 in
lanes 6 and 10. Samples were run either non-reduced conditions
(lanes 2-6) or reduced conditions (lanes 7-10).
[0064] FIG. 9C shows the competitive inhibition of MuCD40L binding
to Murine CD40 receptor immobilized on a biosensor chip by
MuCD40L-specific monovalent (M13) or bivalent tandem scaffolds. The
half maximal inhibitory concentration (IC.sub.50) for the various
constructs is indicated.
[0065] FIG. 9D shows the inhibitory effect of MuCD40L-specific
monovalent (M13) Tn3, bivalent tandem scaffolds, or antibody MR1
(an anti-MuCD40L antibody) on MuCD40L-induced CD86 expression on B
cells.
[0066] FIG. 10 shows the expression levels of soluble monovalent
and TRAIL R2/CD40L-bispecific tandem bivalent Tn3 scaffold
constructs recombinantly expressed in E. coli analyzed by SDS-PAGE
of the bacterial culture media. Monovalent scaffolds, A1 and 79 are
shown in lanes 2 and 3, respectively. Tandem scaffold constructs
comprising A1 and 79, joined in tandem by a Gly.sub.4Ser amino acid
linker of increasing length (cognate to constructs C5, C6, C7 and
C8) are shown in lanes 4-7. The expressed constructs are indicated
on the stained gel by an asterisk.
[0067] FIG. 11A shows the binding of bispecific Tn3 scaffolds to
TRAIL R2 assayed using capture ELISA.
[0068] FIG. 11B shows the binding of bispecific Tn3 scaffolds to
Human CD40L assayed using capture ELISA.
[0069] FIG. 12 shows the simultaneous binding of bispecific tandem
Tn3 scaffolds C5, C6, C7, and C8 to TRAIL R2 and CD40L assayed
using an AlphaScreen.TM. assay.
[0070] FIG. 13 shows the stability of Tn3 scaffolds in the present
of guanidine-HCl. C.sub.m (midpoint value) for each tested scaffold
is indicated.
[0071] FIG. 14 shows the thermostability of three different Tn3
scaffolds with different loop sequences, but the same length FG
loop (nine amino acids) compared to the parental Tn3 scaffold which
has a longer FG loop analyzed by differential scanning calorimetry
(DSC).
[0072] FIG. 15 shows the increase in stability in the presence of
guanidine-HCl of Tn3 scaffolds having a nine amino acid length FG
loop (P1C01, A6, and 71) compared to the parental (WT) Tn3
scaffold.
[0073] FIG. 16 shows a multiple sequence alignment of 103 different
FnIII scaffolds based on structural analysis. Each FnIII sequence
corresponds to a different FnIII three dimensional structure,
identified according to its respective Protein Data Bank (PDB)
structure and chain (e.g., 1V5J_A, corresponds to the sequence of
chain A in the 1V5J PDB structure). The entire sequence for each
FnIII sequence is shown over four consecutive panels starting with
the A strand, and ending with the G strand. The loop regions are
indicated at the top of the alignments with a solid line. The
sequences are displayed in groups with the AB loop of each group
indicated on FIGS. 16A, 16E, 16I, and 16M; the BC and CD loops of
each group are indicated on FIGS. 16B, 16F, 16J, and 16N; the DE
and EF loops are indicated on FIGS. 16C, 16G, 16K, and 16O; and the
FG loop is indicated on FIGS. 16D, 16H, 16L, and 16P.
[0074] FIG. 17A shows a schematic representation and expression of
a trispecific/trivalent Tn3 scaffold. The D1-1E11-79 scaffold
contains a Synagis.RTM.-binding domain (D1), followed by a TRAIL
R2-Fc binding domain (1E11), and a C-terminal Tn3 domain specific
for human CD40L (79). A flexible (Gly.sub.4Ser).sub.3 linker
separates each domain.
[0075] FIG. 17B shows a SDS-PAGE (4-12% Bis-Tris) gel of the
expressed and purified D1-1E11-79 scaffold. The expected molecular
weight of this construct is approximately 34,081 Daltons.
[0076] FIG. 18A shows the simultaneous binding of the
trispecific/trivalent Tn3 scaffold D1-1E11-79 to huCD40L and TRAIL
R2-Fc using AlphaScreen binding analysis. AlphaScreen signal (ASS)
shown as a function of TrailR2-Fc concentration.
[0077] FIG. 18B shows the simultaneous binding of the
trispecific/trivalent Tn3 scaffold D1-1E11-79 to huCD40L and
Synagis.RTM. using AlphaScreen binding analysis. AlphaScreen signal
(ASS) shown as a function of Synagis.RTM. concentration.
[0078] FIG. 19 shows the simultaneous binding of the
trispecific/trivalent Tn3 scaffold D1-1E11-79 to TRAIL R2-Fc and
Synagis.RTM. using ELISA.
[0079] FIG. 20 shows a sequence alignment of parental TRAIL R2
binding clone 1C12 and its affinity matured derivatives. The
position of the engineered disulfide bond is highlighted, the arrow
indicates the location of the one framework mutation, and changes
in the loops that arise during affinity maturation are shown in
highlighted blocks A, B, C, and D.
[0080] FIG. 21 shows a CellTiter-Glo cell viability assay of the
1C12 clone and its affinity matured derivatives.
[0081] FIG. 22 shows concentration of G6 tandems as a function of
time in mouse serum.
[0082] FIG. 23A shows a sequence alignment corresponding to the
engineered enhancement of cyno cross reactivity for clone F4. The
common feature among all of these clones is a mutation from D to G
two amino acids before the DE loop.
[0083] FIG. 23B shows ELISA measurements of the inhibition of
binding of either human or cyno TRAIL R2-Fc to F4 mod 1 coated
plates by F4 or F4 mod 1 monomer.
[0084] FIG. 24A shows a sequence alignment corresponding to
germlining of the clone F4 mod 1, specifically a comparison of F4,
F4 mod 1 and F4 mod 12 to the TN3 germline.
[0085] FIG. 24B shows ELISA measurements of the inhibition of
binding of either human or cyno TRAIL-R2-Fc to F4 mod 1 coated
plates by F4, F4 mod 1, or F4 mod 12 monomer.
[0086] FIG. 24C shows a Colo205 cell killing assay comparing G6
tandem 6 to F4 mod 12 tandem 6.
[0087] FIG. 24D shows a Colo205 cell killing assay comparing G6
tandem 8 to F4 mod 12 tandem 8.
[0088] FIG. 25 shows an HT29 cell killing assay comparing the
activity of G6 tandem 8 to F4 mod 12 tandem 8 in the TRAIL
resistant cell line HT29.
[0089] FIG. 26 shows a sequence alignment corresponding to the
clones tested in Antitope EpiScreen Immunogenicity analyses.
Differences with respect to clone F4 mod 12 are highlighted.
[0090] FIG. 27A shows SEC traces of non-SEC-purified G6 tandem
8.
[0091] FIG. 27B shows SEC traces of SEC-purified G6 tandem 8.
[0092] FIG. 28 shows changes in tumor volume in Colo205 colorectal
cancer xenograft models in response to different doses of the Tn3
TRAIL R2 agonists G6 tandem 6 and G6 tandem 8.
[0093] FIG. 29 shows changes in body weight in Colo205 colorectal
xenograft models in response to different doses of the Tn3 TRAIL R2
agonists G6 tandem 6 and G6 tandem 8.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0094] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such can vary. It must be noted
that, as used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. The terms "a" (or "an"), as
well as the terms "one or more," and "at least one" can be used
interchangeably herein.
[0095] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term "and/or" as
used in a phrase such as "A and/or B" herein is intended to include
"A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the
term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following embodiments: A, B, and
C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B (alone); and C (alone).
[0096] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this invention.
[0097] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, amino acid sequences are written left to right in amino
to carboxy orientation. The headings provided herein are not
limitations of the various aspects or embodiments of the invention,
which can be had by reference to the specification as a whole.
Accordingly, the terms defined immediately below are more fully
defined by reference to the specification in its entirety.
[0098] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0099] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
codes.
[0100] The term "epitope" as used herein refers to a protein
determinant capable of binding to a scaffold of the invention.
Epitopes usually consist of chemically active surface groupings of
molecules such as amino acids or sugar side chains and usually have
specific three dimensional structural characteristics, as well as
specific charge characteristics. Conformational and
non-conformational epitopes are distinguished in that the binding
to the former but not the latter is lost in the presence of
denaturing solvents.
[0101] The terms "fibronectin type III (FnIII) domain," "FnIII
domain" refer to polypeptides homologous to the human fibronectin
type III domain having at least 7 beta strands which are
distributed between two beta sheets, which themselves pack against
each other to form the core of the protein, and further containing
solvent exposed loops which connect the beta strands to each other.
There are at least three such loops at each edge of the beta sheet
sandwich, where the edge is the boundary of the protein
perpendicular to the direction of the beta strands. In certain
embodiments, an FnIII domain comprises 7 beta strands designated A,
B, C, D, E, F, and G linked to six loop regions designated AB, BC,
CD, DE, EF, and FG, wherein a loop region connects each beta
strand. FIG. 16 provides the primary sequence locations for the
beta strands and loops for numerous FnIII domains based on analysis
of their three dimensional structures. It should be noted that
alternative definitions of these regions are known in the art.
However, for these FnIII domains, the definitions in FIG. 16 will
be used herein unless the context clearly dictates otherwise except
that it will be understood that the N-terminus of the A strand
and/or the C-terminus of the G strand may be truncated. The terms
"fibronectin type III (FnIII) domain" and "FnIII domain" also
comprise protein domains recognized to contain the Interpro
IPRO08957 fibronectin type III domain signature as determined using
the InterProScan program, or recognized to contain the Pfam PF00041
fibronectin type III domain signature as determined using
Pfam_scan, HMMER, or any other program known in the art capable of
comparing a protein sequence to a Hidden Markov model describing an
FnIII domain. In addition, the terms include functional fragments
and engineered FnIII domains, e.g., core-engineered FnIII domains
(see, e.g., Ng et al., Nanotechnology 19: 384023, 2008).
[0102] The terms "Fibronectin type III (FnIII) scaffold" or "FnIII
scaffold" refers to a polypeptide comprising an FnIII domain, or
functional fragment thereof, wherein at least one loop is a
non-naturally occurring variant of a FnIII domain/scaffold of
interest, and wherein said FnIII scaffold, or functional fragment
thereof is capable of binding a target, wherein the term "binding"
herein preferably relates to a specific binding. As used herein a
"non-naturally occurring variant" can vary by deletion,
substitution or addition by at least one amino acid from the
cognate sequences in a starting protein sequence (e.g., an FnIII
domain/scaffold of interest), which may be a native FnIII domain
sequence or a previously identified FnIII scaffold sequence. In
certain embodiments, the A beta strand is truncated, for example
one or more N-terminal residues of the A beta strand can be absent.
In certain embodiments, the G beta strand is truncated, for example
one or more C-terminal residues of the G beta strand may be absent.
In certain embodiments, an FnIII scaffold comprises a non-naturally
occurring variant of one or more beta strands. In certain
embodiments, the beta strands of the FnIII scaffold exhibit at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99% or more sequence identity to the primary
sequences of the cognate beta strands of any one of SEQ ID NOs:
1-34, 54, or 69 or to the primary sequences of the beta strands of
any of the FnIII domains shown in FIG. 16; or to the beta strands
of a protein domain recognized to contain the Interpro IPRO08957
fibronectin type III domain signature as determined using the
InterProScan program, or recognized to contain the Pfam PF00041
fibronectin type III domain signature as determined using
Pfam_scan, HMMER, or any other program capable of comparing a
protein sequence to a Hidden Markov model.
[0103] The term "DNA" refers to a sequence of two or more
covalently bonded, naturally occurring or modified
deoxyribonucleotides.
[0104] The term "fusion protein" refers to protein that includes
(i) one or more scaffolds of the invention joined to (ii) a second,
different protein (i.e., a "heterologous" protein).
[0105] The term "heterologous moiety" is used herein to indicate
the addition of a composition to a scaffold of the invention
wherein the composition is not normally part of an FnIII domain.
Exemplary heterologous moieties include proteins, peptides, protein
domains, linkers, drugs, toxins, imaging agents, radioactive
compounds, organic and inorganic polymers, and any other
compositions which might provide an activity that is not inherent
in the FnIII domain itself, including, but are not limited to,
polyethylene glycol (PEG), a cytotoxic agent, a radionuclide,
imaging agent, biotin, a dimerization domain (e.g. leucine zipper
domain), human serum albumin (HSA) or an FcRn binding portion
thereof, a domain or fragment of an antibody (e.g., antibody
variable domain, a CH1 domain, a Ckappa domain, a Clambda domain, a
CH2, or a CH3 domain), a single chain antibody, a domain antibody,
an albumin binding domain, an IgG molecule, an enzyme, a ligand, a
receptor, a binding peptide, a non-FnIII scaffold, an epitope tag,
a recombinant polypeptide polymer, a cytokine, and the like.
[0106] The term "linker" as used herein refers to any molecular
assembly that joins or connects two or more scaffolds. The linker
can be a molecule whose function is to act as a "spacer" between
modules in a scaffold, or it can also be a molecule with additional
function (i.e., a "functional moiety`). A molecule included in the
definition of "heterologous moiety" can also function as a
linker.
[0107] The terms "linked" and "fused" are used interchangeably.
These terms refer to the joining together of two or more scaffolds,
heterologous moieties, or linkers by whatever means including
chemical conjugation or recombinant means.
[0108] The terms "multimer," "multimeric scaffold" and "multivalent
scaffold" refer to a molecule that comprises at least two FnIII
scaffolds in association. The scaffolds forming a multimeric
scaffold can be linked through a linker that permits each scaffold
to function independently. "Multimeric" and "multivalent" can be
used interchangeably herein. A multivalent scaffold can be
monospecific or bispecific.
[0109] The terms "domain" or "protein domain" refer to a region of
a protein that can fold into a stable three-dimensional structure,
often independently of the rest of the protein, and which can be
endowed with a particular function. This structure maintains a
specific function associated with the domain's function within the
original protein, e.g., enzymatic activity, creation of a
recognition motif for another molecule, or to provide necessary
structural components for a protein to exist in a particular
environment of proteins. Both within a protein family and within
related protein superfamilies, protein domains can be
evolutionarily conserved regions. When describing the component of
a multimeric scaffold, the terms "domain," "monomeric scaffold,"
and "module" can be used interchangeably. By "native FnIII domain"
is meant any non-recombinant FnIII domain that is encoded by a
living organism.
[0110] The term ""sequence homology"" in relation to protein
sequences refers to the similarity between two or more protein
sequences, i.e., the percentage of amino acid residues that are
either identical or conservative amino acid substitutions.
[0111] The terms "Percent (%) sequence similarity" and "Percent (%)
homology" as used herein are considered equivalent and are defined
as the percentage of amino acid residues in a candidate sequence
that are identical with or conservative substitutions of the amino
acid residues in a selected sequence, after aligning the amino acid
sequences and introducing gaps in the candidate and/or selected
sequences, if necessary, to achieve the maximum percent sequence
similarity.
[0112] "Percent (%) identity" is defined herein 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 in the candidate and/or selected
sequence, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative amino acid
substitutions as part of the sequence identity.
[0113] The term "conservative substitution" as used herein denotes
the replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative substitutions include the
substitution of one hydrophobic amino acid residue such as
isoleucine, valine, leucine, alanine, cysteine, glycine,
phenylalanine, proline, tryptophan, tyrosine, norleucine, or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine and
vice versa, of glutamic acid for aspartic acid, and vice versa,
glutamine for asparagine, and vice versa, and the like. Neutral
hydrophilic amino acids which can be substituted for one another
include asparagine, glutamine, serine and threonine. The term
"conservative substitution" also includes the use of a substituted
amino acid in place of an unsubstituted parent amino acid provided
that the biologic activity of the peptide is maintained. Biological
similarity between amino acid residues refers to similarities
between properties such as, but not limited to, hydrophobicity,
mutation frequency, charge, side chain length, size chain volume,
pKa, polarity, aromaticity, solubility, surface area, peptide bond
geometry, secondary structure propensity, average solvent
accessibility, etc.
[0114] Alignment for purposes of determining percent homology
(i.e., sequence similarity) or percent identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly or proprietary algorithms. For instance, sequence
similarity can be determined using pairwise alignment methods,
e.g., BLAST, BLAST-2, ALIGN, or ALIGN-2 or multiple sequence
alignment methods such as Megalign (DNASTAR), ClustalW or T-Coffee
software. Those skilled in the art can determine appropriate
scoring functions, e.g., gap penalties or scoring matrices for
measuring alignment, including any algorithms needed to achieve
optimal alignment quality over the full-length of the sequences
being compared. Furthermore, those skilled in the art would
appreciate that methods to identify proteins with a certain fold,
e.g., the FnIII fold, and to align the amino acid sequences of such
proteins, include sequence-sequence methods, sequence-profile
methods, and profile-profile methods. In addition, sequence
alignment can be achieved using structural alignment methods (e.g.,
methods using secondary or tertiary structure information to align
two or more sequences), or hybrid methods combining sequence,
structural, and phylogenetic information to identify and optimally
align candidate protein sequences.
[0115] A "protein sequence" or "amino acid sequence" means a linear
representation of the amino acid constituents in a polypeptide in
an amino-terminal to carboxyl-terminal direction in which residues
that neighbor each other in the representation are contiguous in
the primary structure of the polypeptide.
[0116] The term "nucleic acid" refers to any two or more covalently
bonded nucleotides or nucleotide analogs or derivatives. As used
herein, this term includes, without limitation, DNA, RNA, and PNA.
"Nucleic acid" and "polynucleotide" are used interchangeably
herein.
[0117] The term "polynucleotide" is intended to encompass a
singular nucleic acid as well as plural nucleic acids, and refers
to an isolated nucleic acid molecule or construct, e.g., messenger
RNA (mRNA) or plasmid DNA (pDNA). The term "isolated" nucleic acid
or polynucleotide is intended refers to a nucleic acid molecule,
DNA or RNA, that has been removed from its native environment. For
example, a recombinant polynucleotide encoding, e.g., a scaffold of
the invention contained in a vector is considered isolated for the
purposes of the present invention. Further examples of an isolated
polynucleotide include recombinant polynucleotides maintained in
heterologous host cells or purified (partially or substantially)
polynucleotides in solution. Isolated RNA molecules include in vivo
or in vitro RNA transcripts of polynucleotides of the present
invention. Isolated polynucleotides or nucleic acids according to
the present invention further include such molecules produced
synthetically. In addition, a polynucleotide or a nucleic acid can
be or can include a regulatory element such as a promoter, ribosome
binding site, or a transcription terminator.
[0118] The term "pharmaceutically acceptable" refers to a compound
or protein that can be administered to an animal (for example, a
mammal) without significant adverse medical consequences.
[0119] The term "physiologically acceptable carrier" refers to a
carrier which does not have a significant detrimental impact on the
treated host and which retains the therapeutic properties of the
compound with which it is administered. One exemplary
physiologically acceptable carrier is physiological saline. Other
physiologically acceptable carriers and their formulations are
known to one skilled in the art and are described, for example, in
Remington's Pharmaceutical Sciences, (18.sup.th edition), ed. A.
Gennaro, 1990, Mack Publishing Company, Easton, Pa., incorporated
herein by reference.
[0120] By a "polypeptide" is meant any sequence of two or more
amino acids linearly linked by amide bonds (peptide bonds)
regardless of length, post-translation modification, or function.
"Polypeptide," "peptide," and "protein" are used interchangeably
herein. Thus, peptides, dipeptides, tripeptides, or oligopeptides
are included within the definition of "polypeptide," and the term
"polypeptide" can be used instead of, or interchangeably with any
of these terms. The term "polypeptide" is also intended to refer to
the products of post-expression modifications of the polypeptide,
including without limitation glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or modification
by non-naturally occurring amino acids. A polypeptide can be
derived from a natural biological source or produced by recombinant
technology, but is not necessarily translated from a designated
nucleic acid sequence. A polypeptide can be generated in any
manner, including by chemical synthesis.
[0121] Also included as polypeptides of the present invention are
fragments, derivatives, analogs, or variants of the foregoing
polypeptides, and any combination thereof. Variants can occur
naturally or be non-naturally occurring. Non-naturally occurring
variants can be produced using art-known mutagenesis techniques.
Variant polypeptides can comprise conservative or non-conservative
amino acid substitutions, deletions, or additions. Also included as
"derivatives" are those peptides that contain one or more naturally
occurring amino acid derivatives of the twenty standard amino
acids.
[0122] The term "derived from [e.g., a protein or a
polynucleotide]" means that a protein or polynucleotide is related
to a reference protein or polynucleotide. The relation can be, for
example, one of sequence or structural similarity. A protein or
polynucleotide can be derived from a reference protein or
polynucleotide via one or more of, e.g., mutation (e.g., deletion
or substitution), chemical manipulation (e.g., chemical conjugation
of a scaffold to PEG or to another protein), genetic fusion (e.g.,
genetic fusion of two or more scaffolds to a linker, a heterologous
moiety, or combinations thereof), de novo synthesis based on
sequence or structural similarity, or recombinant production in a
heterologous organism.
[0123] By "randomized" or "mutated" is meant including one or more
amino acid alterations, including deletion, substitution or
addition, relative to a template sequence. By "randomizing" or
"mutating" is meant the process of introducing, into a sequence,
such an amino acid alteration. Randomization or mutation can be
accomplished through intentional, blind, or spontaneous sequence
variation, generally of a nucleic acid coding sequence, and can
occur by any technique, for example, PCR, error-prone PCR, or
chemical DNA synthesis. The terms "randomizing", "randomized",
"mutating", "mutated" and the like are used interchangeably
herein.
[0124] By a "cognate" or "cognate, non-mutated protein" is meant a
protein that is identical in sequence to a variant protein, except
for the amino acid mutations introduced into the variant protein,
wherein the variant protein is randomized or mutated.
[0125] By "RNA" is meant a sequence of two or more covalently
bonded, naturally occurring or modified ribonucleotides. One
example of a modified RNA included within this term is
phosphorothioate RNA.
[0126] The terms "scaffold of the invention" or "scaffolds of the
invention" as used herein, refers to multimeric scaffolds as well
as monomeric FnIII scaffolds. The term "target" refers to a
compound recognized by a specific scaffold of the invention.
Typical targets include proteins, polysaccharides, polynucleotides
and small molecules. The terms "target" and "antigen" are used
interchangeably herein. The term "specificity" as used herein,
e.g., in the terms "specifically binds" or "specific binding,"
refers to the relative affinity by which a scaffold of the
invention binds to one or more antigens via one or more antigen
binding domains, and that binding entails some complementarity
between one or more antigen binding domains and one or more
antigens. According to this definition, a scaffold of the invention
is said to "specifically bind" to an epitope when it binds to that
epitope more readily than it would bind to a random, unrelated
epitope.
[0127] The term "affinity" as used herein refers to a measure of
the strength of the binding of a certain scaffold of the invention
to an individual epitope.
[0128] The term "avidity" as used herein refers to the overall
stability of the complex between a population of scaffolds of the
invention and a certain epitope, i.e., the functionally combined
strength of the binding of a plurality of scaffolds with the
antigen. Avidity is related to both the affinity of individual
antigen-binding domains with specific epitopes, and also the
valency of the scaffold of the invention.
[0129] The term "action on the target" refers to the binding of a
multimeric scaffold of the invention to one or more targets and to
the biological effects resulting from such binding. In this
respect, multiple antigen binding units in a multimeric scaffold
can interact with a variety of targets and/or epitopes and, for
example, bring two targets physically closer, trigger metabolic
cascades through the interaction with distinct targets, etc.
[0130] The term "valency" as used herein refers to the number of
potential antigen-binding modules, e.g., the number of FnIII
modules in a scaffold of the invention. When a scaffold of the
invention comprises more than one antigen-binding module, each
binding module can specifically bind, e.g., the same epitope or a
different epitope, in the same target or different targets.
[0131] The term "disulfide bond" as used herein includes the
covalent bond formed between two sulfur atoms. The amino acid
cysteine comprises a thiol group that can form a disulfide bond or
bridge with a second thiol group.
[0132] The terms "Tn3 module" and "Tn3 scaffold" as used herein,
refers to a FnIII scaffold wherein the A beta strand comprises SEQ
ID NO: 42, the B beta strand comprises SEQ ID NO: 43, the C beta
strand SEQ ID NO: 45 or 131, the D beta strand comprises SEQ ID NO:
46, the E beta strand comprises SEQ ID NO: 47, the F beta strand
comprises SEQ ID NO: 49, and the beta strand G comprises SEQ ID NO:
52, wherein at least one loop is a non-naturally occurring variant
of the loops in the "wild type Tn3 scaffold." In certain
embodiments, one or more of the beta strands of a Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand (e.g., the cysteine in SEQ
ID NOs: 45 or 131) and F beta strands (SEQ ID NO: 49) are not
substituted.
[0133] The term "wild type Tn3 scaffold" as used herein refers to
an FnIII scaffold comprising SEQ ID NO: 1, i.e., an engineered
FnIII scaffold derived from the 3.sup.rd FnIII of human tenascin
C.
[0134] The term "immunoglobulin" and "antibody" comprises various
broad classes of polypeptides that can be distinguished
biochemically. Those skilled in the art will appreciate that heavy
chains are classified as gamma, mu, alpha, delta, or epsilon. It is
the nature of this chain that determines the "class" of the
antibody as IgG, IgM, IgA IgG, or IgE, respectively. Modified
versions of each of these classes are readily discernable to the
skilled artisan. As used herein, the term "antibody" includes but
not limited to an intact antibody, a modified antibody, an antibody
VL or VL domain, a CH1 domain, a Ckappa domain, a Clambda domain,
an Fc domain (see supra), a CH2, or a CH3 domain.
[0135] As used herein, the term "modified antibody" includes
synthetic forms of antibodies which are altered such that they are
not naturally occurring, e.g., antibodies that comprise at least
two heavy chain portions but not two complete heavy chains (as,
e.g., domain deleted antibodies or minibodies); multispecific forms
of antibodies (e.g., bispecific, trispecific, etc.) altered to bind
to two or more antigens or to different epitopes of a single
antigen). In addition, the term "modified antibody" includes
multivalent forms of antibodies (e.g., trivalent, tetravalent,
etc., antibodies that to three or more copies of the same antigen).
(See, e.g., Antibody Engineering, Kontermann & Dubel, eds.,
2010 Springer Protocols, Springer).
[0136] The terms "TRAIL R2" or "TRAIL R2 receptor" are used
interchangeably herein to refer to the full length TRAIL receptor
sequence and soluble, extracellular domain forms of the receptor
described in Sheridan et al., Science, 277:818-821 (1997); Pan et
al., Science, 277:815-818 (1997), U.S. Pat. Nos. 6,072,047 and
6,342,369: PCT Publ. Nos. WO98/51793, WO98/41629, WO98/35986,
WO99/02653, WO99/09165, WO98/46643, and WO99/11791; Screaton et
al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J.,
16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997).
Representative full length TRAIL receptor sequences are available
at GenBank Accession Nos. AAC51778.1 and 014763.2.
[0137] "TRAIL" or "TRAIL polypeptide" refers to a ligand that binds
to one or more TRAIL receptors, including TRAIL R2, as well as
biologically active fragments thereof. Representative TRAIL
sequences are available at GenBank Accession Nos. AAH32722.1 and
P50591.1.
[0138] The term "CD40L" refers to the CD40 ligand protein also
known as CD154, gp39 or TBAM. CD40L a 33 kDa, Type II membrane
glycoprotein. Additionally, shorter 18 kDa CD154 soluble forms
exist, (also known as sCD40L). Representative human CD40L sequences
are available at GenBank Accession No. AAA35662.1 and at UniProt
Accession No. P29965. Representative murine CD40L sequences are
available at GenBank Accession No. AAI19226.1 and at UniProt
Accession No. P27548.
[0139] The term "in vivo half-life" is used in its normal meaning,
i.e., the time at which 50% of the biological activity of a
polypeptide is still present in the body/target organ, or the time
at which the activity of the polypeptide is 50% of its initial
value. As an alternative to determining functional in vivo
half-life, "serum half-life" may be determined, i.e., the time at
which 50% of the polypeptide molecules circulate in the plasma or
bloodstream prior to being cleared. Determination of
serum-half-life is often more simple than determining functional in
vivo half-life and the magnitude of serum-half-life is usually a
good indication of the magnitude of functional in vivo half-life.
Alternative terms to serum half-life include plasma half-life,
circulating half-life, circulatory half-life, serum clearance,
plasma clearance, and clearance half-life. The functionality to be
retained is normally selected from procoagulant, proteolytic,
co-factor binding, receptor binding activity, or other type of
biological activity associated with the particular protein.
[0140] The term "increased" with respect to the functional in vivo
half-life or plasma half-life is used to indicate that the relevant
half-life of the polypeptide is statistically significantly
increased relative to that of a reference molecule (for example an
unmodified polypeptide), as determined under comparable conditions.
For instance the relevant half-life may be increased by at least
about 25%, such as by at least about 50%, e.g., by at least about
100%, at least about 150%, at least about 200%, at least about
250%, or at least about 500% compared to an unmodified reference
molecule. In other embodiments, the half-life may be increased by
about at least 1 fold, at least 2 fold, at least 3 fold, at least 4
fold, at least 5 fold, at least 10 fold, at least 20 fold, or at
least 50 fold as compared to an unmodified reference molecule.
[0141] The term "expression" as used herein refers to a process by
which a gene produces a biochemical, for example, a scaffold of the
invention or a fragment thereof. The process includes any
manifestation of the functional presence of the gene within the
cell including, without limitation, gene knockdown as well as both
transient expression and stable expression. It includes without
limitation transcription of the gene into one or more mRNAs, and
the translation of such mRNAs into one or more polypeptides. If the
final desired product is a biochemical, expression includes the
creation of that biochemical and any precursors.
[0142] An "expression product" can be either a nucleic acid, e.g.,
a messenger RNA produced by transcription of a gene, or a
polypeptide. Expression products described herein further include
nucleic acids with post transcriptional modifications, e.g.,
polyadenylation, or polypeptides with post translational
modifications, e.g., methylation, glycosylation, the addition of
lipids, association with other protein subunits, proteolytic
cleavage, and the like.
[0143] The term "vector" or "expression vector" is used herein to
mean vectors used in accordance with the present invention as a
vehicle for introducing into and expressing a desired expression
product in a host cell. As known to those skilled in the art, such
vectors can easily be selected from the group consisting of
plasmids, phages, viruses and retroviruses. In general, vectors
compatible with the instant invention will comprise a selection
marker, appropriate restriction sites to facilitate cloning of the
desired nucleic acid and the ability to enter and/or replicate in
eukaryotic or prokaryotic cells.
[0144] The term "host cells" refers to cells that harbor vectors
constructed using recombinant DNA techniques and encoding at least
one expression product. In descriptions of processes for the
isolation of an expression product from recombinant hosts, the
terms "cell" and "cell culture" are used interchangeably to denote
the source of the expression product unless it is clearly specified
otherwise, i.e., recovery of the expression product from the
"cells" means either recovery from spun down whole cells, or
recovery from the cell culture containing both the medium and the
suspended cells.
[0145] The terms "treat" or "treatment" as used herein refer to
both therapeutic treatment and prophylactic or preventative
measures, wherein the object is to prevent or slow down (lessen) an
undesired physiological change or disorder in a subject, such as
the progression of an inflammatory disease or condition. Beneficial
or desired clinical results include, but are not limited to,
alleviation of symptoms, diminishment of extent of disease,
stabilized (i.e., not worsening) state of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable.
[0146] The term "treatment" also means prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0147] The terms "subject," "individual," "animal," "patient," or
"mammal" refer to any individual, patient or animal, in
particularly a mammalian subject, for whom diagnosis, prognosis, or
therapy is desired. Mammalian subjects include humans, domestic
animals, farm animals, and zoo, sports, or pet animals such as
dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows,
and so on.
Introduction
[0148] FnIII scaffolds derived from whole, stable, and soluble
structural modules found in human body fluid proteins and from
other sources in nature, including but not limited to, thermophilic
bacteria and archaea, have been engineered to be superior both to
antibody-derived fragments and to non-antibody frameworks. One
particular example of scaffold engineering is the introduction of
at least one non-naturally occurring intramolecular disulfide bond
in an FnIII scaffold. In one embodiment, the multimeric scaffolds
of the invention comprise tandem repeats of these FnIII scaffolds
wherein at least one FnIII scaffold comprises one non-naturally
occurring intramolecular disulfide bond. In some embodiments, the
tandem scaffolds are fused by a peptide linker, thereby allowing
expression as a single construct.
[0149] The FnIII scaffolds that make up the multimeric scaffolds
correctly fold independently of each other, retain their binding
specificity and affinity, and each of the scaffold domains retains
its functional properties. When the FnIII scaffolds that make up
the multimeric scaffolds are assembled in high valency multimeric
scaffolds, e.g., hexavalent or octavalent scaffolds, the scaffolds
correctly fold independently of each other, retain their binding
specificity and affinity, and each of the scaffold domains retains
its functional properties.
[0150] Multimeric scaffolds, including high valency scaffolds
(e.g., hexavalent or octavalent), fold correctly even when the
topology of construct is not linear, e.g., when the monomeric FnIII
or multimeric FnIII scaffolds are assembled in complex branched
structures (e.g., Fc fusion constructs or antibody-like
constructs).
[0151] Native FnIII domains such as the 10th FnIII domain of human
fibronectin (10FnIII) and the vast majority of naturally occurring
FnIII domains contain no disulfide bonds or free cysteines. When
multidomain proteins are engineered by introducing multiple
cysteines, lack of protein expression, precipitation of the
resulting proteins, or production of non-functional proteins, are
common occurrences. These deleterious effects are due to the
incorrect formation of intramolecular intradomain and/or
interdomain disulfide bonds, and/or the incorrect formation of
intermolecular disulfide bonds, which result in incorrect protein
folding. These effects are generally intensified when the number of
cysteines and/or protein domains is increased.
[0152] For example, a linear FnIII scaffold comprising 8 wild type
Tn3 scaffolds (SEQ ID NO: 1) would contain 16 cysteines along a
single polypeptide amino acid sequence. In another exemplary
embodiment, an antibody-like construct comprising 4 Tn3 modules,
wherein two Tn3 modules are linked to IgG heavy chains and two Tn3
are linked to IgG light chains, would comprise 32 cysteines
distributed among 4 different polypeptide chains. Accordingly, it
is highly unexpected that multimeric FnIII scaffolds comprising
such number of cysteines and such structural complexity will fold
correctly and display improved stability and target binding
properties when compared to their respective FnIII monomeric
domains.
[0153] When FnIII scaffolds comprising one or more engineered
disulfide bridges are assembled in high valency multimeric formats,
each individual monomer scaffold folds correctly retaining its
binding specificity and affinity, as well as its functional
properties. In addition, the monomeric scaffolds are capable of
forming stable, functional, and correctly folded multimeric
scaffolds.
[0154] An advantage of the multimeric scaffolds of the invention is
their ability to bind to multiple epitopes, e.g., (i) binding to
multiple epitopes in a single target, (ii) binding to a single
epitope in multiple targets, (iii) binding to multiple epitopes
located on different subunits of one target, or (iv) binding to
multiple epitopes on multiple targets, thus increasing avidity.
[0155] In addition, due to the flexibility of the multimeric
scaffolds and to the possibility of varying the distance between
multiple FnIII modules via linkers, the multimeric scaffolds are
capable of binding to multiple target molecules on a surface
(either on the same cell/surface or in different
cells/surfaces).
[0156] As a result of their ability to bind simultaneously to more
than one target, the multimeric scaffolds of the invention can be
used to modulate multiple pathways, cross-link receptors on a cell
surface, bind cell surface receptors on separate cells, and/or bind
target molecules or cells to a substrate.
[0157] From prior sequence analysis of FnIII domains, large
variations were seen in the BC and FG loops, suggesting that these
loops are not crucial to stability (see, for example, PCT
Publication No: WO 2009/058379). The present invention provides
FnIII scaffolds having improved stability, which vary in amino acid
sequence but which comprise an FG loop having a shorter length than
that of a FnIII domain/scaffold of interest. Although the amino
acids sequences of FnIII domains tend to show low sequence
similarity, their overall three dimensional structure is similar.
Accordingly, using known techniques, such as sequence analysis and
tertiary structure overlay, the specific locations of FG loops of
FnIII scaffolds from different species and different proteins, even
when overall sequence similarity is low, can be identified and be
subjected to mutation. In some embodiments, the engineered FG loop
has an amino acid sequence length that is at least one amino acid
shorter than the length of the starting FG loop. It has been
observed that shortening the FG loops results in a mutated FnIII
scaffold that has increased stability. Consequently, another aspect
of the invention provides FnIII variants having increased protein
stability.
[0158] In certain embodiments, the scaffold of the invention
comprises an FG loop having 9 amino acids and an increased
stability compared to a scaffold comprising the native third FnIII
domain of human tenascin C which has an FG loop length of 10 amino
acids. Additionally the present invention provides libraries of
diverse FnIII scaffolds having specified FG loop lengths which are
useful for isolating FnIII scaffolds having increased stability as
compared to a FnIII domain/scaffold of interest.
[0159] In addition, the present invention provides multispecific
scaffolds that can bind to two or more different targets, affinity
matured scaffolds wherein the affinity of a scaffold for a specific
target is modulated via mutation, and scaffolds whose
immunogenicity and/or cross-reactivity among animal species is
modulated via mutation. Also, the invention provides methods to
produce the scaffolds of the invention as well as methods to
engineer scaffolds with desirable physicochemical, pharmacological,
or immunological properties. Furthermore, the present invention
provides uses for such scaffolds and methods for therapeutic,
prophylactic, and diagnostic use.
The FnIII Structural Motif
[0160] The scaffolds of the present invention are based on the
structure of a fibronectin module of type III (FnIII), a domain
found widely across all three domains of life and viruses, and in
multitude of protein classes. The FnIII domain is found in
fibronectins, multidomain-proteins found in soluble form in blood
plasma and in insoluble form in loose connective tissue and
basement proteins This domain is found in numerous proteins
sequenced to date. The FnIII domain superfamily represents at least
45 different protein families, the majority of which are involved
in cell surface binding in some manner, or function as receptors.
Specific examples of proteins containing FnIII domains include
fibronectins, tenascins, intracellular cytoskeletal proteins,
cytokine receptors, receptor protein tyrosine kinases, and
prokaryotic enzymes (Bork and Doolittle, Proc. Natl. Acad. Sci. USA
89:8990-8894, 1992; Bork et al., Nature Biotechnol. 15:553-557,
1997; Meinke et al., J. Bacteriol. 175:1910-1918, 1993; Watanabe et
al., J. Biol. Chem. 265:15659-15665, 1990).
[0161] Naturally occurring protein sequences comprising FnIII
domains include but are not limited to fibronectin, tenascin C,
growth hormone receptor, .beta.-common receptor, IL-5R, tenascin
XB, and collagen type XIV. Although the domain appears widely
distributed in nature, the percentage of amino acid sequence
similarity between the amino acid sequences of highly divergent
FnIII domains can be very low.
[0162] In specific embodiments, the scaffolds of the invention are
derived from the third FnIII domain of human tenascin C (SEQ ID NO:
4). In one specific embodiment, the scaffolds of the invention
comprise a Tn3 module. The overall three dimensional fold of this
domain is closely related to that of the smallest functional
antibody fragment, the variable region of the heavy chain (VH),
which in the single domain antibodies of camels and camelids (e.g.,
llamas) comprises the entire antigen recognition unit.
[0163] The FnIII scaffolds of the invention and the native FnIII
domains are characterized by the same three dimensional structure,
namely a beta-sandwich structure with three beta strands (A, B, and
E) on one side and four beta strands (C, D, F, and G) on the other
side, connected by six loop regions. These loop regions are
designated according to the beta-strands connected to the N- and
C-terminus of each loop. Accordingly, the AB loop is located
between beta strands A and B, the BC loop is located between
strands B and C, the CD loop is located between beta strands C and
D, the DE loop is located between beta strands D and E, the EF loop
is located between beta strands E and F, and the FG loop is located
between beta strands F and G. FnIII domains possess solvent exposed
loops tolerant of randomization, which facilitates the generation
of diverse pools of protein scaffolds capable of binding specific
targets with high affinity.
[0164] The multiple sequence alignment shown in FIG. 16 identifies
the positions of the beta strands and loops for numerous native
FnIII domains based on the analysis of their three dimensional
structures and amino acid sequences. These FnIII domains can be
utilized to design proteins which are capable of binding to
virtually any target compound, for example, any protein of
interest. One skilled in the art will appreciate that the alignment
shown in FIG. 16 is exemplary and non-limiting. For example, the
alignment of FIG. 16 may be expanded by incorporating protein
domains recognized to contain the Interpro IPRO08957 fibronectin
type III domain signature as determined using the InterProScan
program, or recognized to contain the Pfam PF00041 fibronectin type
III domain signature as determined using Pfam_scan, HMMER, or any
other program capable of comparing a protein sequence to a Hidden
Markov model.
[0165] Thus protein scaffold engineering and design can be based
on, e.g.,
[0166] (i) the aligned sequence set shown in FIG. 16,
[0167] (ii) a subset of aligned sequences derived from FIG. 16,
[0168] (iii) a different aligned set comprising amino acid
sequences of FnIII domains [0169] whose three dimensional structure
has been determined experimentally (e.g., through the use of X-ray
diffraction crystallography or NMR), and/or [0170] (iv) amino acid
sequences of FnIII domains whose three dimensional structure is not
yet available but recognized to contain the Interpro IPRO08957
fibronectin type III domain signature as determined using the
InterProScan program, or recognized to contain the Pfam PF00041
fibronectin type III domain signature as determined using
Pfam_scan, HMMER, or any other program capable of comparing a
protein sequence to a Hidden Markov model.
[0171] In one aspect of the invention, FnIII domains are used as
scaffolds which are subjected to directed evolution designed to
randomize one or more of the loops which are analogous to the
complementarity-determining regions (CDRs) of an antibody variable
region. Such a directed evolution approach results in the
production of antibody-like molecules with high affinities for
targets of interest. In addition, in some embodiments the scaffolds
described herein can be used to display defined exposed loops (for
example, loops previously randomized and selected on the basis of
target binding) in order to direct the evolution of molecules that
bind to such introduced loops. This type of selection can be
carried out to identify recognition molecules for any individual
CDR-like loop or, alternatively, for the recognition of two or all
three CDR-like loops combined into a nonlinear epitope binding
moiety.
[0172] In some embodiments, the scaffolds of the invention are
molecules based on the third FnIII domain of human tenascin C
structural motif described in PCT Publication No: WO 2009/058379. A
set of three loops (designated BC, DE, and FG), which can confer
specific target binding, run between the B and C strands; the D and
E strands, and the F and G beta strands, respectively. The BC, DE,
and FG loops of the third FnIII domain of human tenascin C are 9,
6, and 10 amino acid residues long, respectively. The length of
these loops falls within the narrow range of the cognate
antigen-recognition loops found in antibody heavy chains, that is,
7-10, 4-8, and 4-28 amino acids in length, respectively. Similarly,
a second set of loops, the AB, CD, and EF loops (7, 7, and 8, amino
acids in length respectively) run between the A and B beta strands;
the C and D beta strands; and the E and F beta strands,
respectively.
[0173] In other embodiments, molecules based on the tenth FnIII
("10FnIII") domain derived from human fibronectin (SEQ ID NO: 54)
can be used as scaffolds. As defined in FIG. 16, in the tenth FnIII
domain of human fibronectin the AB loop corresponds to SEQ ID NO:
55, the BC loop corresponds to SEQ ID NO:56, the CD loop
corresponds to SEQ ID NO: 57, the DE loop corresponds to SEQ ID NO:
58, the EF loop corresponds to SEQ ID NO: 59, and the FG loop
corresponds to SEQ ID NO: 60. It will be understood that
alternative definitions for these regions are known in the art, see
for example, Xu et al. Chemistry & Biology 9:933-942, 2002,
which may be used as described herein.
[0174] In still other embodiments, molecules based on the
fourteenth FnIII ("14FnIII") domain derived from human fibronectin
(SEQ ID NO: 69) can be used as scaffolds. As defined in FIG. 16,
the AB loop of 14FnIII corresponds to SEQ ID NO: 70, the BC loop
corresponds to SEQ ID NO: 71, the CD loop corresponds to SEQ ID NO:
72, the DE loop corresponds to SEQ ID NO: 73, the EF loop
corresponds to SEQ ID NO: 74, and the FG loop corresponds to SEQ ID
NO: 75. It will be understood that alternative definitions for
these regions are known in the art, see for example, Cappuccilli et
al. (U.S. Patent Publication No. 2009/0176654) which may be used as
described herein.
[0175] In still other embodiments, molecules based on a consensus
sequence derived from the sequence of FnIII domains of Tenascin
(SEQ ID NO: 256) can be used as scaffolds. The loops of a Tenascin
consensus FnIII are defined in Table 1, the AB loop corresponds to
SEQ ID NO: 257, the BC loop corresponds to SEQ ID NO: 258, the CD
loop corresponds to SEQ ID NO: 259, the DE loop corresponds to SEQ
ID NO: 260, the EF loop corresponds to SEQ ID NO: 261, and the FG
loop corresponds to SEQ ID NO: 262. It will be understood that
alternative definitions for these regions are known in the art, see
for example, Jacobs et al. (International Patent Publication No. WO
2010/093627) which may be used as described herein.
[0176] Once randomized and selected for high affinity binding to a
target, the loops in the FnIII domain may make contacts with
targets equivalent to the contacts of the cognate CDR loops in
antibodies. Accordingly, in some embodiments the AB, CD, and EF
loops, alone or in combination, are randomized and selected for
high affinity binding to one or more targets. In some embodiments,
this randomization and selection process may be performed in
parallel with the randomization of one or more of the BC, DE, and
FG loops, whereas in other embodiments this randomization and
selection process is performed in series.
Monomeric Scaffolds of the Invention
[0177] The invention provides recombinant, non-naturally occurring
FnIII scaffolds comprising, a plurality of beta strand domains
linked to a plurality of loop regions, wherein one or more of said
loop regions vary by deletion, substitution or addition of at least
one amino acid from the cognate loops in a FnIII domain/scaffold of
interest (referred to herein as an "FOI"), and wherein the beta
strands of the FnIII scaffold have at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 99%, or more
homology (i.e., sequence similarity) to the cognate beta strands of
the FOI.
[0178] The FOI is a reference used for comparing sequence,
physicochemical and/or phylogenetic characteristics. It will be
understood that, when comparing the sequence of a scaffold of the
invention to the sequence of an FOI, the same definition of the
beta strands and loops is utilized. The FOI can be a native FnIII
domain, a scaffold comprising a native FnIII domain or a
non-naturally occurring FnIII scaffold. In certain embodiments, the
FOI comprises at least one non-naturally occurring loop. In certain
embodiments, the FOI comprises at least one non-naturally occurring
beta strand. In certain embodiments, the FOI comprises at least one
non-naturally occurring loop and at least one non-naturally
occurring beta strand. In certain embodiments, the FOI comprises at
least one non-naturally occurring disulfide bond. In a specific
embodiment, the FOI comprises a wild type Tn3 scaffold (SEQ ID
NO:1), a scaffold derived from the third FnIII domain of human
tenascin that contains an engineered intramolecular disulfide
bond.
[0179] In a specific embodiment, the monomer scaffolds of the
invention comprise seven beta strands, designated A, B, C, D, E, F,
G, linked to six loop regions, designated AB, BC, CD, DE, EF, FG,
wherein at least one loop is a non-naturally occurring variant of
the cognate loop in an FOI and the beta strands have at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99% or more homology (i.e., sequence similarity) to the
cognate beta strands of the FOI.
[0180] In a specific embodiment, the monomer scaffolds of the
invention comprise seven beta strands, designated A, B, C, D, E, F,
G, linked to six loop regions, designated AB, BC, CD, DE, EF, FG,
wherein at least one loop is a non-naturally occurring variant of
the cognate loop in an FOI and the beta strands have at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99% or more identity to the cognate domain of the FOI.
[0181] In a specific embodiment, the FOI comprises a third FnIII
domain of human tenascin C (SEQ ID NO: 4). In one embodiment, the
scaffolds of the invention comprise a sequence having at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99% or more homology (i.e., sequence similarity) to the third
FnIII domain of human tenascin C (SEQ ID NO:4).
[0182] In one embodiment, the scaffolds of the invention comprise a
sequence having at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 99% or more identity to the third
FnIII domain of human tenascin C (SEQ ID NO:4).
[0183] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKET FTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein n=2-26.
[0184] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nIELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIS(X.sub.FG).sub.nKETF TT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein n=2-26.
[0185] In one embodiment, X.sub.AB consists of SEQ ID NO: 35. In
one embodiment, X.sub.BC consists of SEQ ID NO: 36. In one
embodiment, X.sub.CD consists of SEQ ID NO: 37. In one embodiment,
X.sub.DE consists of SEQ ID NO: 38. In one embodiment, X.sub.EF
consists of SEQ ID NO: 39. In one embodiment, X.sub.FG consists of
SEQ ID NO: 40.
[0186] In one embodiment, X.sub.AB comprises SEQ ID NO: 35. In one
embodiment, X.sub.BC comprises SEQ ID NO: 36. In one embodiment,
X.sub.CD comprises SEQ ID NO: 37. In one embodiment, X.sub.DE
comprises SEQ ID NO: 38. In one embodiment, X.sub.EF comprises SEQ
ID NO: 39. In one embodiment, X.sub.FG comprises SEQ ID NO: 40.
[0187] In certain embodiments, X.sub.AB consists of SEQ ID NO: 35,
X.sub.CD consists of SEQ ID NO: 37, and X.sub.EF consists of SEQ ID
NO: 39. In one embodiment, X.sub.BC consists of SEQ ID NO: 36,
X.sub.DE consists of SEQ ID NO: 38, and X.sub.FG consists of SEQ ID
NO: 40.
[0188] In certain embodiments, X.sub.AB comprises SEQ ID NO: 35,
X.sub.CD comprises SEQ ID NO: 37, and X.sub.EF comprises SEQ ID NO:
39. In one embodiment, X.sub.BC comprises SEQ ID NO: 36, X.sub.DE
comprises SEQ ID NO: 38, and X.sub.FG comprises SEQ ID NO: 40.
[0189] In a specific embodiment, the FOI comprises a wild type
tenth fibronectin type III domain (10FnIII) of human fibronectin
scaffold (SEQ ID NO: 54). In one embodiment, the scaffolds of the
invention comprise a sequence having at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 99% or more
similarity to wild type 10FnIII (SEQ ID NO: 54).
[0190] In one embodiment, the monomer scaffolds of the invention
comprise a sequence having at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99% or more
identity to wild type 10FnIII (SEQ ID NO: 54).
[0191] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence:
LEV(X.sub.AB).sub.nLLISW(X.sub.BC).sub.nYRITYGE(X.sub.CD).sub.nQEFTV(X.su-
b.DE).sub.nATI(X.sub.EF).sub.nYTITVYA (X.sub.FG).sub.nSINYRT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, and wherein n=2-26.
[0192] In one embodiment, X.sub.AB is the amino acid sequence of
loop AB of 10FnIII (SEQ ID NO: 55). In one embodiment, X.sub.BC is
the amino acid sequence of loop BC of wild type 10FnIII (SEQ ID NO:
56). In one embodiment, X.sub.CD is the amino acid sequence of loop
CD of wild type 10FnIII (SEQ ID NO: 57). In one embodiment,
X.sub.DE is the amino acid sequence of loop DE of wild type 10FnIII
(SEQ ID NO: 58). In one embodiment, X.sub.EF is the amino acid
sequence of loop EF of wild type 10FnIII (SEQ ID NO: 59). In one
embodiment, X.sub.FG is the amino acid sequence of loop FG of wild
type 10FnIII (SEQ ID NO: 60).
[0193] In certain embodiments, X.sub.AB is the amino acid sequence
of loop AB of wild type 10FnIII (SEQ ID NO: 55), X.sub.CD is the
amino acid sequence of loop CD of wild type 10FnIII (SEQ ID NO:
57), and X.sub.EF is the amino acid sequence of loop EF of wild
type 10FnIII (SEQ ID NO: 59).
[0194] In one embodiment, X.sub.BC is the amino acid sequence of
loop BC of wild type 10FnIII (SEQ ID NO: 56), X.sub.DE is the amino
acid sequence of loop DE of wild type 10FnIII (SEQ ID NO: 58), and
X.sub.FG is the amino acid sequence of loop FG of wild type 10FnIII
(SEQ ID NO: 60).
[0195] In a specific embodiment, the FOI comprises a wild type
fourteenth type III fibronectin domain (14FnIII) of human
fibronectin scaffold (SEQ ID NO: 69). In one embodiment, the
scaffolds of the invention comprise a sequence having at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99% or more similarity to wild type 14FnIII (SEQ ID NO:
69).
[0196] In one embodiment, the monomer scaffolds of the invention
comprise a sequence having at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99% or more
identity to wild type 14FnIII (SEQ ID NO: 69).
[0197] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence:
ARV(X.sub.AB).sub.nITISW(X.sub.BC).sub.nFQVDAVP(X.sub.CD).sub.nIQRTI(X.su-
b.DE).sub.nYTI(X.sub.EF).sub.nYKIYLYT (X.sub.FG).sub.nVIDAST,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, and wherein n=2-26.
[0198] In one embodiment, X.sub.AB is the amino acid sequence of
loop AB of wild type 14FnIII (SEQ ID NO: 70). In one embodiment,
X.sub.BC is the amino acid sequence of loop BC of wild type 14FnIII
(SEQ ID NO: 71). In one embodiment, X.sub.CD is the amino acid
sequence of loop CD of wild type 14FnIII (SEQ ID NO: 72). In one
embodiment, X.sub.DE is the amino acid sequence of loop DE of wild
type 14FnIII (SEQ ID NO: 73). In one embodiment, X.sub.EF is the
amino acid sequence of loop EF of wild type 14FnIII (SEQ ID NO:
74). In one embodiment, X.sub.FG is the amino acid sequence of loop
FG of wild type 14FnIII (SEQ ID NO: 75).
[0199] In certain embodiments, X.sub.AB is the amino acid sequence
of loop AB of wild type 14FnIII (SEQ ID NO: 70), X.sub.CD is the
amino acid sequence of loop CD of wild type 14FnIII (SEQ ID NO:
72), and X.sub.EF is the amino acid sequence of loop EF of wild
type 14FnIII (SEQ ID NO: 74). In one embodiment, X.sub.BC is the
amino acid sequence of loop BC of wild type 14FnIII (SEQ ID NO:
71), X.sub.DE is the amino acid sequence of loop DE of wild type
14FnIII (SEQ ID NO: 73), and X.sub.FG is the amino acid sequence of
loop FG of wild type 14FnIII (SEQ ID NO: 75).
[0200] In a specific embodiment, the FOI comprises Tenascin
consensus FnIII (SEQ ID NO: 256). In one embodiment, the scaffolds
of the invention comprise a sequence having at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%
or more similarity to Tenascin consensus FnIII (SEQ ID NO:
256).
[0201] In one embodiment, the monomer scaffolds of the invention
comprise a sequence having at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99% or more
identity to Tenascin consensus FnIII (SEQ ID NO: 256).
[0202] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence:
LVV(X.sub.AB).sub.nLRLSW(X.sub.BC).sub.nFLIQYQE(X.sub.CD).sub.nINLTV(X.su-
b.DE).sub.nYDL(X.sub.EF).sub.nYTVSIYG(X.sub.FG).sub.nSA EFTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, and wherein n=2-26.
[0203] In one embodiment, X.sub.AB is the amino acid sequence of AB
loop of Tenascin consensus FnIII (SEQ ID NO: 257). In one
embodiment, X.sub.BC is the amino acid sequence of loop BC of
Tenascin consensus FnIII (SEQ ID NO: 258). In one embodiment,
X.sub.CD is the amino acid sequence of loop CD of Tenascin
consensus FnIII (SEQ ID NO: 259). In one embodiment, X.sub.DE is
the amino acid sequence of loop DE of Tenascin consensus FnIII (SEQ
ID NO: 260). In one embodiment, X.sub.EF is the amino acid sequence
of loop EF of Tenascin consensus FnIII (SEQ ID NO: 261). In one
embodiment, X.sub.FG is the amino acid sequence of loop FG of
Tenascin consensus FnIII (SEQ ID NO: 262).
[0204] In certain embodiments, X.sub.AB is the amino acid sequence
of loop AB of Tenascin consensus FnIII (SEQ ID NO: 257), X.sub.CD
is the amino acid sequence of loop CD of Tenascin consensus FnIII
(SEQ ID NO: 259), and X.sub.EF is the amino acid sequence of loop
EF of Tenascin consensus FnIII (SEQ ID NO: 261). In one embodiment,
X.sub.BC is the amino acid sequence of loop BC of Tenascin
consensus FnIII (SEQ ID NO: 258), X.sub.DE is the amino acid
sequence of loop DE of Tenascin consensus FnIII (SEQ ID NO: 260),
and X.sub.FG is the amino acid sequence of loop FG of Tenascin
consensus FnIII (SEQ ID NO: 262).
[0205] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence selected from the group
consisting of:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nIELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIS(X.sub.FG).sub.nKETF TT,
LEV(X.sub.AB).sub.nLLISW(X.sub.BC).sub.nYRITYGE(X.sub.CD).sub.nQEFTV(X.su-
b.DE).sub.nATI(X.sub.EF).sub.nYTITVYA (X.sub.FG).sub.nSINYRT, ARV
(X.sub.AB).sub.nITISW(X.sub.BC).sub.nFQVDAVP(X.sub.CD).sub.nIQRTI(X.sub.D-
E).sub.nYTI(X.sub.EF).sub.nYKIYLYT(X.sub.FG).sub.nVID AST, and
LVV(X.sub.AB).sub.nLRLSW(X.sub.BC).sub.nFLIQYQE(X.sub.CD).sub.nINLTV(X.su-
b.DE).sub.nYDL(X.sub.EF).sub.nYTVSIYG(X.sub.FG).sub.nSA EFTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, wherein n=2-26, and wherein: X.sub.AB is
selected from the group consisting of SEQ ID NOs: 35, 55, 70, or
257, X.sub.CD is selected from the group consisting of SEQ ID NOs:
37, 57, 72, or 259, and X.sub.EF is selected from the group
consisting of SEQ ID NOs: 39, 59, 74, or 261.
[0206] In another embodiment, the monomer scaffolds of the
invention comprise the amino acid sequence selected from the group
consisting of:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nIELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIS(X.sub.FG).sub.nKETF TT,
LEV(X.sub.AB).sub.nLLISW(X.sub.BC).sub.nYRITYGE(X.sub.CD).sub.nQEFTV(X.su-
b.DE).sub.nATI(X.sub.EF).sub.nYTITVYA (X.sub.FG).sub.nSINYRT,
ARV(X.sub.AB).sub.nITISW(X.sub.BC).sub.nFQVDAVP(X.sub.CD).sub.nIQRTI(X.su-
b.DE).sub.nYTI(X.sub.EF).sub.nYKIYLYT(X.sub.FG).sub.nVID AST, and
LVV(X.sub.AB).sub.nLRLSW(X.sub.BC).sub.nFLIQYQE(X.sub.CD).sub.nINLTV(X.su-
b.DE).sub.nYDL(X.sub.EF).sub.nYTVSIYG(X.sub.FG).sub.nSA EFTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residue present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represent
amino acid residue A or T, wherein n=2-26, and wherein: X.sub.BC is
selected from the group consisting of SEQ ID NOs: 36, 56, 71, or
258, X.sub.DE is selected from the group consisting of SEQ ID NOs:
38, 58, 73, or 260, and X.sub.FG is selected from the group
consisting of SEQ ID NOs: 40, 60, 75, or 262.
[0207] In other embodiments, the scaffolds of the invention
comprise a Tn3 module. In still other embodiments, scaffolds of the
invention comprise a Tn3 module (SEQ ID NO: 1), wherein beta strand
C of a third FnIII domain of human tenascin C (SEQ ID NO; 44) is
replaced by a variant beta strand C (SEQ ID NO: 45, or 131)
comprising an N-terminal cysteine and wherein beta strand F of a
third FnIII domain of human tenascin C (SEQ ID NO: 48) is replaced
by a variant beta strand F (SEQ ID NO: 49) comprising a C-terminal
cysteine. In some embodiments the scaffolds of the invention
comprise a Tn3 module wherein one or more of the beta strands
comprise at least one amino acid substitution except that the
cysteine residues in the C and F beta strands (SEQ ID NOs: 45, or
131 and 49, respectively) may not be substituted. In certain
embodiments, the scaffolds of the invention comprise a variant of a
10FnIII module, wherein one or more of the beta strands comprise at
least one amino acid substitution. In other embodiments, the
scaffolds of the invention comprise a variant of a 14FnIII module,
wherein one or more of the beta strands comprise at least one amino
acid substitution. In still other embodiments, the scaffolds of the
invention comprise a variant of Tenascin consensus FnIII module,
wherein one or more of the beta strands comprise at least one amino
acid substitution.
[0208] In other embodiments, the naturally occurring sequence is a
protein sequence corresponding to an additional FnIII domain from
human tenascin C. In other embodiments, the naturally occurring
sequence is a protein sequence corresponding to a FnIII domain from
another tenascin protein including but not limited to the 29th
FnIII domain from human tenascin XB (SEQ ID NO: 11), the 31st FnIII
domain from human tenascin XB (SEQ ID NO:12), or the 32nd FnIII
domain from human tenascin XB (SEQ ID NO: 13). In other
embodiments, the naturally occurring sequence is a protein sequence
corresponding to an FnIII domain from another organism (such as,
but not limited to, murine, porcine, bovine, or equine
tenascins).
[0209] In additional embodiments, FnIII domains used to generate
scaffolds of the invention, include, e.g., related FnIII domains
from animals, plants, bacteria, archaea, or viruses. Different
FnIII domains from different organisms and parent proteins can be
most appropriate for different applications; for example, in
designing a scaffold stable at a low pH, it can be most desirable
to generate that protein from organism that optimally grows at a
low pH (such as, but not limited to Sulfolobus tokodaii). In
another embodiment, related FnIII domains can be identified and
utilized from thermophilic and hyperthermophilic organisms (e.g.,
hyperthermophilic bacteria or hyperthermophilic archaea). In some
embodiments, FnIII domains used to generate scaffolds of the
invention are FnIII domains from hyperthermophilic archaea such as,
but not limited to, Archaeoglobus fulgidus and Staphylothermus
marinus, each of which exhibit optimal growth at greater than
70.degree. C. In other embodiments, the naturally occurring
sequence corresponds to a predicted FnIII domain from a
thermophilic organism, for example, but not limited to
Archaeoglobus fulgidus, Staphylothermus marinus, Sulfolobus
acidocaldarius, Sulfolobus solfataricus, and Sulfolobus tokodaii.
In yet another embodiment, the scaffolds of the invention comprise
a protein sequence having at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or at least 99% homology (sequence
similarity) to any of the sequences from a sequence corresponding
to a FnIII domain or a predicted FnIII domain from a thermophilic
organism as described above. In some embodiments, the FnIII domains
from thermophilic organisms are selected from the amino acid
sequences of SEQ ID NOs: 20-33.
[0210] The loops connecting the various beta strands of the
scaffolds of the invention can be randomized for length and/or
sequence diversity. In one embodiment, the scaffolds of the
invention have at least one loop that is randomized for length
and/or sequence diversity. In one embodiment, at least one, at
least two, at least three, at least four, at least five or at least
six loops of a scaffold are randomized for length and/or sequence
diversity. In one embodiment, at least one loop of the scaffolds of
the invention is kept constant while at least one additional loop
is randomized for length and/or sequence diversity. In another
embodiment, at least one, at least two, or all three of loops AB,
CD, and EF are kept constant while at least one, at least two, or
all three of loops BC, DE, and FG are randomized for length or
sequence diversity. In another embodiment, at least one, at least
two, or at least all three of loops AB, CD, and EF are randomized
while at least one, at least two, or all three of loops BC, DE, and
FG are randomized for length and/or sequence diversity. In still
another embodiment, at least one, at least two, at least three of
loops, at least 4, at least five, or all six of loops AB, CD, EF,
BC, DE, and FG are randomized for length or sequence diversity.
[0211] In some embodiments, one or more residues within a loop are
held constant while other residues are randomized for length and/or
sequence diversity. In some embodiments, one or more residues
within a loop are held to a predetermined and limited number of
different amino acids while other residues are randomized for
length and/or sequence diversity. Accordingly, scaffolds of the
invention can comprise one or more loops having a degenerate
consensus sequence and/or one or more invariant amino acid
residues. In one embodiment, the scaffolds of the invention
comprise an AB loop which is randomized with the following
consensus sequence: K-X-X-X-X-X-a, wherein X represents asparagine,
aspartic acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine, and wherein
(a) represents serine, threonine, alanine, or glycine. In another
embodiment, the scaffolds of the invention comprise an AB loop
which is randomized with the following consensus sequence:
K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid,
histidine, tyrosine, isoleucine, valine, leucine, phenylalanine,
threonine, alanine, proline, or serine, and wherein (a) represents
serine, threonine, alanine, or glycine.
[0212] In another embodiment, the scaffolds of the invention
comprise a BC loop which is randomized with the following consensus
sequence: S-X-a-X-b-X-X-X-G, wherein X represents any amino acid,
wherein (a) represents proline or alanine and wherein (b)
represents alanine or glycine. In another embodiment, the scaffolds
of the invention comprise a BC loop which is randomized with the
following consensus sequence: S-P-c-X-X-X-X-X-X-T-G, wherein X
represents any amino acid and wherein (c) represents proline,
serine or glycine. In still another embodiment, the scaffolds of
the invention comprise a BC loop which is randomized with the
following consensus sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X
represents any amino acid, wherein (d) represents proline,
glutamate or lysine, wherein (e) represents asparagine or glycine,
and wherein (f) represents serine or glycine.
[0213] In one embodiment, the scaffolds of the invention comprise a
CD loop which is randomized with the following consensus sequence:
X.sub.n, wherein X represents any amino acid, and wherein n=6, 7,
8, 9, or 10. In another embodiment, the scaffolds of the invention
comprise an CD loop which is randomized with the following
consensus sequence: X.sub.n, wherein X represents asparagine,
aspartic acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine, and wherein
n=7, 8, or 9.
[0214] In one embodiment, the scaffolds of the invention comprise
an DE loop which is randomized with the following consensus
sequence: X-X-X-X-X-X, wherein X represents any amino acid.
[0215] In one embodiment, the scaffolds of the invention comprise
an EF loop which is randomized with the following consensus
sequence: X-b-L-X-P-X-c-X, wherein X represents asparagine,
aspartic acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine, wherein (b)
represents asparagine, lysine, arginine, aspartic acid, glutamic
acid, or glycine, and wherein (c) represents isoleucine, threonine,
serine, valine, alanine, or glycine
[0216] In one embodiment, the scaffolds of the invention comprise
an FG loop which is randomized with the following consensus
sequence: X-a-X-X-G-X-X-X-b, wherein X represents any amino acid,
wherein (a) represents asparagine, threonine or lysine, and wherein
(b) represents serine or alanine. In another embodiment, the
scaffolds of the invention comprise an FG loop which is randomized
with the following consensus sequence: X-X-X-X-X-X-X-X-X (X.sub.9),
wherein X represents any amino acid. In still another embodiment,
the scaffolds of the invention comprise an FG loop which is
randomized with the following consensus sequence:
X-a-X-X-X-X-b-N-P-A, wherein X represents any amino acid, wherein
(a) represents asparagine, threonine or lysine and wherein (b)
represents serine or glycine. In a specific embodiment, the
scaffolds of the invention comprise an FG loop which is randomized
with the following consensus sequence: X-a-X-X-G-X-X-S-N-P-A,
wherein X represents any amino acid, and wherein (a) represents
asparagine, threonine or lysine.
[0217] In certain embodiments, the scaffolds of the invention
comprise an FG loop which is held to be at least one amino acid
residue shorter than the cognate FG loop of an FOI and is further
randomized at one or more positions. For example, as defined in
FIG. 16 the native FG loop of the third FnIII domain of human
tenascin C comprises 10 amino acid residues, accordingly, the FG
loop would be held to 9 amino acid residues or less.
[0218] In some embodiments, a scaffold of the invention is a
chimeric scaffold comprising one or more beta strands comprising
amino acid sequences selected from homologous beta strands selected
from a plurality of FOIs. In some embodiments, a scaffold of the
invention is a chimeric scaffold wherein at least one of the loops
BC, DE, and FG are randomized. In some embodiments, a scaffold of
the invention is a chimeric scaffold wherein at least one of loops
AB, CD, and EF is randomized.
[0219] In specific embodiments, at least one of loops BC, DE, and
FG is randomized, wherein the A beta strand comprises SEQ ID NO:41,
42, 61, 62, 76, 77, 248 or 249, the B beta strand comprises SEQ ID
NO:43, 63, 78, or 250, the C beta strand comprises SEQ ID NO:44,
45, 64, 79, 131, or 251, the D beta strand comprises SEQ ID NO:46,
65, 80, or 252, the E beta strand comprises SEQ ID NO:47, 66, 81,
or 253, the F beta strand comprises SEQ ID NO:48, 49, 50, 51, 67,
82, or 254, and the G beta strand comprises SEQ ID NO:52, 53, 68,
83, or 255, the AB loop comprises SEQ ID NO:35, 55, 70, or 242, the
CD loop comprises SEQ ID NO:37, 57, 72, or 244, and the EF loop
comprises SEQ ID NO:39, 59, 74, or 246.
[0220] In other specific embodiments, at least one of loops AB, CD,
and EF are randomized, wherein the A beta strand comprises SEQ ID
NO:41, 42, 61, 62, 76, 77, 248 or 249, the B beta strand comprises
SEQ ID NO:43, 63, 78, or 250, the C beta strand comprises SEQ ID
NO:44, 45, 64, 79, 131, or 251, the D beta strand comprises SEQ ID
NO:46, 65, 80, or 252, the E beta strand comprises SEQ ID NO:47,
66, 81 or 253, the F beta strand comprises SEQ ID NO:48, 49, 50,
51, 67, 82, or 254, and the G beta strand comprises SEQ ID NO:52,
53, 68, 83, or 255, the BC loop comprises SEQ ID NO:36, 56, 71, or
243, the DE loop comprises SEQ ID NO:38, 58, 73, 245 and the FG
loop comprises SEQ ID NO:40, 60, 75, or 247.
Enhanced Scaffold Stability
Non-Naturally Occurring Disulfide Bonds
[0221] The stability of scaffolds of the invention may be increased
by many different approaches. In some embodiments, scaffolds of the
invention can be stabilized by elongating the N- and/or C-terminal
regions. The N- and/or C-terminal regions can be elongated by 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acids. In other
embodiments, the scaffolds of the invention can be stabilized by
introducing an alteration that increases serum half-life, as
described herein. In yet another embodiment, the scaffolds of the
invention comprise an addition, deletion or substitution of at
least one amino acid residue to stabilize the hydrophobic core of
the scaffold.
[0222] Scaffolds of the invention also can be effectively
stabilized by engineering non-natural disulfide bonds. Such
engineered scaffolds can be efficiently expressed as part of
multimeric scaffolds. The correct formation of the disulfide bonds
and the correct folding of the engineered scaffold are evidenced by
the preservation of the biological activity of the scaffold. The
fact that an engineered scaffold comprising non-natural disulfide
bonds can bind simultaneously to at least two targets (see, e.g.,
Example 8) or three targets (see, e.g., Example 12) provides
evidence that the three dimensional structure of the scaffold is
not significantly altered by the engineered disulfide bonds and
that the relative positions of the target-binding loops are
preserved. In some embodiments, scaffolds of the invention comprise
non-naturally occurring disulfide bonds, as described in PCT
Publication No: WO 2009/058379. A bioinformatics approach may be
utilized to identify candidate positions suitable for engineering
disulfide bonds.
[0223] In one embodiment, a monomeric scaffold of the invention
comprise at least one, at least two, at least three, at least four,
or at least five non-naturally occurring intramolecular disulfide
bonds. In a specific embodiment, the invention provides a method of
obtaining a scaffold having increased stability as compared to an
FOI comprising two, three, four, or more engineered intramolecular
disulfide bonds.
[0224] In one embodiment, the scaffolds of the invention comprise
at least one non-naturally occurring intramolecular disulfide bond,
wherein said at least one non-naturally occurring disulfide bond
stabilizes a monomer scaffold. In another embodiment, the scaffolds
of the invention comprise at least one non-naturally occurring
intramolecular disulfide bond located between two beta strands
within the same monomer scaffold. For example, within a monomer
scaffold, at least one non-naturally occurring intramolecular
disulfide bond can form a link between the A strand and B strand,
or between the D strand and E strand, or between the F strand and G
strand, or between the C strand and F strand.
[0225] In another embodiment, non-naturally occurring disulfide
bonds form a first bond between the F strand and the G strand, and
a second link between the C strand and F strand within a single
monomer scaffold. In another embodiment, the scaffolds of the
invention comprise at least one non-naturally occurring
intramolecular disulfide bond located between two loops in the same
monomer scaffold. In another embodiment, the scaffolds of the
invention comprise at least one non-naturally occurring
intramolecular disulfide bond located between a loop and a beta
strand of the same monomer scaffold. In another embodiment,
scaffolds of the invention comprise at least one non-naturally
occurring intramolecular disulfide bond that is located within the
same beta strand in a monomer scaffold. In another embodiment,
scaffolds of the invention comprise at least one non-naturally
occurring intramolecular disulfide bond that is located within the
same loop in a monomer scaffold.
[0226] In another embodiment, scaffolds of the invention comprise
at least one non-naturally occurring disulfide bond, wherein the
bond is located between two distinct monomer scaffolds in a
multimeric scaffold. In yet another embodiment, scaffolds of the
invention comprise at least one non-naturally occurring disulfide
bond, wherein the bond is located between two distinct multimeric
scaffolds, i.e., the disulfide bond is an intermolecular disulfide
bond. For example, a disulfide bond can link distinct scaffolds
(for example, two isolated monomer scaffolds, an isolated monomer
scaffold and a multimeric scaffold, or two multimeric scaffolds), a
scaffold and a linker, a scaffold and an Fn domain, or a scaffold
and an antibody or fragment thereof. In some embodiments, scaffolds
of the invention comprise at least one non-naturally occurring
intermolecular disulfide bond that links a scaffold and an isolated
heterologous moiety, a scaffold and a heterologous moiety fused or
conjugated to the same scaffold, or a scaffold and heterologous
moiety fused or conjugated to a different scaffold.
[0227] In some embodiments, scaffolds of the invention comprise a
disulfide bond that forms a multimeric scaffold of at least 2, at
least 3, at least 4 or more scaffolds.
[0228] In another embodiment, scaffolds of the invention may
comprise an elongation of the N and/or C terminal regions. In one
embodiment, the scaffolds of the invention comprise an alteration
to increase serum half-life, as described herein. In yet another
embodiment, the scaffolds of the invention comprise an addition,
deletion or substitution of at least one amino acid residue to
stabilize the hydrophobic core of the scaffold.
[0229] In one embodiment, scaffolds of the invention comprise at
least one non-naturally occurring intramolecular disulfide bond,
wherein the beta strands of the scaffold of the invention exhibit
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99% or more identity to the cognate beta
strands of any one of SEQ ID NOs: 1-34, 54, 69, or 256, to the beta
strands of any of the FnIII domains shown in FIG. 16, or to the
beta strands of a protein domain recognized to contain the Interpro
IPR008957 fibronectin type III domain signature as determined using
the InterProScan program, or recognized to contain the Pfam PF00041
fibronectin type III domain signature as determined using
Pfam_scan, HMMER, or any other program capable of comparing a
protein sequence to a Hidden Markov model.
[0230] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID
NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID
NO:52. In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:44, the D beta strand comprises SEQ ID NO:46,
the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:50, and the G beta strand comprises SEQ ID
NO:53. In still another specific embodiment, scaffolds of the
invention comprise at least one non-naturally occurring
intramolecular disulfide bond, wherein the A beta strand domain
comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43,
the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand
comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47,
the F beta strand comprises SEQ ID NO:51, and the G beta strand
comprises SEQ ID NO:53.
[0231] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, or 131, the D beta strand consists
of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F
beta strand consists of SEQ ID NO:49, and the G beta strand
consists of SEQ ID NO:52. In another specific embodiment, scaffolds
of the invention consists at least one non-naturally occurring
intramolecular disulfide bond, wherein the A beta strand domain
consists of SEQ ID NO:42, the B beta strand consists of SEQ ID
NO:43, the C beta strand consists of SEQ ID NO:44, the D beta
strand consists of SEQ ID NO:46, the E beta strand consists of SEQ
ID NO:47, the F beta strand consists of SEQ ID NO:50, and the G
beta strand consists of SEQ ID NO:53. In still another specific
embodiment, scaffolds of the invention consists at least one
non-naturally occurring intramolecular disulfide bond, wherein the
A beta strand domain consists of SEQ ID NO:42, the B beta strand
consists of SEQ ID NO:43, the C beta strand consists of SEQ ID
NO:45, or 131, the D beta strand consists of SEQ ID NO:46, the E
beta strand consists of SEQ ID NO:47, the F beta strand consists of
SEQ ID NO:51, and the G beta strand consists of SEQ ID NO:53.
[0232] In another embodiment, scaffolds of the invention comprise
at least one non-naturally occurring intramolecular disulfide bond,
wherein the A beta strand domain consists essentially of SEQ ID
NO:42, the B beta strand consists essentially of SEQ ID NO:43, the
C beta strand consists essentially of SEQ ID NO:45, or 131, the D
beta strand consists essentially of SEQ ID NO:46, the E beta strand
consists essentially of SEQ ID NO:47, the F beta strand consists
essentially of SEQ ID NO:49, and the G beta strand consists
essentially of SEQ ID NO:52. In another specific embodiment,
scaffolds of the invention consists essentially at least one
non-naturally occurring intramolecular disulfide bond, wherein the
A beta strand domain consists essentially of SEQ ID NO:42, the B
beta strand consists essentially of SEQ ID NO:43, the C beta strand
consists essentially of SEQ ID NO:44, the D beta strand consists
essentially of SEQ ID NO:46, the E beta strand consists essentially
of SEQ ID NO:47, the F beta strand consists essentially of SEQ ID
NO:50, and the G beta strand consists essentially of SEQ ID NO:53.
In a specific embodiment, scaffolds of the invention consists
essentially of at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, or 131, the D beta strand consists essentially of SEQ ID
NO:46, the E beta strand consists essentially of SEQ ID NO:47, the
F beta strand consists essentially of SEQ ID NO:51, and the G beta
strand consists essentially of SEQ ID NO:53.
[0233] In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID
NO:46, the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID
NO:52, wherein one or more of the beta strands of the Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand and the F beta strand (SEQ
ID NOs: 45, or 131 and 49, respectively) may not be
substituted.
[0234] In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, or 131, the D beta strand consists
of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F
beta strand consists of SEQ ID NO:49, and the G beta strand
consists of SEQ ID NO:52, wherein one or more of the beta strands
of the Tn3 module comprise at least one amino acid substitution
except that the cysteine residues in the C beta strand and the F
beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not
be substituted.
[0235] In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, or 131, the D beta strand consists essentially of SEQ ID
NO:46, the E beta strand consists essentially of SEQ ID NO:47, the
F beta strand consists essentially of SEQ ID NO:49, and the G beta
strand consists essentially of SEQ ID NO:52, wherein one or more of
the beta strands of the Tn3 module comprise at least one amino acid
substitution except that the cysteine residues in the C beta strand
and the F beta strand (SEQ ID NOs: 45, or 131, and 49,
respectively) may not be substituted.
[0236] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46,
the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID
NO:52, the AB loop comprises SEQ ID NO:35, the CD loop comprises
SEQ ID NO:37 and the EF loop comprises SEQ ID NO:39.
[0237] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, the D beta strand consists of SEQ
ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta
strand consists of SEQ ID NO:49, and the G beta strand consists of
SEQ ID NO:52, the AB loop consists of SEQ ID NO:35, the CD loop
consists of SEQ ID NO:37 and the EF loop consists of SEQ ID
NO:39.
[0238] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, the D beta strand consists essentially of SEQ ID NO:46, the
E beta strand consists essentially of SEQ ID NO:47, the F beta
strand consists essentially of SEQ ID NO:49, and the G beta strand
consists essentially of SEQ ID NO:52, the AB loop consists
essentially of SEQ ID NO:35, the CD loop consists essentially of
SEQ ID NO:37 and the EF loop consists essentially of SEQ ID
NO:39.
[0239] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46,
the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID
NO:52, the BC loop comprises SEQ ID NO:36, the DE loop comprises
SEQ ID NO:38 and the FG loop comprises SEQ ID NO:40.
[0240] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, the D beta strand consists of SEQ
ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta
strand consists of SEQ ID NO:49, and the G beta strand consists of
SEQ ID NO:52, the BC loop consists of SEQ ID NO:36, the DE loop
consists of SEQ ID NO:38 and the FG loop consists of SEQ ID
NO:40.
[0241] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, the D beta strand consists essentially of SEQ ID NO:46, the
E beta strand consists essentially of SEQ ID NO:47, the F beta
strand consists essentially of SEQ ID NO:49, and the G beta strand
consists essentially of SEQ ID NO:52, the BC loop consists
essentially of SEQ ID NO:36, the DE loop consists essentially of
SEQ ID NO:38 and the FG loop consists essentially of SEQ ID
NO:40.
[0242] In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46,
the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, and the G beta strand comprises SEQ ID
NO:52, wherein one or more of the beta strands of the Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand and the F beta strand (SEQ
ID NOs: 45 and 49, respectively) may not be substituted.
[0243] In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, the D beta strand consists of SEQ
ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta
strand consists of SEQ ID NO:49, and the G beta strand consists of
SEQ ID NO:52, wherein one or more of the beta strands of the Tn3
module comprise at least one amino acid substitution except that
the cysteine residues in the C beta strand and the F beta strand
(SEQ ID NOs: 45 and 49, respectively) may not be substituted.
[0244] In another specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, the D beta strand consists essentially of SEQ ID NO:46, the
E beta strand consists essentially of SEQ ID NO:47, the F beta
strand consists essentially of SEQ ID NO:49, and the G beta strand
consists essentially of SEQ ID NO:52, wherein one or more of the
beta strands of the Tn3 module comprise at least one amino acid
substitution except that the cysteine residues in the C beta strand
and the F beta strand (SEQ ID NOs: 45 and 49, respectively) may not
be substituted.
[0245] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46,
the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, the G beta strand comprises SEQ ID NO:52,
the AB loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID
NO:37, and the EF loop comprises SEQ ID NO:39 and, wherein one or
more of the beta strands of the Tn3 module comprise at least one
amino acid substitution except that the cysteine residues in the C
beta strand and the F beta strand (SEQ ID NOs: 45 and 49,
respectively) may not be substituted.
[0246] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, the D beta strand consists of SEQ
ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta
strand consists of SEQ ID NO:49, the G beta strand consists of SEQ
ID NO:52, the AB loop consists of SEQ ID NO:35, the CD loop
consists of SEQ ID NO:37, and the EF loop consists of SEQ ID NO:39
and, wherein one or more of the beta strands of the Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand and the F beta strand (SEQ
ID NOs: 45 and 49, respectively) may not be substituted.
[0247] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, the D beta strand consists essentially of SEQ ID NO:46, the
E beta strand consists essentially of SEQ ID NO:47, the F beta
strand consists essentially of SEQ ID NO:49, the G beta strand
consists essentially of SEQ ID NO:52, the AB loop consists
essentially of SEQ ID NO:35, the CD loop consists essentially of
SEQ ID NO:37, and the EF loop consists essentially of SEQ ID NO:39
and, wherein one or more of the beta strands of the Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand and the F beta strand (SEQ
ID NOs: 45 and 49, respectively) may not be substituted.
[0248] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain comprises SEQ ID
NO:42, the B beta strand comprises SEQ ID NO:43, the C beta strand
comprises SEQ ID NO:45, the D beta strand comprises SEQ ID NO:46,
the E beta strand comprises SEQ ID NO:47, the F beta strand
comprises SEQ ID NO:49, the G beta strand comprises SEQ ID NO:52,
the BC loop comprises SEQ ID NO:36, the DE loop comprises SEQ ID
NO:38, and the FG loop comprises SEQ ID NO:40 and, wherein one or
more of the beta strands of the Tn3 module comprise at least one
amino acid substitution except that the cysteine residues in the C
beta strand and the F beta strand (SEQ ID NOs: 45 and 49,
respectively) may not be substituted.
[0249] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists of SEQ ID
NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, the D beta strand consists of SEQ
ID NO:46, the E beta strand consists of SEQ ID NO:47, the F beta
strand consists of SEQ ID NO:49, the G beta strand consists of SEQ
ID NO:52, the BC loop consists of SEQ ID NO:36, the DE loop
consists of SEQ ID NO:38, and the FG loop consists of SEQ ID NO:40
and, wherein one or more of the beta strands of the Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand and the F beta strand (SEQ
ID NOs: 45 and 49, respectively) may not be substituted.
[0250] In a specific embodiment, scaffolds of the invention
comprise at least one non-naturally occurring intramolecular
disulfide bond, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, the D beta strand consists essentially of SEQ ID NO:46, the
E beta strand consists essentially of SEQ ID NO:47, the F beta
strand consists essentially of SEQ ID NO:49, the G beta strand
consists essentially of SEQ ID NO:52, the BC loop consists
essentially of SEQ ID NO:36, the DE loop consists essentially of
SEQ ID NO:38, and the FG loop consists essentially of SEQ ID NO:40
and, wherein one or more of the beta strands of the Tn3 module
comprise at least one amino acid substitution except that the
cysteine residues in the C beta strand and the E beta strand (SEQ
ID NOs: 45 and 49, respectively) may not be substituted.
[0251] Enhanced Scaffold Stability: FG Loop Length
[0252] The inventors have discovered that the length of the FG loop
plays a role in the stability of FnIII scaffolds. In particular,
FnIII scaffolds comprising non-naturally occurring variant FG loops
which are at least one amino acid shorter than that found in the FG
loop of an FOI are shown to have enhanced stability. Accordingly,
the present invention provides methods for obtaining a fibronectin
type III (FnIII) scaffold variant having increased stability as
compared to an FOI, comprising: engineering a variant of the FOI,
wherein the FG loop of the variant comprises the deletion of at
least 1 amino acid, and wherein the variant exhibits increased
stability as compared to the FOI.
[0253] In certain embodiments, scaffolds of the invention comprise
a non-naturally occurring variant FG loop which is at least one
amino acid residue shorter than the FG loop of an FOI. For example,
as defined herein the native FG loop of the third FnIII domain of
human tenascin C comprises 10 amino acid residues. Accordingly, to
identify an FnIII scaffold having improved stability using the
third FnIII domain of human tenascin C as the FOI the FG loop would
be reduced to 9 or fewer amino acid residues.
[0254] Although the sequence similarity between the amino acids
sequences of the FnIII domains is generally low, the overall three
dimensional structure is similar. Accordingly, using known
techniques, such as sequence analysis and structure overlay, the FG
loops of FnIII domains from multiple FOIs (e.g., FnIII domains from
different species, different proteins, and different FnIII
scaffolds that bind a target) may be determined (see for example
FIG. 16. These loops can then be subjected to mutation to yield an
FG loop that is at least one amino acid shorter than the FG loop
from the FOI.
[0255] Thus, in one embodiment the instant invention encompasses
FnIII scaffolds that comprise a non-naturally occurring variant FG
loop which is at least one amino acid shorter than the FG loop of
FOI regardless of what specific definition of the FG loop is
used.
[0256] In a specific embodiment, the stability of an FOI is
enhanced by deletion of at least one amino acid in the FG loop of
the FOI. In another embodiment, the stability of an FOI is enhanced
by deletion of at least 1, or at least 2, or at least 3, or at
least 4, or at least 5, or at least 6, or at least 7, or at least
8, or at least 9, or at least 10 amino acids in the FG loop. It is
specifically contemplated that the stabilized FOI may comprise at
least one non-naturally occurring disulfide bond. In certain
embodiments, the FOI comprised the non-naturally occurring
intramolecular disulfide bond prior to being stabilized. In other
embodiments, the stabilized FOI is further engineered to introduce
at least one non-naturally occurring intramolecular disulfide
bond.
[0257] In a specific embodiment, the invention provides a method of
obtaining an FnIII scaffold variant having increased stability as
compared to an FOI comprising engineering a variant of the FOI,
wherein the FG loop of the variant comprises the deletion of at
least 1, or at least 2, or at least 3, or at least 4, or at least
5, or at least 6, or at least 7, or at least 8, or at least 9, or
at least 10 amino acids in the FG loop, wherein the variant
exhibits an increased stability as compared to the FOI. In certain
embodiments, the FnIII scaffold variant also comprises at least one
loop, (i.e., AB, BC, CD, DE, EF, and/or FG) that has been
randomized for length and/or sequence. It is specifically
contemplated that the FnIII scaffold variant may comprise at least
one non-naturally occurring disulfide bond. In certain embodiments,
the FOI comprised the non-naturally occurring disulfide bond. In
other embodiments, the FnIII variant is further engineered to
introduce at least one non-naturally occurring disulfide bond.
[0258] In certain embodiments, the scaffold of the invention is an
FnIII scaffold variant (i.e., a stabilized FOI) having increased
stability as compared to an FOI, wherein the FnIII scaffold variant
comprises an FG loop which is at least one, or at least two, or at
least 3, or at least 4, or at least 5, or at least 6, or at least
7, or at least 8, or at least 9, or at least 10 amino acid residues
shorter than the FG loop of the FOI, wherein the FnIII scaffold
variant further comprises at least one amino acid substitution.
Stability Measurements
[0259] The increase in stability of the stabilized FnIII scaffolds
of the invention, isolated or as part of a multimeric scaffold, can
be readily measured by techniques well known in the art, such as
thermal (T.sub.m) and chaotropic denaturation (such as treatment
with urea, or guanidine salts), protease treatment (such as
treatment with thermolysin) or another art accepted methodology to
determine protein stability. A comprehensive review of techniques
used to measure protein stability can be found, for example in
"Current Protocols in Molecular Biology" and "Current Protocols in
Protein Science" by John Wiley and Sons. 2007.
[0260] In one embodiment the stabilized FnIII scaffolds of the
invention exhibit an increase in stability of at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% or more compared
to the same FnIII scaffold prior to engineering (i.e., the FOI), as
measured by thermal tolerance, resistance to chaotropic
denaturation, protease treatment or another stability parameter
well-known in the art.
[0261] The stability of a protein may be measured by the level of
fluorescence exhibited by the protein under varying conditions.
There is a positive correlation between the relative unfoldedness
of a protein and a change in the internal fluorescence the protein
exhibits under stress. Suitable protein stability assays to measure
thermal unfolding characteristics include Differential Scanning
calorimetry (DSC) and Circular Dichroism (CD). When the protein
demonstrates a sizable shift in parameters measured by DSC or CD,
it correlates to an unfolded structure. The temperature at which
this shift is made is termed the melting temperature or
(T.sub.m).
[0262] In one embodiment, the stabilized scaffolds of the invention
exhibit an increased melting temperature (T.sub.m) of at least
1.degree. C., at least 2.degree. C., at least 3.degree. C., at
least 4.degree. C., at least 5.degree. C., at least 10.degree. C.,
at least 15.degree. C., at least 20.degree. C., at least 25.degree.
C., at least 30.degree. C., at least 35.degree. C., at least
45.degree. C., at least 50.degree. C., at least 55.degree. C., at
least 60.degree. C., at least 65.degree. C., at least 70.degree.
C., at least 71.degree. C., at least 72.degree. C., at least
73.degree. C., at least 74.degree. C., at least 75.degree. C., at
least 76.degree. C., at least 77.degree. C., at least 78.degree.
C., at least 79.degree. C., at least 80.degree. C., at least
81.degree. C., at least 82.degree. C., at least 83.degree. C., at
least 84.degree. C., at least 85.degree. C., at least 85.degree.
C., at least 86.degree. C., at least 87.degree. C., at least
88.degree. C., at least 89.degree. C., at least 90.degree. C., at
least 91.degree. C., at least 92.degree. C., at least 93.degree.
C., at least 94.degree. C., at least 94.degree. C., at least, at
least 95.degree. C., at least 96.degree. C., at least 97.degree.
C., at least 98.degree. C., at least 100.degree. C., at least
105.degree. C., at least 110.degree. C., or at least 120.degree. C.
as compared to the FOI under similar conditions.
[0263] In another embodiment, the stabilized FnIII scaffolds of the
invention exhibit an increased melting temperature (T.sub.m) of at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, or at least
95% or more as compared to the FOI under similar conditions.
[0264] Another assay for protein stability involves exposing a
protein to a chaotropic agent, such as urea or guanidine (for
example, guanidine-HCl or guanidine isothiocynate) which acts to
destabilize interactions within the protein. Upon exposing the
protein to increasing levels of urea or guanidine, the relative
intrinsic fluorescence is measured to assess a value in which 50%
of the protein molecules are unfolded. This value is termed the
C.sub.m value and represents a benchmark value for protein
stability. The higher the C.sub.m value, the more stable the
protein. In one embodiment, the stabilized FnIII scaffolds of the
invention exhibit an increased C.sub.m at least 5%, at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95% or more as compared to
the FOI as measured in a urea denaturation experiment under similar
conditions. In another embodiment, the stabilized FnIII scaffolds
of the invention exhibit an increased C.sub.m at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% or more as
compared to the FOI as measured in a guanidinium-HCl denaturation
experiment under similar conditions.
[0265] Another assay used to assay protein stability is a protease
resistance assay. In this assay, a relative level of protein
stability is correlated with the resistance to protease degradation
over time. The more resistant to protease treatment, the more
stable the protein is. In one embodiment, the stabilized FnIII
scaffolds of the invention exhibit increased stability by at least
5%, at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, or at least 95% or
more as compared to the FOI under similar conditions.
Multimeric Scaffolds
[0266] One aspect of the present invention provides multimeric
scaffolds comprising at least two FnIII monomer scaffolds of the
invention joined in tandem. Such multimeric scaffolds can be
assembled in multiple formats. In some embodiments the monomer
scaffolds are assembled in linear formats whereas in other
embodiments the scaffolds are assembled in branched formats (see,
e.g., FIG. 1). In a specific aspect, the invention provides
multimeric scaffolds, wherein at least two FnIII scaffolds are
connected in tandem via a peptide linker. In some embodiments, each
FnIII scaffold in the multimeric scaffolds of the invention binds
to a different target, thereby demonstrating multiple functions,
and/or to the same target, thereby increasing the valency and/or
avidity of target binding. In some embodiments, the increase in
valency and/or avidity of target binding is accomplished when
multiple scaffolds bind to the same target. In some embodiments,
the increase in valency improves a specific action on the target,
such as increasing the dimerization of a target protein.
[0267] In a specific embodiment, the multimeric scaffold of the
invention comprises at least two FnIII monomer scaffolds of the
invention connected in tandem, wherein each scaffold binds at least
one target, and wherein each FnIII scaffold comprises a plurality
of beta strands linked to a plurality of loop regions, wherein at
least one loop is a non-naturally occurring variant of the cognate
loop in an FOI, and wherein the beta strands of the FnIII scaffolds
have at least 50% homology (i.e., sequence similarity) to the
cognate beta strands of the FOI. In certain embodiments, each FnIII
scaffold has at least 50% homology (i.e., sequence similarity) to
the cognate beta strands of the same FOI. In a specific embodiment,
each FnIII scaffold has at least 50% homology (i.e., sequence
similarity) to the cognate beta strands of the wild type Tn3
scaffold (SEQ ID NO:1). It is specifically contemplated that each
FnIII scaffold may have at least 50% homology (i.e., sequence
similarity) to a different FOI. For example, a multimeric scaffold
of the invention may comprise a first FnIII scaffold and a second
FnIII scaffold, wherein the beta strands of the first FnIII
scaffold have at least 50% homology (i.e., sequence similarity) to
the cognate beta strands of the 14th FnIII domain of fibronectin
(SEQ ID NOs:69), and wherein the beta strands of the second FnIII
scaffold have at least 50% homology (i.e., sequence similarity) to
the cognate beta strands of the wild type Tn3 scaffold (SEQ ID
NO:1).
[0268] In some embodiments, a multimeric scaffold of the invention
comprises at least two FnIII monomer scaffolds, wherein the FOI is
the protein sequence corresponding to the third FnIII domain of
human tenascin C. In a specific embodiment, the multimeric scaffold
of the invention comprises at least two FnIII scaffolds, wherein
the FOI is a wild type Tn3 scaffold. In other embodiments, the
multimeric scaffold of the invention comprises at least two FnIII
scaffolds, wherein the FOI is a protein sequence corresponding to
an additional FnIII domain from human tenascin C. In other
embodiments, the multimeric scaffold of the invention comprises at
least two FnIII scaffolds, wherein the FOI is a protein sequence
corresponding to an FnIII domain from another tenascin protein, or
alternatively, a tenascin protein from another organism (such as,
but not limited to, murine, porcine, bovine, or equine tenascins).
In some embodiments, the multimeric scaffold of the invention
comprises at least two FnIII scaffolds, wherein the beta strands of
the FnIII scaffolds have at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 99% homology (i.e.,
sequence similarity) to the cognate beta strands in any of: the
29.sup.th FnIII domain from human tenascin XB (SEQ ID NO: 11), the
31.sup.st FnIII domain from human tenascin XB (SEQ ID NO: 12), the
32.sup.nd FnIII from human tenascin XB (SEQ ID NO: 13), the
3.sup.rd FnIII domain of human fibronectin (SEQ ID NO: 6), the
6.sup.th FnIII domain of human fibronectin (SEQ ID NO: 7), the
10.sup.th FnIII domain of human fibronectin (e.g., SEQ ID NO: 5 and
SEQ ID NO: 54), the 14.sup.th FnIII domain of human fibronectin
(e.g., SEQ ID NO: 69 and SEQ ID NO: 34), an FnIII domain from human
growth hormone receptor (e.g., SEQ ID NO: 8 and SEQ ID NO: 15), an
FnIII domain from beta common receptor (e.g., SEQ ID NO: 9), an
FnIII from IL-5 receptor (e.g., SEQ ID NO: 10), an FnIII from
PTPR-F (e.g., SEQ ID NO: 16 and SEQ ID NO: 17), or an FnIII domain
from collagen type XIV (e.g., SEQ ID NO: 18).
[0269] In yet another embodiment the multimeric scaffold of the
invention comprises at least two FnIII monomer scaffolds, wherein
the FOI is a protein sequence corresponding to an FnIII domain from
any organism. In other embodiments, the multimeric scaffold of the
invention comprises at least two FnIII scaffolds, wherein a
naturally occurring sequence corresponds to a predicted FnIII
domain from a thermophilic or hyperthermophilic organism, for
example, but not limited to Archaeoglobus fulgidus, Staphylothermus
marinus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and
Sulfolobus tokodaii. In some embodiments, the multimeric scaffold
of the invention comprises at least two FnIII scaffolds, wherein
the beta strands of the FnIII scaffolds have at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%
homology (i.e., sequence similarity) to the cognate beta strands in
any of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, or 33.
[0270] In one embodiment, the multimeric scaffold of the invention
comprises at least two FnIII monomer scaffolds, wherein beta
strands of the FnIII scaffolds have at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 99% homology
(sequence similarity) to the cognate beta strands in any one of the
FnIII domains presented in FIG. 16, or to a protein domain
recognized to contain the Interpro IPRO08957 fibronectin type III
domain signature as determined using the InterProScan program, or
recognized to contain the Pfam PF00041 fibronectin type III domain
signature as determined using Pfam_scan, HMMER, or any other
program capable of comparing a protein sequence to a Hidden Markov
model.
Multimeric Tandem Scaffolds
[0271] In one embodiment, the multimeric scaffolds of the invention
comprise two, three, four, five, six, eight or more FnIII monomer
scaffolds of the invention. In some embodiments some of the FnIII
monomer scaffolds are connected in tandem. In yet another
embodiment, some of the FnIII monomer scaffolds are connected in
tandem and some of the FnIII monomer scaffolds are not connected in
tandem. In a specific embodiment, the multimeric scaffolds of the
invention comprise two, or three, or four, or five, or six, or
seven, or eight, or nine, or ten, or more scaffolds of the
invention connected in tandem (see, e.g., FIG. 1 and FIG. 2).
[0272] In one embodiment, the multimeric scaffolds are generated
through covalent binding between FnIII monomer scaffolds, for
example, by directly linking the FnIII scaffolds, or by the
inclusion of a linker, e.g., a peptide linker. In particular
examples, covalently bonded scaffolds are generated by constructing
fasion genes that encode the monomeric FnIII scaffolds or,
alternatively, by engineering codons for cysteine residues into
monomer FnIII scaffolds and allowing disulfide bond formation to
occur between the expression products.
[0273] In one embodiment, the multimeric scaffolds of the invention
comprise at least two FnIII scaffolds that are connected directly
to each other without any additional intervening amino acids. In
another embodiment, the multimeric scaffolds of the invention
comprise at least two FnIII scaffolds that are connected in tandem
via a linker, e.g., a peptide linker. In a specific embodiment, the
multimeric scaffolds of the invention comprise at least two FnIII
scaffolds that are connected in tandem via a peptide linker,
wherein the peptide linker comprises 1 to about 1000, or 1 to about
500, or 1 to about 250, or 1 to about 100, or 1 to about 50, or 1
to about 25, amino acids. In a specific embodiment, the multimeric
scaffolds of the invention comprise at least two FnIII scaffolds
that are connected in tandem via a peptide linker, wherein the
peptide linker comprises 1 to about 20, or 1 to about 15, or 1 to
about 10, or 1 to about 5, amino acids.
[0274] In a specific embodiment, the multimeric scaffolds of the
invention comprise at least two FnIII scaffolds that are connected
in tandem via a linker, e.g., a peptide linker, wherein the linker
is a functional moiety. The functional moiety will be selected
based on the desired function and/or characteristics of the
multimeric scaffold. For example, a functional moiety useful for
purification (e.g., a histidine tag) may be used as a linker.
Functional moieties useful as linkers include, but are not limited
to, polyethylene glycol (PEG), a cytotoxic agent, a radionuclide,
imaging agent, biotin, a dimerization domain (e.g. leucine zipper
domain), human serum albumin (HSA) or an FcRn binding portion
thereof, a domain or fragment of an antibody (e.g., antibody
variable domain, a CH1 domain, a Ckappa domain, a Clambda domain, a
CH2, or a CH3 domain), a single chain antibody, a domain antibody,
an albumin binding domain, an IgG molecule, an enzyme, a ligand, a
receptor, a binding peptide, a non-FnIII scaffold, an epitope tag,
a recombinant polypeptide polymer, a cytokine, and the like.
Specific peptide linkers and functional moieties which may be used
as linkers are disclosed infra.
[0275] In some embodiments, the multimeric scaffolds of the
invention comprise at least two FnIII scaffolds that are connected
via one or more linkers, wherein the linkers interposed between
each FnIII scaffold can be the same linkers or different linkers.
In some embodiments, a linker can comprise multiple linkers, which
can be the same linker or different linkers. In some embodiments,
when a plurality of linkers are concatenated, some or all the
linkers can be functional moieties.
Multimeric Scaffold Binding Stoichiometry
[0276] In some embodiments, the multimeric scaffolds of the
invention comprise scaffolds that are specific for the same
epitope. In other embodiments, multimeric scaffolds of the
invention comprise scaffolds that are specific for different
epitopes, which can be different epitopes on the same or different
targets.
[0277] In a specific embodiment, the scaffolds of the multimeric
scaffolds bind two or more different epitopes (e.g.,
non-overlapping epitopes) on the same target molecule. In another
specific embodiment, the scaffolds of the multimeric scaffolds bind
two or more different epitopes on the different target molecules.
In yet another specific embodiment, the scaffolds of the multimeric
scaffolds bind two or more different epitopes on the same target
and additionally, bind at least one epitope on one or more
different target molecules. In still another specific embodiment,
the scaffolds of the multimeric scaffolds bind to the same epitope
on a multimeric target molecule. In yet another embodiment, the
scaffolds of the multimeric scaffolds bind to the same epitope on
adjacent target molecules. In certain embodiments, the scaffolds of
the multimeric scaffolds bind the same epitope on two or more
copies of a target molecule on an adjacent cell surface. In some
embodiments, the scaffolds of the multimeric scaffolds can bind to
the same epitope or different epitopes in the same target or
different targets with the same or different binding affinities
and/or avidities.
[0278] In another embodiment, the monomer scaffolds in a multimeric
scaffolds of the invention can bind to specific targets according
to a specific binding pattern designed to achieve or enhance (e.g.,
synergistically) a desired effect. For example, the FnIII scaffolds
in a linear multimeric scaffold can bind to a single target or to
multiple targets according to a certain pattern, e.g., FnIII
scaffolds in a 6 module linear multivalent scaffold can bind to two
targets A and B according to an AAABBB pattern, an AABBAA pattern,
an ABABAB pattern, an AAAABB pattern, etc.; to three targets
according to an AABBCC pattern, an ABCABC pattern, and ABCCBA
pattern, etc.; to four targets according to an ABCDDA patterns,
ABCADA pattern, etc.; etc. In addition, when a multimeric scaffold
of the invention comprises a plurality of engineered (e.g.,
disulfide engineered, loop engineered, or both disulfide and loop
engineered) and non-engineered scaffolds, such monomeric scaffolds
can be arranged according to a certain pattern to achieve or
enhance a certain biological effect. Such combinations of monomeric
scaffolds can be combinatorially assembled and subsequently
evaluated using methods known in the art.
[0279] In some embodiments, multimeric scaffolds in branched
constructs, e.g., multimeric scaffolds in an Fc fusion or
antibody-like format, can also bind to a single target or to
multiple targets according to a certain pattern. For instance, in
certain embodiments a linear format scaffold fused to the IgG heavy
chains in an antibody-like format scaffold can bind to a first
target whereas a multivalent linear construct fused to the IgG
light chains in an antibody-like format scaffold can bind to a
second target. In another embodiment, linear format scaffolds fused
to the IgG heavy chains of an antibody-like format scaffold can
bind to a target with a certain affinity whereas the linear format
scaffolds fused to the IgG light chains of an antibody-like format
scaffold can bind to the same target with a different affinity. In
some embodiments, the scaffolds fused to the chains in the left arm
of the "Y" of an antibody can bind to a first target, whereas the
scaffolds fused to the chains of the right of the "Y" of an
antibody can bind to a second target.
Fusions
[0280] The invention further provides multimeric scaffolds
comprising at least two FnIII monomer scaffolds, wherein at least
one monomer scaffold may be fused to a heterologous moiety. In this
context the heterologous moiety is not used to link the scaffolds
as a spacer but may provide additional functionality to the
multimeric scaffold of the invention. For example, in some
embodiments, a multimeric scaffold that binds a target on the
surface of a cell may be fused to a cytotoxic agent to facilitate
target specific cell killing. Additional fusions are disclosed
infra. In some embodiments, a heterologous moiety can function as a
linker.
[0281] The present invention encompasses the use of scaffolds of
the invention conjugated or fused to one or more heterologous
moieties, including but not limited to, peptides, polypeptides,
proteins, fusion proteins, nucleic acid molecules, small molecules,
mimetic agents, synthetic drugs, inorganic molecules, and organic
molecules. The present invention encompasses the use of scaffolds
recombinantly fused or chemically conjugated to a heterologous
protein or polypeptide or fragment thereof. Conjugation includes
both covalent and non-covalent conjugation. In some embodiments, a
scaffold of the invention can be fused or chemically conjugated to
a polypeptide of at least 10, at least 20, at least 30, at least
40, at least 50, at least 60, at least 70, at least 80, at least
90, at least 100, at least 200, at least 300, at least 500, or at
least 1000 amino acids) to generate fusion proteins.
[0282] The fusion or conjugation of a scaffold to one or more
heterologous moieties can be direct, i.e., without a linker
interposed between a scaffold and a heterologous moiety, or via one
or more linker sequences described herein. In some embodiments,
scaffolds can be used to target heterologous polypeptides to
particular cell types, either in vitro or in vivo, by fusing or
conjugating the scaffolds to antibodies specific for particular
cell surface receptors in the target cells. Scaffolds fused or
conjugated to heterologous polypeptides can also be used in in
vitro immunoassays and purification methods using methods known in
the art. See e.g., International Publication No. WO 93/21232;
European Patent No. EP 439,095; Naramura et al. Immunol. Lett.
39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et al., PNAS
89:1428-1432, 1992; and Fell et al., J. Immunol. 146:2446-2452,
1991, which are incorporated by reference in their entireties.
[0283] In some embodiments, the scaffolds can be integrated with
the human immune response by fusing or conjugating a scaffold with
an immunoglobulin or domain thereof including, but not limited to,
the constant region of an IgG (Fc), e.g., through the N or
C-terminus. The Fc fusion molecule activates the complement
component of the immune response and increases the therapeutic
value of the protein scaffold. Similarly, a fusion between a
scaffold and a complement protein, such as CIq, can be used to
target cells. A fusion between scaffold and a toxin can be used to
specifically destroy cells that carry a particular antigen as
described herein.
[0284] Various publications describe methods for obtaining
physiologically active molecules whose half-lives are modified by
introducing an FcRn-binding polypeptide into the molecules (see,
e.g., WO 97/43316; U.S. Pat. No. 5,869,046; U.S. Pat. No.
5,747,035; WO 96/32478; and WO 91/14438), by fusing the molecules
with antibodies whose FcRn-binding affinities are preserved but
affinities for other Fc receptors have been greatly reduced (See,
e.g., WO 99/43713), or by fusing the molecules with FcRn binding
domains of antibodies (see, e.g., WO 00/09560; U.S. Pat. No.
4,703,039). Specific techniques and methods of increasing half-life
of physiologically active molecules can also be found in U.S. Pat.
No. 7,083,784. Specifically, it is contemplated that the scaffolds
of the invention can be fused to an Fc region from an IgG, wherein
the Fc region comprises amino acid residue mutations
M252Y/S254T/T256E or H433K/N434F/Y436H, wherein amino acid
positions are designated according to the Kabat numbering schema.
In some embodiments, the half life of a multimeric scaffold of the
invention is increased by genetically fusing the multimeric
scaffold with an intrinsically unstructured recombinant polypeptide
(e.g., an XTEN.TM. polypeptide) or by conjugation with polyethylene
glycol (PEG).
[0285] In some embodiments, the scaffolds of the invention can be
fused with molecules that increase or extend in vivo or serum half
life. In some embodiments, the scaffolds of the invention are fused
or conjugated with albumin, such as human serum albumin (HSA), a
neonatal Fc receptor (FcRn) binding fragment thereof, polyethylene
glycol (PEG), polysaccharides, immunoglobulin molecules (IgG) or
fragments thereof, complement, hemoglobin, a binding peptide,
lipoproteins and other factors to increase its half-life in the
bloodstream and/or its tissue penetration. Any of these fusions may
be generated by standard techniques, for example, by expression of
the fusion protein from a recombinant fusion gene constructed using
publicly available gene sequences.
[0286] Moreover, the scaffolds of the invention can be fused to
marker sequences, such as a peptide to facilitate purification. In
some embodiments, the marker amino acid sequence is a
poly-histidine peptide (His-tag), e.g., a octa-histidine-tag
(His-8-tag) or hexa-histidine-tag (His-6-tag) such as the tag
provided in a pQE expression vector (QIAGEN, Inc., 9259 Eton
Avenue, Chatsworth, Calif., 91311), among other vectors, many of
which are commercially available. As described in Gentz et al.,
Proc. Natl. Acad. Sci. USA 86:821-824, 1989, for instance,
poly-histidine provides for convenient purification of the fusion
protein. Other peptide tags useful for purification include, but
are not limited to, a hemagglutinin ("HA") tag, which corresponds
to an epitope derived from the influenza hemagglutinin protein
(see, e.g., Wilson et al., Cell 37:767, 1984), a FLAG tag, a
Strep-tag, a myc-tag, a V5 tag, a GFP-tag, an AU1-tag, an AU5-tag,
an ECS-tag, a GST-tag, or an OLLAS tag.
[0287] Additional fusion proteins comprising scaffolds of the
invention may be generated through the techniques of
gene-shuffling, motif-shuffling, exon-shuffling, and/or
codon-shuffling (collectively referred to as "DNA shuffling"). DNA
shuffling may be employed to alter the activities of scaffolds of
the invention (e.g., scaffolds with higher affinities and lower
dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793;
5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al.,
Curr. Opinion Biotechnol. 8:724-33, 1997; Harayama, Trends
Biotechnol. 16(2):76-82, 1998; Hansson, et al., J. Mol. Biol.
287:265-76, 1999; and Lorenzo and Blasco, 1998, Biotechniques
24(2):308-313 (each of these patents and publications is hereby
incorporated by reference in its entirety). Scaffolds, or the
encoded scaffolds thereof, may be altered by being subjected to
random mutagenesis by error-prone PCR, random nucleotide insertion
or other methods prior to recombination. One or more portions of a
polynucleotide encoding a scaffold, which bind to a specific target
may be recombined with one or more components, motifs, sections,
parts, domains, fragments, etc. of one or more heterologous
molecules.
Antibody-Like Multimeric Scaffolds
[0288] In some embodiments, the multimeric scaffold of the
invention comprise at least two FnIII, wherein at least one
scaffold is fused to a domain or fragment of an antibody (e.g., an
IgG), including but not limited to an intact antibody, an antibody
variable domain, a CH1 domain, a Ckappa domain, a Clambda domain,
an Fc domain, a CH2, or a CH3 domain.
[0289] In some embodiments, scaffolds of the invention can be fused
to a domain or fragment of an antibody. The domain or fragment of
an antibody further enhances the avidity and/or affinity of the
multimeric scaffold by providing, similarly to the Fc domain
described below, a dimerization or multimerization domain which
facilitates the formation of multimeric scaffolds of the
invention.
[0290] In some embodiments, only one multimeric tandem scaffold
comprising two FnIII domains is conjugated or fused to a domain or
fragment of an antibody. For instance, a single multimeric tandem
scaffold can be fused to the N-terminus of a polypeptide of a
domain or fragment of an antibody (e.g., a heavy chain or a light
chain of an antibody). In some embodiments, multivalent scaffolds
are created by fusing or conjugating one or more FnIII scaffolds to
the N-terminus and/or the C-terminus a polypeptide of a domain or
fragment of an antibody (e.g., a heavy chain and/or a light chain
of an antibody. In some embodiments, some or all the scaffolds
fused to a domain or fragment of an antibody are identical. In some
other embodiments, some or all the scaffolds fused to a domain or
fragment of an antibody are different.
[0291] In some embodiments, the scaffolds of the invention used to
generate an antibody-like multivalent scaffold can contain the same
number of FnIII modules. In other embodiments, the scaffolds of the
invention used to generate an antibody-like multivalent scaffold
can contain a different number of FnIII modules. For example, a
tetravalent FnIII scaffold can be formed, e.g., by fusing a linear
format tetravalent FnIII scaffold to a single position, or by
fusing one FnIII monomer scaffold in one position and a trimeric
linear format FnIII scaffold to another position, or by fusing two
dimeric FnIII linear format scaffolds to two different positions,
or by fusing 4 FnIII monomer scaffolds, each one to a single
position.
[0292] In a specific embodiment, multimeric FnIII scaffolds of the
invention comprise four multimeric linear scaffolds fused to a
domain or fragment of an antibody wherein each multimeric linear
scaffold comprises two FnIII monomer scaffolds that are connected
in tandem via a linker (FIG. 1). In other embodiments, multimeric
FnIII scaffolds of the invention comprise at least one, at least
two, at least three, at least four, at least five, at least six, at
least seven or at least eight monomeric or multimeric FnIII
scaffolds of the invention fused to a domain or fragment of an
antibody.
[0293] In one specific embodiment, a tetravalent FnIII scaffold can
be generated by fusing one scaffold to the N-terminus of each of
the light chains and heavy chains of a domain or fragment of an
antibody (see, e.g., A9 construct in FIG. 2).
[0294] An antibody-like format multivalent FnIII scaffold can be
generated by fusing any combination of scaffolds of the invention
to a domain or fragment of an antibody or modified antibody.
Examples of modified antibodies include domain deleted antibodies,
minibodies (see, e.g., U.S. Pat. No. 5,837,821), tetravalent
minibodies, tetravalent antibodies (see, e.g., Coloma &
Morrison, Nature Biotechnol. 15:159-163, 1997; PCT Publication No.
WO 95/09917), thermally stabilized antibodies, humanized
antibodies, etc.
[0295] Each of the linear scaffolds of the invention used to
generate an antibody-like multivalent scaffold according to FIG. 1
can contain the same linkers and linker distributions, or different
linkers and different linker distributions.
Fc-Fusion Multimeric Scaffolds
[0296] In some embodiments, a multimeric scaffold of the invention
comprises a plurality of monomeric or multimeric scaffolds
connected to an Fc domain. The fusion of a multimeric scaffold of
the invention to an antibody fragment comprising an Fc domain
further enhances the avidity and/or activity of the multimeric
FnIII scaffold by providing a dimerization domain which facilitates
the formation of dimers of the multimeric FnIII scaffolds.
[0297] In some embodiments, only one multimeric FnIII scaffold is
fused to an Fc domain. In a specific embodiment, multimeric
scaffolds of the invention comprise two multimeric FnIII scaffolds
fused to an Fc domain wherein each multimeric FnIII scaffold
comprises two or more FnIII scaffolds that are connected via one or
more linkers (FIG. 1). In one specific embodiment, the multimeric
FnIII scaffolds fused to the Fc domain are linear format
scaffolds.
[0298] In one specific embodiment, two linear format FnIII
scaffolds comprising two FnIII domains in tandem are fused to an Fc
domain to yield a multimeric scaffold with a valency of 4 (see,
e.g., A7 construct in FIG. 2). In another specific embodiment, two
linear format scaffolds, each one of them comprising four FnIII
monomer scaffolds are fused to an Fc domain to yield an FnIII
multimeric scaffold with a valency of 8 (see, e.g., A8 construct in
FIG. 2).
[0299] In some embodiments, the FnIII scaffolds fused to the Fc
domain comprise the same number of FnIII modules. In some
embodiments, the FnIII scaffolds fused to the Fc domain comprise a
different number of FnIII modules. In some embodiments, the FnIII
scaffolds fused to the Fc domain comprise the same linkers. In
other embodiments, the FnIII scaffolds fused to the Fc domain
comprise different linkers.
[0300] In some embodiments, different multimeric FnIII scaffolds of
the invention can be dimerized by the use of Fc domain mutations
which favor the formation of heterodimers. See, for example,
WO96/27011 which describes a method, in which one or more small
amino acid side chains from the interface of a first Fc domain are
replaced with larger side chains (e.g., tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of a second Fc domain by
replacing large amino acid side chains with smaller ones (e.g.,
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
[0301] It is known that variants of the Fc region (e.g., amino acid
substitutions and/or additions and/or deletions) enhance or
diminish effector function of the antibody (see, e.g., U.S. Pat.
Nos. 5,624,821; 5,885,573; 6,538,124; 7,317,091; 5,648,260;
6,538,124; International Publications Nos. WO 03/074679; WO
04/029207; WO 04/099249; WO 99/58572; US Publication No.
2006/0134105; 2004/0132101; 2006/0008883) and can alter the
pharmacokinetic properties (e.g. half-life) of the antibody (see,
U.S. Pat. Nos. 6,277,375 and 7,083,784). Thus, in certain
embodiments, the multispecific FnIII scaffolds of the invention
comprise Fc domain(s) that comprise an altered Fc region in which
one or more alterations have been made in the Fc region in order to
change functional and/or pharmacokinetic properties of the
multimeric FnIII scaffolds.
[0302] It is also known that the glycosylation of the Fc region can
be modified to increase or decrease effector function and/or
anti-inflammatory activity (see, e.g., Umana et al., Nat.
Biotechnol. 17:176-180, 1999; Davies et al. Biotechnol. Bioeng.
74:288-294, 2001; Shields et al., J. Biol. Chem. 277:26733-26740,
2002; Shinkawa et al., J. Biol. Chem. 278:3466-3473, 2003; U.S.
Pat. Nos. 6,602,684; 6,946,292; 7,064,191; 7,214,775; 7,393,683;
7,425,446; 7,504,256; U.S. Publication. Nos. 2003/0157108;
2003/0003097; 2009/0010921; Potillegent.TM. technology (Biowa, Inc.
Princeton, N.J.); GlycoMAb.TM. glycosylation engineering technology
(GLYCART biotechnology AG, Zurich, Switzerland); Keneko et al.,
Science 313:670-673, 2006; Scallon et al., Mol. Immuno.
44(7):1524-34, 2007). Accordingly, in one embodiment the Fc regions
of the multimeric FnIII scaffolds of the invention comprise altered
glycosylation of amino acid residues in order to change cytotoxic
and/or anti-inflammatory properties of the multimeric
scaffolds.
Multimeric Scaffold Topologies
[0303] One skilled in the art would appreciate that multimeric
scaffolds discussed above, in FIG. 1 and FIG. 2, and throughout the
specification are just illustrative examples. The construct
topologies or formats shown in FIG. 1 and FIG. 2 illustrate that in
some embodiments the scaffolds of the invention are fused to the
N-termini of the constituent polypeptides of Fc domains and
antibodies. The scaffolds of the invention can be fused to the
C-terminus of the Fc domains, antibody light chains, and antibody
heavy chains in any suitable spatial arrangement. For example, an
some embodiments a tetravalent scaffold can be created by fusing an
FnIII monomer scaffold to the N-terminus of each heavy chain and an
FnIII monomer scaffold to the C-terminus domain of each light chain
of an antibody, by fusing an FnIII monomer scaffold to the
N-terminus of each light chain and an FnIII monomer scaffold to the
C-terminus of each heavy chain of an antibody, or by fusing an
FnIII monomer scaffold to the N-terminus of each heavy chain and an
FnIII monomer scaffold to the N-terminus of each light chain of an
antibody. Monomeric and/or multimeric FnIII scaffolds can be fused
to full length heavy and/or light chains comprising both variable
regions and constant regions. Alternatively, monomeric and/or
multimeric FnIII scaffolds can be fused to truncated heavy and/or
light chains comprising only constant regions (e.g., as in the A9
construct shown in FIG. 2).
[0304] Multimeric scaffolds can be created by using the formats
shown in FIG. 1 as building blocks. For example, the antibody-like
and Fc fusion formats are combinations comprising more simple
linear format modules. Accordingly, in some embodiments more
complex multimeric scaffolds formats can be created by combining
the building blocks shown in FIG. 1.
[0305] FIGS. 1 and 2 also illustrate that in some embodiments the
multimeric scaffolds of the invention can be linear or branched and
exhibit different levels of branching. For example, the Fc format
provides an example of first order branched format, whereas the
antibody-like format provides an example of a second-order branched
format. Higher order branched constructs can be obtained by
replacing the linear format building blocks in the antibody-like
format or the Fc fusion format with Fc fusion format building
blocks or antibody-like building blocks, and connect them to either
the C-termini or N-termini of the constituent polypeptides of the
Fc or antibody.
[0306] FIGS. 1 and 2, and TABLE 1 illustrate the fact that in some
embodiments the linkers in a multimeric scaffold can be
structurally and functionally diverse and can provide a plurality
of attachment points. For example, all the FnIII modules in the A4
and A5 constructs are connected by (Gly-4-Ser).sub.3Ala linkers,
except for the 4th and 5th FnIII modules, which are connected by a
(Gly.sub.4-Ser)-2-Gly-Thr-Gly-Ser-Ala-Met-Ala-Ser-(Gly.sub.4-Ser).sub.1-A-
la linker. For example, in the A7 construct, the first and second
FnIII domains and the third and fourth FnIII domain are connected
by (Gly.sub.4-Ser).sub.3Ala linkers, whereas the second and third
FnIII domains are connected by an Fc domain as a functional moiety
linker.
[0307] The Fc fusion shown in FIG. 1 exemplifies that in some
embodiments monomeric or multimeric FnIII scaffolds can be fused to
the N-termini of the polypeptides of the functional moiety linker.
In some embodiments, monomeric or multimeric FnIII scaffolds of the
invention can readily be fused to the C-terminus of the Fc domain
in an Fc fusion format construct.
[0308] Similarly, the antibody or modified antibody in an
antibody-like format construct is also a functional moiety linker.
In this case, instead of two attachment points as in a
(Gly.sub.4-Ser).sub.3Ala or
(Gly.sub.4-Ser).sub.2Gly-Thr-Gly-Ser-Ala-Met-Ala-Ser-(Gly.sub.4-Ser).sub.-
1-Ala, or four possible attachment points as in the Fc domain case,
the antibody shown in the antibody-like example of FIG. 1 provides
6 possible attachment points. The antibody-like format shown in
FIG. 1 exemplifies that in some embodiments only the N-terminal
attachment points in the functional moiety linker are occupied by
FnIII domains of the invention. In an antibody-like format
construct some or all that scaffolds of the invention can readily
be fused to the C-termini of the heavy chains and the light chains
of an antibody or modified antibody domain. Other fusion
stoichiometries can be applied, i.e., one, two, three, four, five,
six, seven, eight, or more scaffolds of the invention can be fused
to an antibody or modified antibody.
[0309] FIGS. 1 and 2 also illustrate that in some embodiments
multimeric FnIII scaffolds can be generated by combining other
FnIII multimeric scaffolds. For example, the Fc format A6, A7, and
A8 scaffolds of FIG. 2 are homodimeric FnIII scaffolds wherein the
multimeric scaffold is formed by two polypeptide chains, each one
comprising a linear format FnIII scaffold fused to an Fc domain,
which in turn are connected via intermolecular disulfide bonds. The
antibody-like format scaffold of FIGS. 1 and 2 exemplifies a
heterotetrameric FnIII scaffold wherein 4 polypeptides
corresponding to two different types of scaffolds (2 FnIII
scaffolds formed by fusing an FnIII monomer scaffold to an IgG
light chain constant region, and 2 FnIII scaffolds formed by fusing
an FnIII monomer scaffold to an CH1-hinge-region-Fc region of an
IgG) are connected via intermolecular disulfide bonds.
Generation of Scaffolds of the Invention
[0310] The FnIII scaffolds described herein may be used in any
technique for evolving new or improved target binding proteins. In
one particular example, the target is immobilized on a solid
support, such as a column resin or microtiter plate well, and the
target contacted with a library of candidate scaffold-based binding
proteins. Such a library may consist of clones constructed from an
FnIII domain, including without limitation the Tn3 module, through
randomization of the sequence and/or the length of the CDR-like
loops. In one embodiment, the library may be a phage, phagemid,
virus, bacterial, yeast, or mammalian cell display or a ribosome
display library. If desired, this library may be an RNA-protein
fusion library generated, for example, by the techniques described
in Szostak et al., U.S. Pat. Nos. 6,258,558; 6,261,804; 5,643,768;
and 5,658,754. Alternatively, it may be a DNA-protein library (for
example, as described in PCT Publ. No. WO 2000/032823).
[0311] In this regard, bacteriophage (phage) display is one well
known technique which allows one to screen large oligopeptide
libraries to identify member(s) of those libraries which are
capable of specifically binding to a target. Phage display is a
technique by which variant polypeptides are displayed as fusion
proteins to the coat protein on the surface of bacteriophage
particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386).
The utility of phage display lies in the fact that large libraries
of selectively randomized protein variants (or randomly cloned
cDNAs) can be rapidly and efficiently sorted for those sequences
that bind to a target molecule with high affinity. Display of
peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA,
87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been
used for screening millions of polypeptides or oligopeptides for
ones with specific binding properties (Smith, G. P. (1991) Current
Opin. Biotechnol., 2:668). Sorting phage libraries of random
mutants requires a strategy for constructing and propagating a
large number of variants, a procedure for affinity purification
using the target receptor, and a means of evaluating the results of
binding enrichments (see for example, U.S. Pat. Nos. 5,223,409,
5,403,484, 5,571,689, and 5,663,143).
[0312] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No.
5,627,024), T4 phage display systems (Ren et al., Gene, 215: 439
(1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998);
Jiang et al., Infection & Immunity, 65(11): 4770-4777 (1997);
Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5: 1833
(1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage
display systems (Smith and Scott, Methods in Enzymology, 217:
228-257 (1993); U.S. Pat. No. 5,766,905) are also known.
[0313] Many other improvements and variations of the basic phage
display concept have now been developed. These improvements enhance
the ability of display systems to screen peptide libraries for
binding to selected target molecules and to display functional
proteins with the potential of screening these proteins for desired
properties. Combinatorial reaction devices for phage display
reactions have been developed (WO 98/14277) and phage display
libraries have been used to analyze and control bimolecular
interactions (WO 98/20169; WO 98/20159) and properties of
constrained helical peptides (WO 98/20036). WO 97/35196 describes a
method of isolating an affinity ligand in which a phage display
library is contacted with one solution in which the ligand will
bind to a target molecule and a second solution in which the
affinity ligand will not bind to the target molecule, to
selectively isolate binding ligands. WO 97/46251 describes a method
of biopanning a random phage display library with an affinity
purified antibody and then isolating binding phage, followed by a
micropanning process using microplate wells to isolate high
affinity binding phage. The use of Staphylococcus aureus protein A
as an affinity tag has also been reported (Li et al. (1998) Mol.
Biotech., 9:187). WO 97/47314 describes the use of substrate
subtraction libraries to distinguish enzyme specificities using a
combinatorial library which may be a phage display library. A
method for selecting enzymes suitable for use in detergents using
phage display is described in WO 97/09446. Additional methods of
selecting specific binding proteins are described in U.S. Pat. Nos.
5,498,538, 5,432,018, and WO 98/15833.
[0314] Methods of generating peptide libraries and screening these
libraries are also disclosed in U.S. Pat. Nos. 5,723,286,
5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018,
5,698,426, 5,763,192, and 5,723,323.
[0315] A bioinformatics approach may be employed to determine the
loop length and diversity preferences of naturally occurring FnIII
domains. Using this analysis, the preferences for loop length and
sequence diversity may be employed to develop a "restricted
randomization" approach. In this restricted randomization, the
relative loop length and sequence preferences are incorporated into
the development of a library strategy. Integrating the loop length
and sequence diversity analysis into library development results in
a restricted randomization (i.e. certain positions within the
randomized loop are limited in which amino acid could reside in
that position).
[0316] The invention also provides recombinant libraries
(hereinafter referred to as "libraries of the invention")
comprising diverse populations of non-naturally occurring FnIII
scaffolds of the invention. In one embodiment, the libraries of the
invention comprise non-naturally occurring FnIII scaffolds
comprising, a plurality of beta strand domains linked to a
plurality of loop regions, wherein one or more of said loops vary
by deletion, substitution or addition by at least one amino acid
from the cognate loops in an FOI, and wherein the beta strands of
the FnIII scaffold have at least 50% homology (i.e., sequence
similarity) to the cognate beta strand sequences of the FOI.
Non-limiting examples of FOI sequences useful for the generation of
recombinant libraries are provided in TABLE 1 and in FIG. 16.
[0317] In some embodiments, libraries of the invention comprise
non-naturally occurring FnIII scaffolds, wherein the FOI is the
protein sequence corresponding to the third FnIII domain of human
tenascin C. In some embodiments, libraries of the invention
comprise non-naturally occurring FnIII scaffolds, wherein the FOI
is the protein sequence corresponding to the tenth FnIII domain of
human fibronectin. In some embodiments, libraries of the invention
comprise non-naturally occurring FnIII scaffolds, wherein the FOI
is the protein sequence corresponding to the fourteenth FnIII
domain of human fibronectin.
[0318] In a specific embodiment, the libraries of the invention
comprise non-naturally occurring FnIII scaffolds, wherein the FOI
is a wild type Tn3 scaffold. In other embodiments, libraries of the
invention comprise non-naturally occurring FnIII scaffolds, wherein
the FOI is a protein sequence corresponding to an additional FnIII
domain from human tenascin C. In other embodiments, libraries of
the invention comprise non-naturally occurring FnIII scaffolds,
wherein the FOI is a protein sequence corresponding to a FnIII
domain from another tenascin protein, or alternatively, a tenascin
protein from another organism (such as, but not limited to, murine,
porcine, bovine, or equine tenascins).
[0319] In yet another embodiment, libraries of the invention
comprise non-naturally occurring FnIII scaffolds, wherein the FOI
is a protein sequence corresponding to a FnIII domain from any
organism. In other embodiments, libraries of the invention comprise
non-naturally occurring FnIII scaffolds, wherein the naturally
occurring sequence corresponds to a predicted FnIII domain from a
thermophilic or hyperthermophilic organism. For example, the
hyperthermophilic organism can be a hyperthermophilic archaea such
as Archaeoglobus fulgidus, Staphylothermus marinus, Sulfolobus
acidocaldarius, Sulfolobus solfataricus, and Sulfolobus
tokodaii.
[0320] In some embodiments, the libraries of the invention comprise
FnIII scaffolds, wherein the beta strands of the FnIII scaffold
have at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99% homology (sequence similarity) to the cognate
beta strain domain in any of SEQ ID NOs: 1-34, 54, 69 or those
presented in FIG. 16, or to the beta strands of a domain recognized
to contain the Pfam PF00041 fibronectin type III domain signature
as determined using Pfam_scan, HMMER, or any other program capable
of comparing a protein sequence to a Hidden Markov model.
[0321] As detailed above, the loops connecting the various beta
strands of the scaffolds may be randomized for length and/or
sequence diversity. In one embodiment, the libraries of the
invention comprise FnIII scaffolds having at least one loop that is
randomized for length and/or sequence diversity. In one embodiment,
at least one, at least two, at least three, at least four, at least
five or at least six loops of the FnIII scaffolds are randomized
for length and/or sequence diversity. In one embodiment, at least
one loop is kept constant while at least one additional loop is
randomized for length and/or sequence diversity. In another
embodiment, at least one, at least two, or all three of loops AB,
CD, and EF are kept constant while at least one, at least two, or
all three of loops BC, DE, and FG are randomized for length or
sequence diversity. In another embodiment, at least one, at least
two, or at least all three of loops AB, CD, and EF are randomized
while at least one, at least two, or all three of loops BC, DE, and
FG are randomized for length and/or sequence diversity.
[0322] As detailed above, it has been surprisingly found that FG
loops which are at least one amino acid shorter than that found in
the FG loop of an FOI are shown to have enhanced stability.
Accordingly the present invention provides libraries of the
invention comprising FnIII scaffolds, wherein at least one loop is
randomized for length and/or sequence diversity, except that length
of the FG loops are held to be at least one amino acid shorter than
the cognate FG loop of an FOI. For example, as defined in FIG. 16,
the native FG loop of the third FnIII domain of human tenascin C
comprises 10 amino acid residues, accordingly, where the third
FnIII domain of human tenascin C is the FOI the FG loop would be
held to 9 amino acid residues or less although the sequence of the
FG loop may be randomized.
[0323] In some embodiments, the libraries of the invention comprise
FnIII scaffolds, wherein each scaffold comprises seven beta strands
designated A, B, C, D, E, F, and G linked to six loop regions,
wherein a loop region connects each beta strand and is designated
AB, BC, CD, DE, EF, and FG; and wherein at least one loop is a
non-naturally occurring variant of a FOI loop, and wherein the FG
loop is at least one amino acid shorter than the cognate FG loop in
the FOI.
[0324] In one embodiment, libraries of the invention comprise FnIII
scaffold, wherein the amino acid sequence of one or more loops
(i.e., AB, BC, CD, DE, EF, FG) has been randomized for length
and/or sequence diversity, except that the length of the FG loops
are held to be at least one, or at least two, or at least 3, or at
least 4, or at least 5, or at least 6, or at least 7, or at least
8, or at least 9, or at least 10 amino acid residue shorter than
the cognate FG loop of an FOI.
[0325] In certain embodiments, the libraries of the invention
comprise FnIII scaffolds, wherein each beta strand has at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 99% or more homology (sequence similarity) to the cognate
beta strands of any one of SEQ ID NOs: 1-34, 54, or 69, to the beta
strands of any of the FnIII domains shown in FIG. 16, or to the
beta strands of a domain recognized to contain the Pfam PF00041
fibronectin type III domain signature as determined using
Pfam_scan, HMMER, or any other program capable of comparing a
protein sequence to a Hidden Markov model.
[0326] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand comprises SEQ
ID NO: 42, the B beta strand comprises SEQ ID NO: 43, the C beta
strand comprises SEQ ID NO: 45, or 131, the D beta strand comprises
SEQ ID NO: 46, the E beta strand comprises SEQ ID NO: 47, the F
beta strand comprises SEQ ID NO: 49, and the G beta strand
comprises SEQ ID NO: 52. In another specific embodiment, the
libraries of the invention comprise FnIII scaffolds, wherein the A
beta strand comprises SEQ ID NO:42, the B beta strand comprises SEQ
ID NO:43, the C beta strand comprises SEQ ID NO:44, the D beta
strand comprises SEQ ID NO:46, the E beta strand comprises SEQ ID
NO:47, the F beta strand comprises SEQ ID NO:50, and the G beta
strand comprises SEQ ID NO:53. In still another specific
embodiment, the libraries of the invention comprise FnIII
scaffolds, wherein the A beta strand comprises SEQ ID NO:42, the B
beta strand comprises SEQ ID NO:43, the C beta strand comprises SEQ
ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E
beta strand comprises SEQ ID NO:47, the F beta strand comprises SEQ
ID NO:51, and the G beta strand comprises SEQ ID NO:53.
[0327] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand consists of SEQ
ID NO: 42, the B beta strand consists of SEQ ID NO: 43, the C beta
strand consists of SEQ ID NO: 45, or 131, the D beta strand
consists of SEQ ID NO: 46, the E beta strand consists of SEQ ID NO:
47, the F beta strand consists of SEQ ID NO: 49, and the G beta
strand consists of SEQ ID NO: 52. In another specific embodiment,
the libraries of the invention comprise FnIII scaffolds, wherein
the A beta strand consists of SEQ ID NO:42, the B beta strand
consists of SEQ ID NO:43, the C beta strand consists of SEQ ID
NO:44, the D beta strand consists of SEQ ID NO:46, the E beta
strand consists of SEQ ID NO:47, the F beta strand consists of SEQ
ID NO:50, and the G beta strand consists of SEQ ID NO:53. In still
another specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand consists of SEQ
ID NO:42, the B beta strand consists of SEQ ID NO:43, the C beta
strand consists of SEQ ID NO:45, or 131, the D beta strand consists
of SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F
beta strand consists of SEQ ID NO:51, and the G beta strand
consists of SEQ ID NO:53.
[0328] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand consists
essentially of SEQ ID NO: 42, the B beta strand consists
essentially of SEQ ID NO: 43, the C beta strand consists
essentially of SEQ ID NO: 45, or 131, the D beta strand consists
essentially of SEQ ID NO: 46, the E beta strand consists
essentially of SEQ ID NO: 47, the F beta strand consists
essentially of SEQ ID NO: 49, and the G beta strand consists
essentially of SEQ ID NO: 52. In another specific embodiment, the
libraries of the invention comprise FnIII scaffolds, wherein the A
beta strand consists essentially of SEQ ID NO:42, the B beta strand
consists essentially of SEQ ID NO:43, the C beta strand consists
essentially of SEQ ID NO:44, the D beta strand consists essentially
of SEQ ID NO:46, the E beta strand consists essentially of SEQ ID
NO:47, the F beta strand consists essentially of SEQ ID NO:50, and
the G beta strand consists essentially of SEQ ID NO:53. In still
another specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, or 131, the D beta strand consists essentially of SEQ ID
NO:46, the E beta strand consists essentially of SEQ ID NO:47, the
F beta strand consists essentially of SEQ ID NO:51, and the G beta
strand consists essentially of SEQ ID NO:53.
[0329] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain
comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43,
the C beta strand comprises SEQ ID NO:45, the D beta strand
comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47,
the F beta strand comprises SEQ ID NO:49, and the G beta strand
comprises SEQ ID NO:52, the AB loop comprises SEQ ID NO:35, the CD
loop comprises SEQ ID NO:37 and the EF loop comprises SEQ ID
NO:39.
[0330] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C
beta strand consists of SEQ ID NO:45, the D beta strand consists of
SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F
beta strand consists of SEQ ID NO:49, and the G beta strand
consists of SEQ ID NO:52, the AB loop consists of SEQ ID NO:35, the
CD loop consists of SEQ ID NO:37 and the EF loop consists of SEQ ID
NO:39.
[0331] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, the D beta strand consists essentially of SEQ ID NO:46, the
E beta strand consists essentially of SEQ ID NO:47, the F beta
strand consists essentially of SEQ ID NO:49, and the G beta strand
consists essentially of SEQ ID NO:52, the AB loop consists
essentially of SEQ ID NO:35, the CD loop consists essentially of
SEQ ID NO:37 and the EF loop consists essentially of SEQ ID
NO:39.
[0332] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain
comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43,
the C beta strand comprises SEQ ID NO:45, the D beta strand
comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47,
the F beta strand comprises SEQ ID NO:49, and the G beta strand
comprises SEQ ID NO:52, the BC loop comprises SEQ ID NO:36, the DE
loop comprises SEQ ID NO:38 and the FG loop comprises SEQ ID
NO:40.
[0333] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C
beta strand consists of SEQ ID NO:45, the D beta strand consists of
SEQ ID NO:46, the E beta strand consists of SEQ ID NO:47, the F
beta strand consists of SEQ ID NO:49, and the G beta strand
consists of SEQ ID NO:52, the BC loop consists of SEQ ID NO:36, the
DE loop consists of SEQ ID NO:38 and the FG loop consists of SEQ ID
NO:40.
[0334] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, or 131, the D beta strand consists essentially of SEQ ID
NO:46, the E beta strand consists essentially of SEQ ID NO:47, the
F beta strand consists essentially of SEQ ID NO:49, and the G beta
strand consists essentially of SEQ ID NO:52, the BC loop consists
essentially of SEQ ID NO:36, the DE loop consists essentially of
SEQ ID NO:38 and the FG loop consists essentially of SEQ ID
NO:40.
[0335] In another specific embodiment, the libraries of the
invention comprise FnIII scaffolds, wherein the A beta strand
comprises SEQ ID NO: 42, the B beta strand comprises SEQ ID NO: 43,
the C beta strand comprises SEQ ID NO: 45, or 131, the D beta
strand comprises SEQ ID NO: 46, the E beta strand comprises SEQ ID
NO: 47, the F beta strand comprises SEQ ID NO: 49, and beta strand
G comprises SEQ ID NO: 52, and wherein one or more of the beta
strands comprise at least one amino acid substitution except that
the cysteine in the C beta strand and the cysteine in the F beta
strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be
substituted.
[0336] In another specific embodiment, the libraries of the
invention comprise FnIll scaffolds, wherein the A beta strand
consists of SEQ ID NO: 42, the B beta strand consists of SEQ ID NO:
43, the C beta strand consists of SEQ ID NO: 45, or 131, the D beta
strand consists of SEQ ID NO: 46, the E beta strand consists of SEQ
ID NO: 47, the F beta strand consists of SEQ ID NO: 49, and beta
strand G consists of SEQ ID NO: 52, and wherein one or more of the
beta strands comprise at least one amino acid substitution except
that the cysteine in the C beta strand and the cysteine in the F
beta strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not
be substituted.
[0337] In another specific embodiment, the libraries of the
invention comprise FnIII scaffolds, wherein the A beta strand
consists essentially of SEQ ID NO: 42, the B beta strand consists
essentially of SEQ ID NO: 43, the C beta strand consists
essentially of SEQ ID NO: 45, or 131, the D beta strand consists
essentially of SEQ ID NO: 46, the E beta strand consists
essentially of SEQ ID NO: 47, the F beta strand consists
essentially of SEQ ID NO: 49, and beta strand G consists
essentially of SEQ ID NO: 52, and wherein one or more of the beta
strands comprise at least one amino acid substitution except that
the cysteine in the C beta strand and the cysteine in the F beta
strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be
substituted.
[0338] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain
comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43,
the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand
comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47,
the F beta strand comprises SEQ ID NO:49, the G beta strand
comprises SEQ ID NO:52, the AB loop comprises SEQ ID NO:35, the CD
loop comprises SEQ ID NO:37, and the EF loop comprises SEQ ID NO:39
and, wherein one or more of the beta strands comprise at least one
amino acid substitution except that the cysteine in the C beta and
the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49,
respectively) may not be substituted.
[0339] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C
beta strand consists of SEQ ID NO:45, or 131, the D beta strand
consists of SEQ ID NO:46, the E beta strand consists of SEQ ID
NO:47, the F beta strand consists of SEQ ID NO:49, the G beta
strand consists of SEQ ID NO:52, the AB loop consists of SEQ ID
NO:35, the CD loop consists of SEQ ID NO:37, and the EF loop
consists of SEQ ID NO:39 and, wherein one or more of the beta
strands comprise at least one amino acid substitution except that
the cysteine in the C beta and the cysteine in the F beta strand
(SEQ ID NOs: 45, or 131, and 49, respectively) may not be
substituted.
[0340] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, or 131, the D beta strand consists essentially of SEQ ID
NO:46, the E beta strand consists essentially of SEQ ID NO:47, the
F beta strand consists essentially of SEQ ID NO:49, the G beta
strand consists essentially of SEQ ID NO:52, the AB loop consists
essentially of SEQ ID NO:35, the CD loop consists essentially of
SEQ ID NO:37, and the EF loop consists essentially of SEQ ID NO:39
and, wherein one or more of the beta strands comprise at least one
amino acid substitution except that the cysteine in the C beta and
the cysteine in the F beta strand (SEQ ID NOs: 45, or 131, and 49,
respectively) may not be substituted.
[0341] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain
comprises SEQ ID NO:42, the B beta strand comprises SEQ ID NO:43,
the C beta strand comprises SEQ ID NO:45, or 131, the D beta strand
comprises SEQ ID NO:46, the E beta strand comprises SEQ ID NO:47,
the F beta strand comprises SEQ ID NO:49, the G beta strand
comprises SEQ ID NO:52, the BC loop comprises SEQ ID NO:36, the DE
loop comprises SEQ ID NO:38, and the FG loop comprises SEQ ID NO:40
and, wherein one or more of the beta strands comprise at least one
amino acid substitution except that the cysteine in the C beta
strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or
131, and 49, respectively) may not be substituted.
[0342] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
of SEQ ID NO:42, the B beta strand consists of SEQ ID NO:43, the C
beta strand consists of SEQ ID NO:45, or 131, the D beta strand
consists of SEQ ID NO:46, the E beta strand consists of SEQ ID
NO:47, the F beta strand consists of SEQ ID NO:49, the G beta
strand consists of SEQ ID NO:52, the BC loop consists of SEQ ID
NO:36, the DE loop consists of SEQ ID NO:38, and the FG loop
consists of SEQ ID NO:40 and, wherein one or more of the beta
strands comprise at least one amino acid substitution except that
the cysteine in the C beta strand and the cysteine in the F beta
strand (SEQ ID NOs: 45, or 131, and 49, respectively) may not be
substituted.
[0343] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the A beta strand domain consists
essentially of SEQ ID NO:42, the B beta strand consists essentially
of SEQ ID NO:43, the C beta strand consists essentially of SEQ ID
NO:45, or 131, the D beta strand consists essentially of SEQ ID
NO:46, the E beta strand consists essentially of SEQ ID NO:47, the
F beta strand consists essentially of SEQ ID NO:49, the G beta
strand consists essentially of SEQ ID NO:52, the BC loop consists
essentially of SEQ ID NO:36, the DE loop consists essentially of
SEQ ID NO:38, and the FG loop consists essentially of SEQ ID NO:40
and, wherein one or more of the beta strands comprise at least one
amino acid substitution except that the cysteine in the C beta
strand and the cysteine in the F beta strand (SEQ ID NOs: 45, or
131, and 49, respectively) may not be substituted.
[0344] As detailed above, one or more residues within a loop may be
held constant while other residues are randomized for length and/or
sequence diversity. Optionally or alternatively, one or more
residues within a loop may be held to a predetermined and limited
number of different amino acids while other residues are randomized
for length and/or sequence diversity. Accordingly, libraries of the
invention comprise FnIII scaffolds that may comprise one or more
loops having a degenerate consensus sequence and/or one or more
invariant amino acid residues. In one embodiment, the libraries of
the invention comprise FnIII scaffolds having AB loops which are
randomized with the following consensus sequence: K-X-X-X-X-X-a,
wherein X represents asparagine, aspartic acid, histidine,
tyrosine, isoleucine, valine, leucine, phenylalanine, threonine,
alanine, proline, or serine, and wherein (a) represents serine,
threonine, alanine, or glycine. In another embodiment, the
libraries of the invention comprise FnIII scaffolds having AB loops
which are randomized with the following consensus sequence:
K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid,
histidine, tyrosine, isoleucine, valine, leucine, phenylalanine,
threonine, alanine, proline, or serine, and wherein (a) represents
serine, threonine, alanine, or glycine.
[0345] In another embodiment, the libraries of the invention
comprise FnIII scaffolds having BC loops which are randomized with
the following consensus sequence: S-X-a-X-b-X-X-X-G, wherein X
represents any amino acid, wherein (a) represents proline or
alanine and wherein (b) represents alanine or glycine. In another
embodiment, the libraries of the invention comprise FnIII scaffolds
having BC loops which are randomized with the following consensus
sequence: S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino
acid and wherein (c) represents proline, serine or glycine. In
still another embodiment, the libraries of the invention comprise
FnIII scaffolds having BC loops which are randomized with the
following consensus sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X
represents any amino acid, wherein (d) represents proline,
glutamate or lysine, wherein (e) represents asparagine or glycine,
and wherein (f) represents serine or glycine.
[0346] In one embodiment, the libraries of the invention comprise
FnIII scaffolds having CD loops which are randomized with the
following consensus sequence: X.sub.n, wherein X represents any
amino acid, and wherein n=6, 7, 8, 9, or 10. In another embodiment,
the scaffolds of the invention comprise an CD loop which is
randomized with the following consensus sequence: X.sub.n, wherein
X represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein n=7, 8, or 9.
[0347] In one embodiment the libraries of the invention comprise
FnIII scaffolds having DE loops which are randomized with the
following consensus sequence: X-X-X-X-X-X, wherein X represents any
amino acid.
[0348] In one embodiment, the libraries of the invention comprise
FnIII scaffolds having EF loops which are randomized with the
following consensus sequence: X-b-L-X-P-X-c-X, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine,
wherein (b) represents asparagine, lysine, arginine, aspartic acid,
glutamic acid, or glycine, and wherein (c) represents isoleucine,
threonine, serine, valine, alanine, or glycine.
[0349] In one embodiment, the libraries of the invention comprise
FnIII scaffolds having FG loops which are randomized with the
following consensus sequence: X-a-X-X-G-X-X-X-b, wherein X
represents any amino acid, wherein (a) represents asparagine,
threonine or lysine, and wherein (b) represents serine or alanine.
In another embodiment, the libraries of the invention comprise
FnIII scaffolds having FG loops which are randomized with the
following consensus sequence: X-X-X-X-X-X-X-X-X (X.sub.9), wherein
X represents any amino acid.
[0350] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the FnIII scaffolds comprise a
Tn3 module. In another specific embodiment, the libraries of the
invention comprise FnIII scaffolds, wherein the FnIII scaffolds
comprise a Tn3 module and wherein one or more of the beta strands
of the Tn3 module comprise at least one amino acid substitution
except that the cysteine in the C beta strand and the cysteine in
the F beta strand (SEQ ID NOs: 45, or 131, and 49, respectively)
may not be substituted.
[0351] In a specific embodiment, the libraries of the invention
comprise FnIII scaffolds, wherein the scaffolds comprise the amino
acid sequence:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKET FTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein n=2-26 and m=1-9.
[0352] The invention further provides methods for identifying a
recombinant FnIII scaffold that binds a target and has increased
stability as compared to an FOI by screening the libraries of the
invention, in particular the libraries comprising FnIII scaffolds
wherein the FG loops are held to be at least one amino acid shorter
than the cognate FG loop of the FOI.
[0353] In certain embodiments, the method for identifying a
recombinant FnIII scaffold having increased protein stability as
compared to an FOI, and which specifically binds a target,
comprising: [0354] a. contacting the target ligand with a library
of the invention under conditions suitable for forming a
scaffold:target ligand complex, wherein the libraries comprise
FnIII scaffolds having FG loops that are held to be at least one
amino acid shorter than the cognate FG loop of the POI; [0355] b.
obtaining from the complex, the scaffold that binds the target
ligand; [0356] c. determining if the stability of the scaffold
obtained in step (b) is greater than that of the FOI.
[0357] In one embodiment, in step (a) the scaffold library of the
invention is incubated with immobilized target. In one embodiment,
in step (b) the scaffold:target ligand complex is washed to remove
non-specific binders, and the tightest binders are eluted under
very stringent conditions and subjected to PCR to recover the
sequence information. Methods useful for the determination of
stability in step (c) have been described supra. It is specifically
contemplated that the binders and/or sequence information obtained
in step (b) can be used to create a new library using the methods
disclosed herein or known to one of skill in the art, which may be
used to repeat the selection process, with or without further
mutagenesis of the sequence. In some embodiments, a number of
rounds of selection may be performed until binders of sufficient
affinity for the antigen are obtained.
[0358] A further embodiment of the invention is a collection of
isolated nucleic acid molecules encoding a library comprising the
scaffolds of the invention and as described above.
[0359] Scaffolds of the invention may comprise codons encoded by
the NHT codon scheme described in PCT Publication No: WO
2009/058379 or, alternatively, may comprise codons encoded by the
NNK mixed codon scheme.
[0360] The scaffolds of the invention may be subjected to affinity
maturation. In this art-accepted process, a specific binding
protein is subject to a scheme that selects for increased affinity
for a specific target (see Wu et al., Proc Natl Acad Sci USA.
95(11):6037-42). The resultant scaffolds of the invention may
exhibit binding characteristics at least as high as compared to the
scaffolds prior to affinity maturation.
[0361] The invention also provides methods of identifying the amino
acid sequence of a protein scaffold capable of binding to target so
as to form a scaffold:target complex. In one embodiment, the method
comprises: a) contacting a library of the invention with an
immobilized or separable target; b) separating the scaffold:target
complexes from the free scaffolds; c) causing the replication of
the separated scaffolds of (b) so as to result in a new polypeptide
display library distinguished from that in (a) by having a lowered
diversity and by being enriched in displayed scaffolds capable of
binding the target; d) optionally repeating steps (a), and (b) with
the new library of (c); and e) determining the nucleic acid
sequence of the region encoding the displayed scaffold of a species
from (d) and hence deducing the peptide sequence capable of binding
to the target.
[0362] In another embodiment, the scaffolds of the invention may be
further randomized after identification from a library screen. In
one embodiment, methods of the invention comprise further
randomizing at least one, at least two, at least three, at least
four, at least five or at least six loops of a scaffold identified
from a library using a method described herein. In another
embodiment, the further randomized scaffold is subjected to a
subsequent method of identifying a scaffold capable of binding a
target. This method comprises (a) contacting said further
randomized scaffold with an immobilized or separable target, (b)
separating the further randomized scaffold:target complexes from
the free scaffolds, (c) causing the replication of the separated
scaffolds of (b), optionally repeating steps (a)-(c), and (d)
determining the nucleic acid sequence of the region encoding said
further randomized scaffold and hence, deducing the peptide
sequence capable of binding to the target.
[0363] In a further embodiment, the further randomized scaffolds
comprise at least one, at least two, at least three, at least four,
at least five, or at least six randomized loops which were
previously randomized in the first library. In an alternate further
embodiment, the further randomized scaffolds comprise at least one,
at least two, at least three, at least four, at least five, or at
least six randomized loops which were not previously randomized in
the first library.
[0364] The invention also provides a method for obtaining at least
two FnIII scaffolds that bind to at least one or more targets. This
method allows for the screening of agents that act cooperatively to
elicit a particular response. It may be advantageous to use such a
screen when an agonistic activity requiring the cooperation of more
than one scaffold is required (for example, but not limited to,
agonism of a receptor belonging to the TNF receptor family). This
method allows for the screening of cooperative agents without the
reformatting of the library to form multimeric complexes. In one
embodiment, the method of the invention comprises contacting a
target ligand with a library of the invention under conditions that
allow a scaffold:target ligand complex to form, engaging said
scaffolds with a crosslinking agent (defined as an agent that
brings together, in close proximity, at least two identical or
distinct scaffolds) wherein the crosslinking of the scaffolds
elicits a detectable response and obtaining from the complex, said
scaffolds that bind the target. In a further embodiment, the
crosslinking agent is a scaffold specific antibody, or fragment
thereof, an epitope tag specific antibody of a fragment thereof, a
dimerization domain, such as Fc region, a coiled coil motif (for
example, but not limited to, a leucine zipper), a chemical
crosslinker, or another dimerization domain known in the art.
[0365] The invention also provides methods of detecting a compound
utilizing the scaffolds of the invention. Based on the binding
specificities of the scaffolds obtained by library screening, it is
possible to use such scaffolds in assays to detect the specific
target in a sample, such as for diagnostic methods. In one
embodiment, the method of detecting a compound comprises contacting
said compound in a sample with a scaffold of the invention, under
conditions that allow a compound: scaffold complex to form and
detecting said scaffold, thereby detecting said compound in a
sample. In further embodiments, the scaffold is labeled (i.e.,
radiolabel, fluorescent, enzyme-linked or colorimetric label) to
facilitate the detection of the compound.
[0366] The invention also provides methods of capturing a compound
utilizing the scaffolds of the invention. Based on the binding
specificities of the scaffolds obtained by library screening, it is
possible to use such scaffolds in assays to capture the specific
target in a sample, such as for purification methods. In one
embodiment, the method of capturing a compound in a sample
comprises contacting said compound in a sample with a scaffold of
the invention under conditions that allow the formation of a
compound:scaffold complex and removing said complex from the
sample, thereby capturing said compound in said sample. In further
embodiments, the scaffold is immobilized to facilitate the removing
of the compound:scaffold complex.
[0367] In some embodiments, scaffolds isolated from libraries of
the invention comprise at least one, at least two, at least four,
at least five, at least six, or more randomized loops. In some
embodiments, isolated scaffold loop sequences may be swapped from a
donor scaffold to any loop in a receiver scaffold (for example, an
FG loop sequence from a donor scaffold may be transferred to any
loop in a receiver scaffold). In specific embodiments, an isolated
loop sequences may be transferred to the cognate loop in the
receiving scaffold (for example, an FG loop sequence from a donor
scaffold may be transferred to a receiver scaffold in the FG loop
position). In some embodiments, isolated loop sequences may be "mix
and matched" randomly with various receiver scaffolds.
[0368] In other embodiments, isolated scaffolds sequences may be
identified by the loop sequence. For example, a library is used to
pan against a particular target and an collection of specific
binders are isolated. The randomized loop sequences may be
characterized as specific sequences independently of the scaffold
background (i.e., the scaffold that binds target X wherein said
scaffold comprises an FG loop sequence of SEQ ID NO:X). In
alternative embodiments, where a scaffold exhibits two loop
sequences that bind target X, the loop sequences may be
characterized as binding target X in the absence of the scaffold
sequence. In other words, it is contemplated that scaffolds
isolated from a library that bind a particular target may be
expressed as the variable loop sequences that bind that target
independent of the scaffold backbone. This process would be
analogous to the concept of CDRs in variable regions of
antibodies.
Generation of Tandem Repeats
[0369] Linking of tandem constructs may be generated by ligation of
oligonucleotides at restriction sites using restriction enzymes
known in the art, including but not limited to type II and type IIS
restriction enzymes. Type II restriction enzymes cut within their
recognition sequence while type IIS restriction enzymes cut outside
their recognition sequence to one side. In one embodiment for
generating tandem repeats, type IIS enzymes are oriented so that
cutting with them cleaves off their recognition site and leaves
ends that can be joined together without generating recognition
sites at the junction of two subunits. After ligation, both type II
and type IIS sites remain at the ends. Additional subunits may be
added by cutting with a type IIS restriction enzyme again and
ligating. Alternatively, the clone may be cut with a type II
restriction enzyme and ligated into a vector.
[0370] The multimeric scaffolds of the invention may comprise a
linker at the C-terminus and/or the N-terminus and/or between
domains as described herein. Further, scaffolds of the invention
comprising at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7, at least 8 or polypeptide scaffolds may
be fused or conjugated to a dimerization domain, including but not
limited to an antibody moiety selected from: [0371] (i) a Fab
fragment, having VL, CL, VH and CH1 domains; [0372] (ii) a Fab'
fragment, which is a Fab fragment having one or more cysteine
residues at the C-terminus of the CH1 domain; [0373] (iii) a Fd'
fragment having VH and CH1 domains; [0374] (iv) a Fd' fragment
having VH and CH1 domains and one or more cysteine residues at the
C-terminus of the CH1 domain; [0375] (v) a Fv fragment having the
VL and VH domains of a single arm of an antibody; [0376] (vi) a dAb
fragment (Ward et al., Nature 341, 544-546 (1989)) which consists
of a VH domain; [0377] (vii) isolated CDR regions; [0378] (viii)
F(ab').sub.2 fragments, a bivalent fragment including two Fab'
fragments linked by a disulphide bridge at the hinge region; [0379]
(ix) single chain antibody molecules (e.g., single chain Fv; scFv)
(Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS
(USA) 85:5879-5883 (1988)); [0380] (x) a "diabody" with two antigen
binding sites, comprising a heavy chain variable domain (VH)
connected to a light chain variable domain (VL) in the same
polypeptide chain (see, e.g., EP Patent Publication No. 404,097;
WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993)); [0381] (xi) a "linear antibody" comprising a
pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995);
and U.S. Pat. No. 5,641,870); [0382] (xii) a full length antibody;
and [0383] (xiii) an Fc region comprising CH2-CH3, which may
further comprise all or a portion of a hinge region and/or a CH1
region. Various valency, affinity, and spatial orientation schemes
are exemplified below in the Examples.
Scaffold Production
[0384] Recombinant expression of a scaffold of the invention
requires construction of an expression vector containing a
polynucleotide that encodes the scaffold. Once a polynucleotide
encoding a scaffold has been obtained, the vector for the
production of scaffold may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods
for preparing a protein by expressing a polynucleotide containing a
scaffold encoding nucleotide sequence are described herein. Methods
that are well known to those skilled in the art can be used to
construct expression vectors containing scaffold polypeptide coding
sequences and appropriate transcriptional and translational control
signals. These methods include, for example, in vitro recombinant
DNA techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding a scaffold of the
invention, operably linked to a promoter.
[0385] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce a scaffold of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding a scaffold of the invention, operably linked to a
heterologous promoter. Suitable host cells include, but are not
limited to, microorganisms such as bacteria (e.g., E. colit and B.
subtilis).
[0386] A variety of host-expression vector systems may be utilized
to express the scaffolds of the invention. Such host-expression
systems represent vehicles by which the coding sequences of
interest may be produced and subsequently purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, express a scaffold of the
invention in situ. These include but are not limited to
microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing scaffold coding sequences;
yeast (e.g., Saccharomyces, Pichia) transformed with recombinant
yeast expression vectors containing scaffold coding sequences;
insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus) containing scaffold coding sequences;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing scaffold coding sequences; or
mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0387] Expression vectors containing inserts of a gene encoding a
scaffold of the invention can be identified by three general
approaches: (a) nucleic acid hybridization, (b) presence or absence
of "marker" gene functions, and (c) expression of inserted
sequences. In the first approach, the presence of a gene encoding a
peptide, polypeptide, protein or a fusion protein in an expression
vector can be detected by nucleic acid hybridization using probes
comprising sequences that are homologous to an inserted gene
encoding the peptide, polypeptide, protein or the fusion protein,
respectively. In the second approach, the recombinant vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, transformation phenotype,
occlusion body formation in baculovirus, etc.) caused by the
insertion of a nucleotide sequence encoding an antibody or fusion
protein in the vector. For example, if the nucleotide sequence
encoding the scaffold is inserted within the marker gene sequence
of the vector, recombinants containing the gene encoding the
scaffold insert can be identified by the absence of the marker gene
function. In the third approach, recombinant expression vectors can
be identified by assaying the gene product (e.g., scaffold or
multimer thereof) expressed by the recombinant. Such assays can be
based, for example, on the physical or functional properties of the
protein in in vitro assay systems, e.g., binding, agonistic or
antagonistic properties of the scaffold.
[0388] Methods useful for the production of scaffolds of the
invention are disclosed, for example, in Publication No: WO
2009/058379.
Scaffold Purification
[0389] Once a scaffold of the invention has been produced by
recombinant expression, it may be purified by any method known in
the art for purification of a protein, for example, by
chromatography (e.g., metal-chelate chromatography, ion exchange,
affinity, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins.
[0390] The highly stable nature of the scaffolds of the invention
allow for variations on purification schemes. For example, the
thermal stability exhibited by the scaffolds of the invention allow
for the heating of the crude lysate comprising the scaffolds to
remove the bulk of the host cell proteins by denaturation. The high
protease resistance exhibited by the scaffolds of the invention
allows for the rapid degradation of host cell proteins in crude
lysates prior to any purification steps. Also, the pH tolerance
exhibited by some scaffolds of the invention allows for the
selective precipitation of host cell proteins in the crude lysate
by lowering or raising the pH prior to any purification steps. A
combination of any of the above may be used in an effort to remove
bulk host cell proteins from the crude lysate.
[0391] Production of the scaffolds of the invention in the research
laboratory can be scaled up to produce scaffolds in analytical
scale reactors or production scale reactors, as described in U.S.
Patent Application Publ. No. US 2010/0298541 A1.
Scalable Production of Secreted Scaffolds
[0392] The scaffolds of the invention may be produced
intracellularly or as a secreted form. In some embodiments, the
secreted scaffolds are properly folded and fully functional. The
production of secreted scaffolds comprises the use of a Ptac
promoter and an oppA signal. The scaffold expressed in a
prokaryotic host cell is secreted into the periplasmic space of the
prokaryotic host cell into the media. Scaffolds of the invention
may act as carrier molecules for the secretion of peptides and/or
proteins into the cell culture media or periplasmic space of a
prokaryotic cell.
[0393] In an effort to obtain large quantities, scaffolds of the
invention may be produced by a scalable process (hereinafter
referred to as "scalable process of the invention"). In some
embodiments, scaffolds may be produced by a scalable process of the
invention in the research laboratory that may be scaled up to
produce the scaffolds of the invention in analytical scale
bioreactors (for example, but not limited to 5 L, 10 L, 15 L, 30 L,
or 50 L bioreactors). In other embodiments, the scaffolds may be
produced by a scalable process of the invention in the research
laboratory that may be scaled up to produce the scaffolds of the
invention in production scale bioreactors (for example, but not
limited to 75 L, 100 L, 150 L, 300 L, or 500 L). In some
embodiments, the scalable process of the invention results in
little or no reduction in production efficiency as compared to the
production process performed in the research laboratory.
[0394] In some embodiments, the scalable process of the invention
produces multimeric scaffolds at production efficiency of about 10
mg/L, about 20 m/L, about 30 mg/L, about 50 mg/L, about 75 mg/L,
about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L,
about 200 mg/L, about 250 mg/L, about 300 mg/L or higher.
[0395] In other embodiments, the scalable process of the invention
produces multimeric scaffolds at a production efficiency of at
least about 10 mg/L, at least about 20 m/L, at least about 30 mg/L,
at least about 50 mg/L, at least about 75 mg/L, at least about 100
mg/L, at least about 125 mg/L, at least about 150 mg/L, at least
about 175 mg/L, at least about 200 mg/L, at least about 250 mg/L,
at least about 300 mg/L or higher.
[0396] In other embodiments, the scalable process of the invention
produces multimeric scaffolds at a production efficiency from about
10 mg/L to about 300 mg/L, from about 10 mg/L to about 250 mg/L,
from about 10 mg/L to about 200 mg/L, from about 10 mg/L to about
175 mg/L, from about 10 mg/L to about 150 mg/L, from about 10 mg/L
to about 100 mg/L, from about 20 mg/L to about 300 mg/L, from about
20 mg/L to about 250 mg/L, from about 20 mg/L to about 200 mg/L,
from 20 mg/L to about 175 mg/L, from about 20 mg/L to about 150
mg/L, from about 20 mg/L to about 125 mg/L, from about 20 mg/L to
about 100 mg/L, from about 30 mg/L to about 300 mg/L, from about 30
mg/L to about 250 mg/L, from about 30 mg/L to about 200 mg/L, from
about 30 mg/L to about 175 mg/L, from about 30 mg/L to about 150
mg/L, from about 30 mg/L to about 125 mg/L, from about 30 mg/L to
about 100 mg/L, from about 50 mg/L to about 300 mg/L, from about 50
mg/L to about 250 mg/L, from about 50 mg/L to about 200 mg/L, from
50 mg/L to about 175 mg/L, from about 50 mg/L to about 150 mg/L,
from about 50 mg/L to about 125 mg/L, or from about 50 mg/L to
about 100 mg/L.
[0397] In some embodiments, the scalable process of the invention
produces scaffolds at production efficiency of about 1 g/L, about 2
g/L, about 3 g/L, about 5 g/L, about 7.5 g/L, about 10 g/L, about
12.5 g/L, about 15.0 g/L, about 17.5 g/L, about 20 g/L, about 25
g/L, about 30 g/L, or higher.
[0398] In other embodiments, the scalable process of the invention
produces scaffolds at a production efficiency of at least about 1
g/L, at least about 2 g/L, at least about 3 g/L, at least about 5
g/L, at least about 7.5 g/L, at least about 10 g/L, at least about
12.5 g/L, at least about 15 g/L, at least about 17.5 g/L, at least
about 20 g/L, at least about 25 g/L, at least about 30 g/L, or
higher.
Linkers
[0399] The scaffolds of the invention are linked by protein and/or
nonprotein linkers, wherein each linker is fused to at least two
scaffolds of the invention. Choosing a suitable linker for a
specific case where two or more scaffolds of the invention are to
be connected depends on a variety of parameters including, e.g.,
the nature of the FnIII monomer domains, the stability of the
peptide linker towards proteolysis and oxidation, conformational
constrains to guide multimer folding, and/or conformational
constraints related to the desired biological activity of the
scaffold.
[0400] A suitable linker can consist of a protein linker, a
nonprotein linker, and combinations thereof. Combinations of
linkers can be homomeric or heteromeric. In some embodiments, a
multimeric FnIII scaffold of the invention comprises a plurality of
FnIII scaffolds of the invention wherein are all the linkers are
identical. In other embodiments, a multimeric FnIII scaffold of the
invention comprises a plurality of FnIII scaffolds of the invention
wherein at least one of the linkers is functionally or structurally
different from the rest of the linkers. In some embodiments,
linkers can themselves contribute to the activity of a multimeric
FnIII scaffold by participating directly in the binding to a
target.
[0401] In some embodiments, the protein linker is a polypeptide. In
some embodiments, a linker polypeptide predominantly includes amino
acid residues selected from the group consisting of Gly, Ser, Ala
and Thr. For example, in some embodiments the peptide linker
contains at least 75% (calculated on the basis of the total number
of amino acid residues present in the peptide linker), at least
80%, at least 85% or at least 90% of amino acid residues selected
from the group consisting of Gly, Ser, Ala and Thr. In some
embodiments, the peptide linker consists of Gly, Ser, Ala and/or
Thr residues only.
[0402] The linker polypeptide should have a length, which is
adequate to link two or more monomer scaffolds of the invention or
two or more multimeric scaffolds of the invention in such a way
that they assume the correct conformation relative to one another
so that they retain the desired activity.
[0403] In one embodiment, the polypeptide linker comprises 1 to
about 1000 amino acids residues, 1 to about 50 amino acid residues,
1-25 amino acid residues, 1-20 amino acid residues, 1-15 amino acid
residues, 1-10 amino acid residues, 1-5 amino acid residues, 1-3
amino acid residues. The invention further provides nucleic acids,
such as DNA, RNA, or combinations of both, encoding the polypeptide
linker sequence. The amino acid residues selected for inclusion in
the polypeptide linker should exhibit properties that do not
interfere significantly with the activity or function of the
multimeric scaffold of the invention. Thus, a polypeptide linker
should on the whole not exhibit a charge which would be
inconsistent with the activity or function of the multimeric
scaffold of the invention, or interfere with internal folding, or
form bonds or other interactions with amino acid residues in one or
more of the FnIII monomer domains which would seriously impede the
binding of the multimeric scaffold of the invention to specific
targets.
[0404] In some embodiments, randomization is used to obtain linkers
that afford maximum stability and/or activity of a multimeric
scaffold. In this process, conformationally flexible linkers are
first used to find suitable combination of scaffolds of the
invention, and the resulting multimeric scaffold is subsequently
optimized by randomizing the amino acids residues in the
polypeptide linkers.
[0405] The use of naturally occurring as well as artificial peptide
linkers to connect polypeptides into novel linked fusion
polypeptides is well known in the literature (Hallewell et al.
(1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995),
Protein Eng 8, 725-731; Robinson & Sauer (1996), Biochemistry
35, 109-116; Khandekar et al. (1997), J. Biol. Chem. 272,
32190-32197; Fares et al. (1998), Endocrinology 139, 2459-2464;
Smallshaw et al. (1999), Protein Eng. 12, 623-630; U.S. Pat. No.
5,856,456).
[0406] Accordingly, the linkers fusing two or more scaffolds of the
invention are natural linkers (see, e.g., George & Hering a,
Protein Eng. 11:871-879, 2002), artificial linkers, or combinations
thereof. In some embodiments, the amino acid sequences of all
peptide linkers present in a multimeric scaffold of the invention
are identical. In other embodiments, the amino acid sequences of at
least two of the peptide linkers present in a multimeric scaffold
of the invention are different.
[0407] In some embodiments, a polypeptide linker possesses
conformational flexibility. In some embodiments, a polypeptide
linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15
glycine residues or 8-12 glycine residues. In some embodiments, a
polypeptide linker comprises at least 50% glycine residues, at
least 75% glycine residues, at least 80% glycine residues, or at
least 85% glycine residues. In some embodiments, a polypeptide
linker sequence comprises glycine residues only. In a specific
embodiment, a polypeptide linker sequence comprises a
(G-G-G-G-S).sub.x amino acid sequence where x is a positive
integer. In another specific embodiment, a polypeptide linker
sequence comprises a (G-A).sub.x sequence where x is a positive
integer. In another specific embodiment, a polypeptide linker
sequence comprises a (G-G-G-T-P-T).sub.x sequence where x is a
positive integer. In still another specific embodiment, a
polypeptide linker sequence comprises a
(G-G-G-G-S-G-T-G-S-A-M-A-S).sub.x sequence where x is a positive
integer.
[0408] In some embodiments, a polypeptide linker is an inherently
unstructured natural or artificial polypeptide (see, e.g.,
Schellenberger et al., Nature Biotechnol. 27:1186-1190, 2009; see
also, Sickmeier et al., Nucleic Acids Res. 35:D786-93, 2007).
[0409] In some embodiments, the conformational flexibility of a
polypeptide linker is restricted by including one or more proline
amino acid residues in the amino acid sequence of the polypeptide
linker. Thus, in another embodiment of the invention, the
polypeptide linker may comprise at least one proline residue in the
amino acid sequence of the polypeptide linker. For example, the
polypeptide linker has an amino acid sequence, wherein at least
25%, at least 50%, at least 75%, of the amino acid residues are
proline residues. In one particular embodiment of the invention,
the polypeptide linker comprises proline residues only.
[0410] In some embodiments, alpha-helix-forming linkers can be
used, e.g., the Ala-(Glu-Ala-Ala-Ala-Lys)n-Ala linear linker
(n=2-5) (see, e.g., Arai et al., Protein Eng 14:529-532, 2001) or
alpha-helix-bundle linkers (see, e.g., Maeda et al., Anal. Biochem.
249:147-152, 1997). In other embodiments, Ser-rich linkers can be
used, e.g., (Ser-4-Gly).sub.n (n>1) or (X4-Gly).sub.n (wherein
up to two X's are Thr, the remaining X's are Ser, and n>1) (see
U.S. Pat. No. 5,525,491). In other embodiments, (Gly-Ser).sub.n,
(Gly-Gly-Ser-Gly).sub.n, or Gly-Ser-Ala-Thr linkers are used.
[0411] The peptide linker can be modified in such a way that an
amino acid residue comprising an attachment group for a
non-polypeptide moiety is introduced. Examples of such amino acid
residues may be a cysteine residue (to which the non-polypeptide
moiety is then subsequently attached) or the amino acid sequence
may include an in vivo N-glycosylation site (thereby attaching a
sugar moiety (in vivo) to the peptide linker). An additional option
is to genetically incorporate non-natural amino acids using evolved
tRNAs and tRNA synthetases (see, e.g., U.S. Patent Appl. Publ. No.
2003/0082575) into the monomer domains or linkers. For example,
insertion of keto-tyrosine allows for site-specific coupling to
expressed monomer domains or multimers.
[0412] In some embodiments, the amino acid sequences of all peptide
linkers present in the polypeptide multimer are identical.
Alternatively, the amino acid sequences of all peptide linkers
present in the polypeptide multimer may be different.
Labeling or Conjugation of Scaffolds
[0413] The scaffolds of the invention can be used in non-conjugated
form or conjugated to at least one of a variety of heterologous
moieties to facilitate target detection or for imaging or therapy.
The scaffolds of the can be labeled or conjugated either before or
after purification, when purification is performed.
[0414] Many heterologous moieties lack suitable functional groups
to which scaffolds of the invention can be linked. Thus, in some
embodiments, the effector molecule is attached to the scaffold
through a linker, wherein the linker contains reactive groups for
conjugation. In some embodiments, the heterologous moiety
conjugated to a scaffold of the invention can function as a linker.
In other embodiments, the moiety is conjugated to the scaffold via
a linker that can be cleavable or non-cleavable. In one embodiment,
the cleavable linking molecule is a redox cleavable linking
molecule, such that the linking molecule is cleavable in
environments with a lower redox potential, such as the cytoplasm
and other regions with higher concentrations of molecules with free
sulfhydryl groups. Examples of linking molecules that may be
cleaved due to a change in redox potential include those containing
disulfides.
[0415] In some embodiments, scaffolds of the invention are
engineered to provide reactive groups for conjugation. In such
scaffolds, the N-terminus and/or C-terminus can also serve to
provide reactive groups for conjugation. In other embodiments, the
N-terminus can be conjugated to one moiety (such as, but not
limited to PEG) while the C-terminus is conjugated to another
moiety (such as, but not limited to biotin), or vice versa.
[0416] The term "polyethylene glycol" or "PEG" means a polyethylene
glycol compound or a derivative thereof, with or without coupling
agents, coupling or activating moieties (e.g., with thiol,
triflate, tresylate, aziridine, oxirane, N-hydroxysuccinimide or a
maleimide moiety). The term "PEG" is intended to indicate
polyethylene glycol of a molecular weight between 500 and 150,000
Da, including analogues thereof, wherein for instance the terminal
OH-group has been replaced by a methoxy group (referred to as
mPEG).
[0417] The scaffolds of the invention can be derivatized with
polyethylene glycol (PEG). PEG is a linear, water-soluble polymer
of ethylene oxide repeating units with two terminal hydroxyl
groups. PEGs are classified by their molecular weights which
typically range from about 500 daltons to about 40,000 daltons. In
a specific embodiment, the PEGs employed have molecular weights
ranging from 5,000 daltons to about 20,000 daltons. PEGs coupled to
the scaffolds of the invention can be either branched or
unbranched. (See, for example, Monfardini, C. et al. 1995
Bioconjugate Chem 6:62-69). PEGs are commercially available from
Nektar Inc., Sigma Chemical Co. and other companies. Such PEGs
include, but are not limited to, monomethoxypolyethylene glycol
(MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S),
monomethoxypolyethylene glycol-succinimidyl succinate
(MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2),
monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and
monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).
[0418] Briefly, the hydrophilic polymer which is employed, for
example, PEG, is capped at one end by an unreactive group such as a
methoxy or ethoxy group. Thereafter, the polymer is activated at
the other end by reaction with a suitable activating agent, such as
cyanuric halides (for example, cyanuric chloride, bromide or
fluoride), carbonyldiimidazole, an anhydride reagent (for example,
a dihalo succinic anhydride, such as dibromosuccinic anhydride),
acyl azide, p-diazoniumbenzyl ether,
3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The
activated polymer is then reacted with a polypeptide as described
herein to produce a polypeptide derivatized with a polymer.
Alternatively, a functional group in the scaffolds of the invention
can be activated for reaction with the polymer, or the two groups
can be joined in a concerted coupling reaction using known coupling
methods. It will be readily appreciated that the polypeptides of
the invention can be derivatized with PEG using a myriad of other
reaction schemes known to and used by those of skill in the
art.
[0419] In other embodiments, scaffolds of the invention, analogs or
derivatives thereof may be conjugated to a diagnostic or detectable
agent. Such scaffolds can be useful for monitoring or prognosing
the development or progression of a disease as part of a clinical
testing procedure, such as determining the efficacy of a particular
therapy. Such diagnosis and detection can be accomplished by
coupling the scaffold to detectable substances including, but not
limited to various enzymes, such as but not limited to horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; prosthetic groups, such as but not limited to
streptavidin/biotin and avidin/biotin; fluorescent materials, such
as but not limited to, umbelliferone, fluorescein, fluorescein
isothiocynate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin; luminescent materials, such as,
but not limited to, luminol; bioluminescent materials, such as but
not limited to, luciferase, luciferin, and aequorin; radioactive
materials, such as but not limited to iodine (.sup.131I, .sup.125I,
.sup.123I, .sup.121I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In,
.sup.111In,), and technetium (.sup.99Tc), thallium (.sup.201Ti),
gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), samarium
(.sup.153Sm), lutetium (.sup.177Lu), gadolinium (.sup.159Gd,
.sup.153Gd), promethium (.sup.149Pm), lanthanum (.sup.140La),
ytterbium (.sup.175Yb, .sup.169Yb), holmium (.sup.166Ho), yytrium
(.sup.90Y), scandium (.sup.47Sc), rhenium (.sup.186Re, .sup.188Re),
praseodymium (.sup.142Pr), rhodium (.sup.105Rh) ruthenium
(.sup.97Ru), germanium (.sup.68Ge), cobalt (.sup.57Co), zinc
(.sup.65Zn), strontium (.sup.85Sr), phosphorus (.sup.32P), chromium
(.sup.51Cr), manganese (.sup.54Mn), selenium (.sup.75Se), tin
(.sup.113Sn), and indium (.sup.117In); positron emitting metals
using various positron emission topographies, nonradioactive
paramagnetic metal ions, and molecules that are radiolabeled or
conjugated to specific radioisotopes.
[0420] The present invention further encompasses uses of scaffolds
conjugated to a therapeutic moiety. A scaffold may be conjugated to
a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Therapeutic moieties include,
but are not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa
chlorambucil, melphalan, carmustihe (BCNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and
doxorubicin), antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),
Auristatin molecules (e.g., auristatin PHE, bryostatin 1, and
solastatin 10; see Woyke et al., Antimicrob. Agents Chemother.
46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother.
45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40
(2001), Wall et al., Biochem. Biophys. Res. Commun. 266:76-80
(1999), Mohammad et al., Int. J. Oncol. 15:367-72 (1999), all of
which are incorporated herein by reference), hormones (e.g.,
glucocorticoids, progestins, androgens, and estrogens), DNA-repair
enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors
(e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin
Cancer Res. 8(7):2167-76 (2002)), cytotoxic agents (e.g.,
paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracindione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, procaine, tetracaine, lidocaine,
propranolol, and puromycin and analogs or homologs thereof) and
those compounds disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633,
6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372,
6,057,300 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844,
5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239,
5,587,459), farnesyl transferase inhibitors (e.g., R1 15777,
BMS-214662 and those disclosed by, for example, U.S. Pat. Nos.
6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387,
6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905,
6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501,
6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865,
6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096,
6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295,
6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935,
6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and
6,040,305), topoisomerase inhibitors (e.g., camptothecin;
irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI
147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000;
saintopin; UCE6; UCE1022; TAN-1518A; TAN-1518B; KT6006; KT6528;
ED-110; NB-506; ED-110; NB-506; and rebeccamycin); bulgarein; DNA
minor groove binders such as Hoescht dye 33342 and Hoechst dye
33258; nitidine; fagaronine; epiberberine; coralyne;
beta-lapachone; BC-4-1; and pharmaceutically acceptable salts,
solvates, clathrates, and prodrugs thereof. See, e.g., Rothenberg,
M. L., Annals of Oncology 8:837-855 (1997); and Moreau, P., et al.,
J. Med. Chem. 41:1631-1640 (1998); bisphosphonates (e.g.,
alendronate, cimadronte, clodronate, tiludronate, etidronate,
ibandronate, neridronate, olpandronate, risedronate, piridronate,
pamidronate, zolendronate) HMG-CoA reductase inhibitors, statins
(e.g., lovastatin, simvastatin, atorvastatin (Lipitor.TM.),
pravastatin, fluvastatin (Lescol.TM., cerivastatin, and
rosuvastatin)), antisense oligonucleotides (e.g., those disclosed
in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033,
and 5,618,709), immunomodulators (e.g., antibodies and cytokines),
and adenosine deaminase inhibitors (e.g., fludarabine phosphate and
2-chlorodeoxyadenosine).
[0421] Further, a scaffold may be conjugated to a therapeutic
moiety or drug moiety that modifies a given biological response.
Therapeutic moieties or drug moieties are not to be construed as
limited to classical chemical therapeutic agents. For example, the
drug moiety may be a protein or polypeptide possessing a desired
biological activity. Such proteins may include, for example, an
enzyme, an antibody, a toxin (e.g., abrin, ricin A, Pseudomonas
exotoxin, cholera toxin, or diphtheria toxin; a protein such as a
tumor necrosis factor (e.g., TNF-alpha, TNF-beta), an interferon
(e.g., .alpha.-interferon, .beta.-interferon), a nerve growth
factor, a platelet derived growth factor, a tissue plasminogen
activator, an apoptotic agent (e.g., TNF-alpha, TNF-beta, AIM I
(see, International publication No. WO 97/33899), AIM II (see,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., 1994, J. Immunol., 6:1567-1574), and VEGI (see,
International publication No. WO 99/23105)), a thrombotic agent or
an anti-angiogenic agent (e.g., angiostatin, endostatin or a
component of the coagulation pathway (e.g., tissue factor)); or, a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), and granulocyte colony stimulating factor
("G-CSF")), a growth factor (e.g., growth hormone ("GH")), or a
coagulation agent (e.g., calcium, vitamin K, tissue factors, such
as but not limited to, Hageman factor (factor XII),
high-molecular-weight kininogen (HMWK), prekallikrein (PK),
coagulation proteins-factors II (prothrombin), factor V, XIIa,
VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, fibrinopeptides A
and B from the .alpha. and .beta. chains of fibrinogen, fibrin
monomer).
[0422] Moreover, a scaffold can be conjugated to therapeutic
moieties such as a radioactive metal ion, such as alpha-emitters
such as .sup.213Bi or macrocyclic chelators useful for conjugating
radiometal ions, including but not limited to, .sup.131In,
.sup.131Lu, .sup.131Y, .sup.131Ho, .sup.131Sm, to polypeptides. In
certain embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N''''-tetraacetic acid
(DOTA) which can be attached to the scaffold via a linker molecule.
Such linker molecules are commonly known in the art and described
in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson
et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al.,
1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference
in their entireties.
[0423] Techniques for conjugating therapeutic moieties to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies 84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol.
Rev. 62: 119-58. Similar approaches may be adapted for use with
scaffolds of the invention.
[0424] The therapeutic moiety or drug conjugated to a scaffold of
the invention should be chosen to achieve the desired prophylactic
or therapeutic effect(s) for a particular disorder in a subject. A
clinician or other medical personnel should consider the following
when deciding on which therapeutic moiety or drug to conjugate to a
scaffold: the nature of the disease, the severity of the disease,
and the condition of the subject.
Assaying Scaffolds
[0425] The scaffolds of the invention may be assayed for specific
binding to a target by any method known in the art. Representative
assays which can be used, include but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitation reactions, gel diffusion precipitin
reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, to name but a few. Such assays are routine and known
in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York).
[0426] The binding affinity and other binding properties of a
scaffold to an antigen may be determined by a variety of in vitro
assay methods known in the art including for example, equilibrium
methods (e.g., enzyme-linked immunoabsorbent assay (ELISA; or
radioimmunoassay (RIA)), or kinetics (e.g., BIACORE.RTM. analysis),
and other methods such as indirect binding assays, competitive
binding assays, gel electrophoresis and chromatography (e.g., gel
filtration). These and other methods may utilize a label on one or
more of the components being examined and/or employ a variety of
detection methods including but not limited to chromogenic,
fluorescent, luminescent, or isotopic labels. A detailed
description of binding affinities and kinetics can be found in
Paul, W. E., ed., Fundamental Immunology, 4th Ed.,
Lippincott-Raven, Philadelphia (1999).
[0427] In some embodiments, scaffolds of the invention specifically
bind a target with specific kinetics. In some embodiments,
scaffolds of the invention may have a dissociation constant or
K.sub.d (k.sub.off/k.sub.on) of less than 1.times.10.sup.-2 M,
1.times.10.sup.-M, 1.times.10.sup.-4M, 1.times.10.sup.-5M,
1.times.10.sup.-6M, 1.times.10.sup.-7M, 1.times.10.sup.-8M,
1.times.10.sup.-9M, 1.times.10.sup.-10M, 1.times.10.sup.-11M,
1.times.10.sup.-12M, 1.times.10.sup.-13M, 1.times.10.sup.-14M or
less than 1.times.10.sup.-15M. In specific embodiments, scaffolds
of the invention have a K.sub.d of 500 .mu.M, 100 .mu.M, 100 .mu.M,
500 nM, 100 nM, 1 nM, 500 pM, 100 pM or less as determined by a
BIAcore Assay.RTM. or by other assays known in the art. In an
alternative embodiment, the affinity of the scaffolds of the
invention is described in terms of the association constant
(K.sub.a), which is calculated as the ratio k.sub.on/k.sub.off, of
at least 1.times.10.sup.2M.sup.-1, 1.times.10.sup.3M.sup.-1,
1.times.10.sup.4M.sup.-1, 1.times.10.sup.5M.sup.-1,
1.times.10.sup.6M.sup.-1, 1.times.10.sup.7M.sup.-1,
1.times.10.sup.8M.sup.-1, 1.times.10.sup.9M.sup.-1,
1.times.10.sup.10M.sup.-1 1.times.10.sup.11M.sup.-1
1.times.10.sup.12M.sup.-1, 1.times.10.sup.13M.sup.-1,
1.times.10.sup.14M.sup.-1, 1.times.10.sup.15M.sup.-1, or at least
5.times.10.sup.15 M.sup.-1.
[0428] In certain embodiments the rate at which the scaffolds of
the invention dissociate from a target epitope may be more relevant
than the value of the K.sub.d or the K.sub.a. In some embodiments,
the scaffolds of the invention have a k.sub.off of less than
10.sup.-3 s.sup.-1, less than 5.times.10.sup.-3 s.sup.-1, less than
10.sup.-4 s.sup.-1, less than 5.times.10.sup.-4 s.sup.-1, less than
10.sup.-5 s.sup.-1, less than 5.times.10.sup.-5 s.sup.-1, less than
10.sup.-6 s.sup.-1, less than 5.times.10.sup.-6 s.sup.-1, less than
10.sup.-7 s.sup.-1, less than 5.times.10.sup.-7 s.sup.-1, less than
10.sup.-8 s.sup.-1, less than 5.times.10.sup.-8 s.sup.-1, less than
10.sup.-9 s.sup.-1, less than 5.times.10.sup.-9 s.sup.-1, or less
than 10.sup.-10 s.sup.-1.
[0429] In certain other embodiments, the rate at which the
scaffolds of the invention associate with a target epitope may be
more relevant than the value of the K.sub.d or the K.sub.a. In this
instance, the scaffolds of the invention bind to a target with a
k.sub.on rate of at least 10.sup.5 M.sup.-1s.sup.-1, at least
5.times.10.sup.5M.sup.-1s.sup.-1, at least 10.sup.6 M.sup.-1
s.sup.-1, at least 5.times.10.sup.6 M.sup.-1s.sup.-1, at least
10.sup.7 M.sup.-1s.sup.-1, at least
5.times.10.sup.7M.sup.-1s.sup.-1, or at least
10.sup.8M.sup.-1s.sup.-1, or at least 10.sup.9
M.sup.-1s.sup.-1.
[0430] Scaffolds of the invention may also be attached to solid
supports, which are particularly useful for immunoassays or
purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
Assays for Detecting Soluble, Secreted Polypeptides
[0431] In a specific embodiment, the invention provides an improved
ELISA method for detecting soluble recombinant polypeptides
secreted in culture media. In some embodiments, the recombinant
polypeptide is a recombinant Fn type III variant. In one
embodiment, the method for detecting a soluble recombinant
fibronectin type III variant comprises: [0432] (a) reacting culture
media containing an expressed polypeptide with an immobilized
antibody which binds to the polypeptide, [0433] (b) reacting the
polypeptide with a target conjugated to an enzyme under conditions
suitable for binding, [0434] (c) reacting the bound conjugate
target to a substrate wherein a signal is generated, and [0435] (d)
measuring the signal intensity.
[0436] In another embodiment, the method comprises: [0437] (a)
reacting culture media containing an expressed variant with an
antibody which binds the variant, wherein the antibody is
immobilized to a solid support; [0438] (b) washing the immobilized
support with buffer solution; [0439] (c) reacting the variant with
a target conjugated to a first member of a binding pair; [0440] (d)
washing the immobilized support with buffer solution; [0441] (e)
reacting said first member with a second member of a binding pair,
wherein said second member is conjugated to an enzyme; [0442] (f)
reacting said enzyme with a substrate, wherein a signal is
generated; and [0443] (g) measuring the intensity of said signal,
wherein signal intensity correlates with binding affinity. In one
embodiment, the method comprises detecting a secreted polypeptide
or secreted variant in crude culture media.
[0444] In a specific embodiment, the method comprises detecting a
secreted polypeptide or secreted variant in crude culture media. In
a specific embodiment, the signal intensity varies by less than
40%, less than 30%, less than 20%, or less than 19%.
[0445] In one embodiment, the ELISA method is performed in a high
throughput or ultrahigh throughput format using assay plates of at
least 96 wells. In a specific embodiment, a 384 well assay plate or
a 1536 well assay plate is used.
[0446] In another embodiment, the method detects a variant
comprising a heterologous amino acid sequence, including but not
limited to: a poly(his) tag, a hemagglutinin (HA) tag, a FLAG tag,
a Strep-tag, a myc tag, or a V5 tag.
[0447] In yet another embodiment, the first member of the binding
pair is biotin and the second member of the binding pair is
streptavidin or avidin.
Pharmaceutical Compositions
[0448] In another aspect, the present invention provides a
composition, for example, but not limited to, a pharmaceutical
composition, containing one or a combination of scaffolds or
multimeric scaffolds of the present invention, formulated together
with a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of, for example, but not limited to
two or more different scaffolds of the invention. For example, a
pharmaceutical composition of the invention may comprise a
combination of scaffolds that bind to different epitopes on the
target antigen or that have complementary activities. In a specific
embodiment, a pharmaceutical composition comprises a multimeric
scaffold of the invention.
[0449] Pharmaceutical compositions of the invention also can be
administered in combination therapy, such as, combined with other
agents. For example, the combination therapy can include a scaffold
of the present invention combined with at least one other therapy
wherein the therapy may be immunotherapy, chemotherapy, radiation
treatment, or drug therapy.
[0450] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. Examples of such
salts include acid addition salts and base addition salts. Acid
addition salts include those derived from nontoxic inorganic acids,
such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0451] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable antioxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0452] Examples of suitable aqueous and non-aqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0453] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0454] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
suitable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0455] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0456] In one embodiment the compositions (e.g., liquid
formulations) of the invention are pyrogen-free formulations which
are substantially free of endotoxins and/or related pyrogenic
substances. Endotoxins include toxins that are confined inside a
microorganism and are released when the microorganisms are broken
down or die. Pyrogenic substances also include fever-inducing,
thermostable substances (glycoproteins) from the outer membrane of
bacteria and other microorganisms. Both of these substances can
cause fever, hypotension and shock if administered to humans. Due
to the potential harmful effects, it is advantageous to remove even
low amounts of endotoxins from intravenously administered
pharmaceutical drug solutions. The Food & Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight it is advantageous
to remove even trace amounts of endotoxin. In one embodiment,
endotoxin and pyrogen levels in the composition are less than 10
EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1
EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg. In
another embodiment, endotoxin and pyrogen levels in the composition
are less than about 10 EU/mg, or less than about 5 EU/mg, or less
than about 1 EU/mg, or less than about 0.1 EU/mg, or less than
about 0.01 EU/mg, or less than about 0.001 EU/mg.
Pharmaceutical Dosing and Administration
[0457] To prepare pharmaceutical or sterile compositions including
a scaffold of the invention, a scaffold is mixed with a
pharmaceutically acceptable carrier or excipient. Formulations of
therapeutic and diagnostic agents can be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous
solutions, lotions, or suspensions (see, e.g., Hardman, et al.
(2001) Goodman and Gilman's The Pharmacological Basis of
Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000)
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.;
Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel
Dekker, Inc., New York, N.Y.).
[0458] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. In certain embodiments, an administration
regimen maximizes the amount of therapeutic delivered to the
patient consistent with an acceptable level of side effects.
Accordingly, the amount of biologic delivered depends in part on
the particular entity and the severity of the condition being
treated. Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)
(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New
Engl J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh,
et al. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000)
New Engl. J. Med. 343:1594-1602).
[0459] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0460] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0461] Scaffolds of the invention can be provided by continuous
infusion, or by doses at intervals of, e.g., one day, one week, or
1-7 times per week. Doses may be provided intravenously,
subcutaneously, topically, orally, nasally, rectally,
intramuscular, intracerebrally, or by inhalation. A specific dose
protocol is one involving the maximal dose or dose frequency that
avoids significant undesirable side effects. A total weekly dose
may be at least 0.05 .mu.g/kg body weight, at least 0.2 .mu.g/kg,
at least 0.5 .mu.g/kg, at least 1 .mu.g/kg, at least 10 .mu.g/kg,
at least 100 .mu.g/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at
least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or at least
50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med.
349:427-434; Herold, et al. (2002) New Engl J. Med. 346:1692-1698;
Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456;
Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).
The desired dose of a small molecule therapeutic, e.g., a peptide
mimetic, protein scaffold, natural product, or organic chemical, is
about the same as for an antibody or polypeptide, on a moles/kg
body weight basis.
[0462] The desired plasma concentration of a small molecule or
scaffold therapeutic is about the same as for an antibody, on a
moles/kg body weight basis. The dose may be at least 15 .mu.g, at
least 20 .mu.g, at least 25 .mu.g, at least 30 .mu.g, at least 35
.mu.g, at least 40 .mu.g, at least 45 .mu.g, at least 50 .mu.g, at
least 55 .mu.g, at least 60 .mu.g, at least 65 .mu.g, at least 70
.mu.g, at least 75 .mu.g, at least 80 .mu.g, at least 85 .mu.g, at
least 90 .mu.g, at least 95 .mu.g, or at least 100 .mu.g. The doses
administered to a subject may number at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, or 12, or more.
[0463] For scaffolds of the invention, the dosage administered to a
patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body
weight. The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001
mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg,
0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and
0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001
to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to
0.10 mg/kg of the patient's body weight.
[0464] The dosage of the scaffolds of the invention may be
calculated using the patient's weight in kilograms (kg) multiplied
by the dose to be administered in mg/kg. The dosage of the
scaffolds of the invention may be 150 .mu.g/kg or less, 125
.mu.g/kg or less, 100 .mu.g/kg or less, 95 .mu.g/kg or less, 90
.mu.g/kg or less, 85 .mu.g/kg or less, 80 .mu.g/kg or less, 75
.mu.g/kg or less, 70 .mu.g/kg or less, 65 .mu.g/kg or less, 60
.mu.g/kg or less, 55 .mu.g/kg or less, 50 .mu.g/kg or less, 45
.mu.g/kg or less, 40 .mu.g/kg or less, 35 .mu.g/kg or less, 30
.mu.g/kg or less, 25 .mu.g/kg or less, 20 .mu.g/kg or less, 15
.mu.g/kg or less, 10 .mu.g/kg or less, 5 .mu.g/kg or less, 2.5
.mu.g/kg or less, 2 .mu.g/kg or less, 1.5 .mu.g/kg or less, 1
.mu.g/kg or less, 0.5 .mu.g/kg or less, or 0.5 .mu.g/kg or less of
a patient's body weight.
[0465] Unit dose of the scaffolds of the invention may be 0.1 mg to
20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to
8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20
mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25
mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg
to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg,
1 mg to 5 mg, or 1 mg to 2.5 mg.
[0466] The dosage of the scaffolds of the invention may achieve a
serum titer of at least 0.1 .mu.g/ml, at least 0.5 .mu.g/ml, at
least 1 .mu.g/ml, at least 2 .mu.g/ml, at least 5 .mu.g/ml, at
least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15 .mu.g/ml, at
least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50 .mu.g/ml, at
least 100 .mu.g/ml, at least 125 .mu.g/ml, at least 150 .mu.g/ml,
at least 175 .mu.g/ml, at least 200 .mu.g/ml, at least 225
.mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml, at least
300 .mu.g/ml, at least 325 .mu.g/ml, at least 350 .mu.g/ml, at
least 375 .mu.g/ml, or at least 400 .mu.g/ml in a subject.
Alternatively, the dosage of the scaffolds of the invention may
achieve a serum titer of at least 0.1 .mu.g/ml, at least 0.5
.mu.g/ml, at least 1 .mu.g/ml, at least, 2 .mu.g/ml, at least 5
.mu.g/ml, at least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50
.mu.g/ml, at least 100 .mu.g/ml, at least 125 .mu.g/ml, at least
150 .mu.g/ml, at least 175 .mu.g/ml, at least 200 .mu.g/ml, at
least 225 .mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml,
at least 300 .mu.g/ml, at least 325 .mu.g/ml, at least 350
.mu.g/ml, at least 375 .mu.g/ml, or at least 400 .mu.g/ml in the
subject.
[0467] Doses of scaffolds of the invention may be repeated and the
administrations may be separated by at least 1 day, 2 days, 3 days,
5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months.
[0468] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side effects (see, e.g.,
Maynard, et al. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0469] A composition of the present invention may also be
administered via one or more routes of administration using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
Selected routes of administration for scaffolds of the invention
include without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracerebral, intraocular, intraocular, intraarterial,
intracerebrospinal, intralesional intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion, or by sustained release
systems or an implant (see, e.g., Sidman et al. (1983) Biopolymers
22:547-556; Langer, et al. (1981) J. Biomed. Mater. Res.
15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein, et al.
(1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al.
(1980) Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos.
6,350,466 and 6,316,024). Alternatively, a composition of the
invention can be administered via a non-parenteral route, such as a
topical, epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lidocaine to ease
pain at the site of the injection. In addition, pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent. See, e.g.,
U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,
5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication
Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO
99/66903, each of which is incorporated herein by reference their
entirety. In one embodiment, an antibody, combination therapy, or a
composition of the invention is administered using Alkermes AIR.TM.
pulmonary drug delivery technology (Alkermes, Inc., Cambridge,
Mass.).
[0470] If the scaffolds of the invention are administered in a
controlled release or sustained release system, a pump may be used
to achieve controlled or sustained release (see Langer, Chem. Tech.
12:98-105, 1982; Seflon, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;
Buchwald et al, 1980, Surgery 88:507; Saudek et al., 1989, N. Engl.
J. Med. 321:51 A). Polymeric materials can be used to achieve
controlled or sustained release of the therapies of the invention
(see e.g., Medical Applications of Controlled Release, Langer and
Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol Sd. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253.
[0471] Examples of polymers used in sustained release formulations
include, but are not limited to, poly(2-hydroxy ethyl
methacrylate), poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol),
polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and
polyorthoesters. In one embodiment, the polymer used in a sustained
release formulation is inert, free of leachable impurities, stable
on storage, sterile, and biodegradable. A controlled or sustained
release system can be placed in proximity of the prophylactic or
therapeutic target, thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)).
[0472] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more scaffolds of the invention.
See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548,
PCT publication WO 96/20698, Ning et al., 1996, "Intratumoral
Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a
Sustained-Release Gel," Radiotherapy & Oncology 39: 179-189,
Song et al, 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. ReI. Bioact. Mater.
24:853-854, and Lam et al, 1997, "Microencapsulation of Recombinant
Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l.
Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is
incorporated herein by reference in their entirety.
[0473] The scaffolds of the invention can be formulated for topical
administration in the form of an ointment, cream, transdermal
patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or
other form well-known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences and Introduction to
Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.
(1995). For non-sprayable topical dosage forms, viscous to
semi-solid or solid forms comprising a carrier or one or more
excipients compatible with topical application and having a dynamic
viscosity, in some instances, greater than water are typically
employed. Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, in some instances, in combination with a
solid or liquid inert carrier, is packaged in a mixture with a
pressurized volatile (e.g., a gaseous propellant, such as freon) or
in a squeeze bottle. Moisturizers or humectants can also be added
to pharmaceutical compositions and dosage forms if desired.
Examples of such additional ingredients are well-known in the
art.
[0474] If the scaffolds of the invention are administered
intranasally, it can be formulated in an aerosol form, spray, mist
or in the form of drops. In particular, prophylactic or therapeutic
agents for use according to the present invention can be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0475] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art (see,
e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill,
New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice: A Practical Approach,
Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., Pa.).
[0476] An effective amount of therapeutic may decrease the symptoms
by at least 10%; by at least 20%; at least about 30%; at least 40%,
or at least 50%.
[0477] Additional therapies (e.g., prophylactic or therapeutic
agents), which can be administered in combination with the
scaffolds of the invention may be administered to a subject
concurrently. The term "concurrently" is not limited to the
administration of therapies (e.g., prophylactic or therapeutic
agents) at exactly the same time, but rather it is meant that a
pharmaceutical composition comprising scaffolds of the invention
are administered to a subject in a sequence and within a time
interval such that the scaffolds of the invention can act together
with the other therapy or therapies to provide an increased benefit
than if they were administered otherwise. For example, each therapy
may be administered to a subject at the same time or sequentially
in any order at different points in time; however, if not
administered at the same time, they should be administered
sufficiently close in time so as to provide the desired therapeutic
or prophylactic effect. Each therapy can be administered to a
subject separately, in any appropriate form and by any suitable
route. In various embodiments, the therapies (e.g., prophylactic or
therapeutic agents) are administered to a subject less than 15
minutes, less than 30 minutes, less than 1 hour apart, at about 1
hour apart, at about 1 hour to about 2 hours apart, at about 2
hours to about 3 hours apart, at about 3 hours to about 4 hours
apart, at about 4 hours to about 5 hours apart, at about 5 hours to
about 6 hours apart, at about 6 hours to about 7 hours apart, at
about 7 hours to about 8 hours apart, at about 8 hours to about 9
hours apart, at about 9 hours to about 10 hours apart, at about 10
hours to about 11 hours apart, at about 11 hours to about 12 hours
apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week
apart. The two or more therapies may be administered within one
same patient visit.
[0478] The scaffolds of the invention and the other therapies may
be cyclically administered. Cycling therapy involves the
administration of a first therapy (e.g., a first prophylactic or
therapeutic agent) for a period of time, followed by the
administration of a second therapy (e.g., a second prophylactic or
therapeutic agent) for a period of time, optionally, followed by
the administration of a third therapy (e.g., prophylactic or
therapeutic agent) for a period of time and so forth, and repeating
this sequential administration, i.e., the cycle in order to reduce
the development of resistance to one of the therapies, to avoid or
reduce the side effects of one of the therapies, and/or to improve
the efficacy of the therapies.
[0479] In certain embodiments, the scaffolds of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannosides
(Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134); pI20 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J J. Killion; I J. Fidler (1994; Immunomethods 4:273.
[0480] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
Methods of Using Scaffolds
[0481] The scaffolds of the present invention have in vitro and in
vivo diagnostic and therapeutic utilities. For example, these
molecules can be administered to cells in culture, e.g. in vitro or
ex vivo, or in a subject, e.g., in vivo, to treat, prevent or
diagnose a variety of disorders.
[0482] The invention also provides methods of using the scaffolds
of the invention. The present invention also encompasses the use of
the scaffolds of the invention for the prevention, diagnosis,
management, treatment or amelioration of one or more symptoms
associated with diseases, disorders of diseases or disorders,
including but not limited to cancer, inflammatory and autoimmune
diseases, infectious diseases either alone or in combination with
other therapies. The invention also encompasses the use of the
scaffolds of the invention conjugated or fused to a moiety (e.g.,
therapeutic agent or drug) for prevention, management, treatment or
amelioration of one or more symptoms associated with diseases,
disorders or infections, including but not limited to cancer,
inflammatory and autoimmune diseases, infectious diseases either
alone or in combination with other therapies.
[0483] Also, many cell surface receptors activate or deactivate as
a consequence of cross-linking of sub units. The proteins of the
invention may be used to stimulate or inhibit a response in a
target cell by cross-linking of cell surface receptors. In another
embodiment, the scaffolds of the invention of the invention may be
used to block the interaction of multiple cell surface receptors
with antigens. In another embodiment, the scaffolds of the
invention may be used to strengthen the interaction of multiple
cell surface receptors with antigens. In another embodiment, it may
be possible to crosslink homo- or heterodimers of a cell surface
receptor using the scaffolds of the invention containing binding
domains that share specificity for the same antigen, or bind two
different antigens. In another embodiment, the proteins of the
invention could be used to deliver a ligand, or ligand analogue to
a specific cell surface receptor.
[0484] The invention also provides methods of targeting epitopes
not easily accomplished with traditional antibodies. For example,
in one embodiment, the scaffolds and of the invention may be used
to first target an adjacent antigen and while binding, another
binding domain may engage the cryptic antigen.
[0485] The invention also provides methods of using the scaffolds
to bring together distinct cell types. In one embodiment, the
proteins of the invention may bind a target cell with one binding
domain and recruit another cell via another binding domain. In
another embodiment, the first cell may be a cancer cell and the
second cell is an immune effector cell such as an NK cell. In
another embodiment, the scaffolds of the invention may be used to
strengthen the interaction between two distinct cells, such as an
antigen presenting cell and a T cell to possibly boost the immune
response.
[0486] The invention also provides methods of using the scaffolds
to ameliorate, treat, or prevent cancer or symptoms thereof. In one
embodiment, methods of the invention are useful in the treatment of
cancers of the head, neck, eye, mouth, throat, esophagus, chest,
skin, bone, lung, colon, rectum, colorectal, stomach, spleen,
kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma,
endometrial, prostate, breast, ovaries, testicles, thyroid, blood,
lymph nodes, kidney, liver, pancreas, brain, or central nervous
system.
[0487] The invention also provides methods of using the scaffolds
to deplete a cell population. In one embodiment, methods of the
invention are useful in the depletion of the following cell types:
eosinophil, basophil, neutrophil, T cell, B cell, mast cell,
monocytes and tumor cell.
[0488] The invention also provides methods of using scaffolds to
inactivate, inhibit, or deplete cytokines. In one embodiment,
methods of the invention are useful in the inactivation,
inhibition, or depletion of at least one of the following
cytokines: TNF-.alpha., TGF-.beta., C5a, fMLP, Interferon alpha
(including subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17
and 21), Interferon beta, Interferon omega, Interferon gamma,
interleukins IL-1-33, CCL1-28, CXCL 1-17, and CX3CL1.
[0489] The invention also provides methods of using the scaffolds
to inactivate various infections agents such as viruses, fungi,
eukaryotic microbes, and bacteria. In some embodiments the
scaffolds of the invention may be used to inactivate RSV, hMPV,
PIV, or influenza viruses. In other embodiments, the scaffolds of
the invention may be used to inactivate fungal pathogens, such as,
but not limited to members of Naegleria, Aspergillus, Blastomyces,
Histoplasma, Candida or Tinea genera. In other embodiments, the
scaffolds of the invention may be used to inactivate eukaryotic
microbes, such as, but not limited to members of Giardia,
Toxoplasma, Plasmodium, Trypanosoma, and Entamoeba genera. In other
embodiments, the scaffolds of the invention may be used to
inactivate bacterial pathogens, such as but not limited to members
of Staphylococcus, Streptococcus, Pseudomonas, Clostridium,
Borrelia, Vibrio and Neisseria genera.
[0490] The invention also provides methods of using scaffolds
proteins as diagnostic reagents. The proteins of the invention may
be useful in kits or reagents where different antigens need to be
efficiently captured concurrently.
[0491] The proteins of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, cancer. Examples of cancers that can
be prevented, managed, treated or ameliorated in accordance with
the methods of the invention include, but are not limited to,
cancer of the head, neck, eye, mouth, throat, esophagus, chest,
bone, lung, colon, rectum, stomach, prostate, breast, ovaries,
kidney, liver, pancreas, and brain. Additional cancers include, but
are not limited to, the following: leukemias such as but not
limited to, acute leukemia, acute lymphocytic leukemia, acute
myelocytic leukemias such as myeloblasts, promyelocytic,
myelomonocytic, monocytic, erythroleukemic leukemias and
myclodysplastic syndrome, chronic leukemias such as but not limited
to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic
leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as
but not limited to Hodgkin's disease, non-Hodgkin's disease;
multiple myelomas such as but not limited to smoldering multiple
myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell
leukemia, solitary plasmacytoma and extramedullary plasmacytoma;
Waldenstrom's macroglobulinemia; monoclonal gammopathy of
undetermined significance; benign monoclonal gammopathy; heavy
chain disease; bone cancer and connective tissue sarcomas such as
but not limited to bone sarcoma, myeloma bone disease, multiple
myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease
of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, and synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, and
primary brain lymphoma; breast cancer including but not limited to
adenocarcinoma, lobular (small cell) carcinoma, intraductal
carcinoma, medullary breast cancer, mucinous breast cancer, tubular
breast cancer, papillary breast cancer, Paget's disease (including
juvenile Paget's disease) and inflammatory breast cancer; adrenal
cancer such as but not limited to pheochromocytom and
adrenocortical carcinoma; thyroid cancer such as but not limited to
papillary or follicular thyroid cancer, medullary thyroid cancer
and anaplastic thyroid cancer; pancreatic cancer such as but not
limited to, insulinoma, gastrinoma, glucagonoma, vipoma,
somatostatin-secreting tumor, and carcinoid or islet cell tumor;
pituitary cancers such as but limited to Cushing's disease,
prolactin-secreting tumor, acromegaly, and diabetes insipius; eye
cancers such as but not limited to ocular melanoma such as iris
melanoma, choroidal melanoma, and ciliary body melanoma, and
retinoblastoma; vaginal cancers such as squamous cell carcinoma,
adenocarcinoma, and melanoma; vulvar cancer such as squamous cell
carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,
and Paget's disease; cervical cancers such as but not limited to,
squamous cell carcinoma, and adenocarcinoma; uterine cancers such
as but not limited to endometrial carcinoma and uterine sarcoma;
ovarian cancers such as but not limited to, ovarian epithelial
carcinoma, borderline tumor, germ cell tumor, and stromal tumor;
esophageal cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers
such as but not limited to, adenocarcinoma, fungating (polyploid),
ulcerating, superficial spreading, diffusely spreading, malignant
lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon
cancers; rectal cancers; liver cancers such as but not limited to
hepatocellular carcinoma and hepatoblastoma, gallbladder cancers
such as adenocarcinoma; cholangiocarcinomas such as but not limited
to papillary, nodular, and diffuse; lung cancers such as non-small
cell lung cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers such as but not limited to germinal tumor,
seminoma, anaplastic, classic (typical), spermatocyte, nonseminoma,
embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac
tumor), prostate cancers such as but not limited to,
adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal
cancers; oral cancers such as but not limited to squamous cell
carcinoma; basal cancers; salivary gland cancers such as but not
limited to adenocarcinoma, mucoepidermoid carcinoma, and
adenoidcystic carcinoma; pharynx cancers such as but not limited to
squamous cell cancer, and verrucous; skin cancers such as but not
limited to, basal cell carcinoma, squamous cell carcinoma and
melanoma, superficial spreading melanoma, nodular melanoma, lentigo
malignant melanoma, acral lentiginous melanoma; kidney cancers such
as but not limited to renal cell cancer, adenocarcinoma,
hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis
and/or ureter); Wilms' tumor; bladder cancers such as but not
limited to transitional cell carcinoma, squamous cell cancer,
adenocarcinoma, carcinosarcoma. In addition, cancers include
myxosarcoma, osteogenic sarcoma, endotheliosarcoma,
lymphangioendotheliosarcoma, mesothelioma, synovioma,
hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,
bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma and papillary adenocarcinomas (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997,
Informed Decisions: The Complete Book of Cancer Diagnosis,
Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,
inc., United States of America).
[0492] It is also contemplated that cancers caused by aberrations
in apoptosis can also be treated by the methods and compositions of
the invention. Such cancers may include, but not be limited to,
follicular lymphomas, carcinomas with p53 mutations, hormone
dependent tumors of the breast, prostate and ovary, and
precancerous lesions such as familial adenomatous polyposis, and
myelodysplastic syndromes.
[0493] The proteins of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, autoimmune and/or inflammatory
diseases. The compositions and methods of the invention described
herein are useful for the prevention or treatment of autoimmune
disorders and/or inflammatory disorders. Examples of autoimmune
and/or inflammatory disorders include, but are not limited to,
antiphospholipid syndrome, arthritis, atherosclerosis, anaphylactic
shock, autoimmune Addison's disease, alopecia greata, autoimmune
diseases of the adrenal gland, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune oophoritis, autoimmune orchitis,
autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune
dysfunction syndrome, chronic inflammatory demyelinating
polyneuropathy, chronic inflammation, Churg-Strauss syndrome,
cicatrical pemphigoid, cold agglutinin disease, corneal and other
tissue transplantation, CREST syndrome, Crohn's disease, cystic
fibrosis, diabetic retinopathies, discoid lupus, endocarditis,
endotoxic shock, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, hemangiomas, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenia purpura, IgA
neuropathy, juvenile arthritis, lichen planus, lupus erythematosus,
Meniere's disease, mixed connective tissue disease, multiple
sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia
gravis, neovascular glaucoma, organ ischemia, pemphigus vulgaris,
peritonitis, pernicious anemia, polyarteritis nodosa,
polychrondritis, polyglandular syndromes, polymyalgia rheumatica,
polymyositis and dermatomyositis, primary agammaglobulinemia,
primary biliary cirrhosis, psoriasis, psoriatic arthritis,
Raynauld's phenomenon, Reiter's syndrome, reperfusion injury,
retrolental fibroplasia, rheumatoid arthritis, sarcoidosis,
scleroderma, sepsis, septicemia, Sjogren's syndrome, spinal cord
injury, stiff-man syndrome, systemic lupus erythematosus, takayasu
arteritis, temporal arteritis/giant cell arteritis, thyroid
hyperplasias, ulcerative colitis, uveitis, vasculitides such as
dermatitis herpetiformis vasculitis, vitiligo, and Wegener's
granulomatosis.
[0494] Examples of inflammatory disorders include, but are not
limited to, asthma, encephalitis, inflammatory bowel disease,
chronic obstructive pulmonary disease (COPD), allergic disorders,
septic shock, pulmonary fibrosis, undifferentiated
spondyloarthropathy, undifferentiated arthropathy, arthritis,
inflammatory osteolysis, and chronic inflammation resulting from
chronic viral or bacteria infections. The compositions and methods
of the invention can be used with one or more conventional
therapies that are used to prevent, manage or treat the above
diseases.
[0495] The proteins of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, infectious disease, including viral,
bacterial and fungal diseases.
[0496] Examples of viral pathogens include but are not limited to:
adenovirdiae (e.g., mastadenovirus and aviadeno virus),
herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus
2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae
(e.g., levivirus, enterobacteria phase MS2, allolevirus),
poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus,
capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and
entomopoxyirinae), papovaviridae (e.g., polyomavirus and
papillomavirus), paramyxoviridae (e.g., paramyxovirus,
parainfluenza virus 1, mobillivirus (e.g., measles virus),
rubulavirus (e.g., mumps virus), pneumonovirinae (e.g.,
pneumovirus, human respiratory syncytial virus), and metapneumo
virus (e.g., avian pneumovirus and human metapneumo virus)),
picornaviridae (e.g., enterovirus, rhino virus, hepato virus (e.g.,
human hepatits A virus), cardiovirus, and apthovirus), reoviridae
(e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus,
phytoreo virus, and oryzavirus), retroviridae (e.g., mammalian type
B retroviruses, mammalian type C retroviruses, avian type C
retroviruses, type D retrovirus group, BLV-HTLV retroviruses,
lentivirus (e.g. human immunodeficiency virus 1 and human
immunodeficiency virus 2), spumavirus), flaviviridae (e.g.,
hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus),
togaviridae (e.g., alphavirus (e.g., sindbis virus) and rubivirus
(e.g., rubella virus)), rhabdoviridae (e.g., vesiculovirus,
lyssavirus, ephemerovirus, cytorhabdo virus, and necleorhabdo
virus), arenaviridae (e.g., arenavirus, lymphocytic
choriomeningitis virus, Ippy virus, and lassa virus), and
coronaviridae (e.g., coronavirus and torovirus).
[0497] Examples of bacterial pathogens include but are not limited
to: but not limited to, the Aquaspirillum family, Azospirillum
family, Azotobacteraceae family, Bacteroidaceae family, Bartonella
species, Bdellovibrio family, Campylobacter species, Chlamydia
species (e.g., Chlamydia pneumoniae), Clostridium,
Enterobacteriaceae family (e.g., Citrobacter species, Edwardsiella,
Enterobacter aerogenes, Erwinia species, Escherichia coli, Hafnia
species, Klebsiella species, Morganella species, Proteus vulgaris,
Providencia, Salmonella species, Serratia marcescens, and Shigella
flexneri), Gardinella family, Haemophilus influenzae,
Halobacteriaceae family, Helicobacter family, Legionallaceae
family, Listeria species, Methylococcaceae family, mycobacteria
(e.g., Mycobacterium tuberculosis), Neisseriaceae family,
Oceanospirillum family, Pasteurellaceae family, Pneumococcus
species, Pseudomonas species, Rhizobiaceae family, Spirillum
family, Spirosomaceae family, Staphylococcus (e.g., methicillin
resistant Staphylococcus aureus and Staphylococcus pyrogenes),
Streptococcus (e.g., Streptococcus enteritidis, Streptococcus
fasciae, and Streptococcus pneumoniae), Vampirovibrio, Helicobacter
family, and Vampirovibrio family.
[0498] Examples of fungal pathogens include, but are not limited
to: Absidia species (e.g., Absidia corymbifera and Absidia ramosa),
Aspergillus species, (e.g., Aspergillus flavus, Aspergillus
fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus
terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida
species (e.g., Candida albicans, Candida glabrata, Candida kerr,
Candida krusei, Candida parapsilosis, Candida pseudotropicalis,
Candida quillermondii, Candida rugosa, Candida stellatoidea, and
Candida tropicalis), Coccidioides immitis, Conidiobolus species,
Cryptococcus neoforms, Cunninghamella species, Dermatophytes,
Histoplasma capsulatum, Microsporum gypseum, Mucor pusillus,
Paracoceidioides brasiliensis, Pseudallescheria boydii,
Rhinosporidium seeberi, Pneumocystis carinii, Rhizopus species
(e.g., Rhizopus arrhizus, Rhizopus oryzae, and Rhizopus
microsporus), Saccharomyces species, Sporothrix schenckii, and
classes such as Zygomycetes, Ascomycetes, the Basidiomycetes,
Deuteromycetes, and Oomycetes.
[0499] In another embodiment, the invention provides methods for
preventing, managing, treating or ameliorating cancer, autoimmune,
inflammatory or infectious diseases or one or more symptoms
thereof, said methods comprising administering to a subject in need
thereof a dose of a prophylactically or therapeutically effective
amount of one or more scaffolds of the invention in combination
with surgery, alone or in further combination with the
administration of a standard or experimental chemotherapy, a
hormonal therapy, a biological therapy/immunotherapy and/or a
radiation therapy. In accordance with these embodiments, the
scaffolds of the invention utilized to prevent, manage, treat or
ameliorate cancer, autoimmune, inflammatory or infectious diseases
or one or more symptoms or one or more symptoms thereof may or may
not be conjugated or fused to a moiety (e.g., therapeutic agent or
drug).
[0500] The invention provides methods for preventing, managing,
treating or ameliorating cancer, autoimmune, inflammatory or
infectious diseases or one or more symptoms or one or more symptoms
thereof, said methods comprising administering to a subject in need
thereof one or more scaffolds of the invention in combination with
one or more of therapeutic agents that are not cancer therapeutics
(a.k.a., non-cancer therapies). Examples of such agents include,
but are not limited to, anti-emetic agents, anti-fungal agents,
anti-bacterial agents, such as antibiotics, anti-inflammatory
agents, and anti-viral agents. Non-limiting examples of anti-emetic
agents include metopimazin and metoclopramide. Non-limiting
examples of antifungal agents include azole drugs, imidazole,
triazoles, polyene, amphotericin and yrimidine. Non-limiting
examples of anti-bacterial agents include dactinomycin, bleomycin,
erythromycin, penicillin, mithramycin, cephalosporin, imipenem,
axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol,
clindamycin, tetracycline, streptomycin, tobramycin, gentamicin,
amikacin, kanamycin, neomycin, spectinomycin, trimethoprim,
norfloxacin, refampin, polymyxin, amphotericin B, nystatin,
ketocanazole, isoniazid, metronidazole and pentamidine.
Non-limiting examples of antiviral agents include nucleoside
analogs (e.g., zidovudine, acyclivir, gangcyclivir, vidarbine,
idoxuridine, trifluridine and ribavirin), foscaret, amantadine,
rimantadine, saquinavir, indinavir, ritonavir, interferon
("IFN")-.alpha.,.beta. or .gamma. and AZT. Non-limiting examples of
anti-inflammatory agents include non-steroidal anti-inflammatory
drugs ("NSAIDs"), steroidal anti-inflammatory drugs, beta-agonists,
anti-cholingenic agents and methylxanthines.
[0501] In another embodiment, the invention comprises compositions
capable of inhibiting a cancer cell phenotype. In one embodiment,
the cancer cell phenotype is cell growth, cell attachment, loss of
cell attachment, decreased receptor expression (such as, for
example, but not limited to Eph receptors), increased receptor
expression (such as, for example, but not limited to Eph
receptors), metastatic potential, cell cycle inhibition, receptor
tyrosine kinase activation/inhibition or others.
[0502] In one embodiment, the invention comprises compositions
capable of treating chronic inflammation. The compositions can be
used in the targeting of immune cells for destruction or
deactivation. The compositions are useful in targeting activated T
cells, dormant T cells, B cells, neutrophils, eosiniphils,
basophils, mast cells, or dendritic cells. The compositions may be
capable of decreasing or ablating immune cell function.
[0503] In another embodiment, the invention comprises compositions
capable of inhibiting or reducing angiogenesis. In another
embodiment, the angiogenesis is related to tumor growth, rheumatoid
arthritis, SLE, Sjogren's syndrome or others.
[0504] In another embodiment, the invention comprises compositions
useful for treatment of diseases of the gastrointestinal tract. The
scaffolds of the invention exhibit a high level of stability under
low pH conditions. The stability at low pH suggests that the
composition will be suitable for oral administration for a variety
of gastrointestinal disorders, such as irritable bowel syndrome,
gastroesophageal reflux, intestinal pseudo-obstructions, dumping
syndrome, intractable nausea, peptic ulcer, appendicitis, ischemic
colitis, ulcerative colitis, gastritis, Helicobacter pylori
disease, Crohn's disease, Whipple's disease, celiac sprue,
diverticulitis, diverticulosis, dysphagia, hiatus hernia,
infections esophageal disorders, hiccups, rumination and
others.
[0505] The invention further provides combinatorial compositions
and methods of using such compositions in the prevention,
treatment, reduction, or amelioration of disease or symptoms
thereof. The scaffolds of the invention may be combined with
conventional therapies suitable for the prevention, treatment,
reduction or amelioration of disease or symptoms thereof. Exemplary
conventional therapies can be found in the Physician's Desk
Reference (56th ed., 2002 and 57th ed., 2003). In some embodiments,
scaffolds of the invention may be combined with chemotherapy,
radiation therapy, surgery, immunotherapy with a biologic (antibody
or peptide), small molecules, or another therapy known in the art.
In some embodiments, the combinatorial therapy is administered
together. In other embodiments, the combinatorial therapy is
administered separately.
[0506] The invention also provides methods of diagnosing diseases.
The scaffolds of the invention which bind a specific target
associated with a disease may be implemented in a method used to
diagnose said disease. In one embodiment, the scaffolds of the
invention are used in a method to diagnose a disease in a subject,
said method comprising obtaining a sample from the subject,
contacting the target with the scaffold in said sample under
conditions that allow the target:scaffold interaction to form,
identifying the target: scaffold complex and thereby detecting the
target in the sample.
[0507] In some embodiments, the target is an antigen associated
with disease. In another embodiment, the target is a cytokine,
inflammatory mediator, and intracellular antigen, a self-antigen, a
non-self antigen, an intranuclear antigen, a cell-surface antigen,
a bacterial antigen, a viral antigen or a fungal antigen. In other
embodiments, the disease to be diagnosed is described herein.
[0508] The invention also provides methods of imaging specific
targets. In one embodiment, scaffolds of the invention conjugated
to imaging agents such as green-fluorescent proteins, other
fluorescent tags (Cy3, Cy5, Rhodamine and others), biotin, or
radionuclides may be used in methods to image the presence,
location, or progression of a specific target. In some embodiments,
the method of imaging a target comprising a scaffold of the
invention is performed in vitro. In other embodiments, the method
of imaging a target comprising a scaffold of the invention is
performed in vivo. In other embodiments, the method of imaging a
target comprising a scaffold of the invention is performed by MRI,
PET scanning, X-ray, fluorescence detection or by other detection
methods known in the art.
[0509] The invention also provides methods of monitoring disease
progression, relapse, treatment, or amelioration using the
scaffolds of the invention. In one embodiment, methods of
monitoring disease progression, relapse, treatment, or amelioration
is accomplished by the methods of imaging, diagnosing, or
contacting a compound/target with a scaffold of the invention as
presented herein.
Kits
[0510] Also within the scope of the invention are kits comprising
the compositions of the invention (e.g. scaffolds,) and
instructions for use. The kit can further contain at least one
additional reagent, or one or more additional scaffolds of the
invention. Kits typically include a label indicating the intended
use of the contents of the kit. The term label includes any
writing, or recorded material supplied on or with the kit, or which
otherwise accompanies the kit.
EQUIVALENTS
[0511] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0512] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference. This application
claims the benefit of priority to U.S. Provisional Application No.
61/323,708 filed Apr. 13, 2010, the entire contents of which are
incorporated herein by reference. Additionally, PCT Application No.
PCT/US2008/012398, filed on Oct. 10, 2008 and published as
International Publication No. WO 2009/058379 is hereby incorporated
by reference herein in its entirety for all purposes.
Exemplary Embodiments
[0513] 1. A multimeric scaffold comprising at least two fibronectin
type III (FnIII) scaffolds connected in tandem, wherein each FnIII
scaffold binds a target, and wherein each FnIII scaffold comprises:
[0514] I. seven beta strand domains designated A, B, C, D, E, F,
and G; [0515] II. linked to six loop regions, wherein a loop region
connects each beta strand and is designated AB, BC, CD, DE, EF, and
FG; [0516] wherein each beta strand has at least 50% homology to
the cognate beta strand of a FnIII domain of interest (FOI) and at
least one loop is a non-naturally occurring variant of the cognate
loop in the FOI, and [0517] wherein the binding affinity and/or
avidity for said target, and/or a biological activity of the
multimeric scaffold is improved over that of the corresponding
monomeric FnIII scaffolds. [0518] 2. The multimeric scaffold of
embodiment 1, wherein the binding affinity and/or avidity for said
target is improved. [0519] 3. The multimeric scaffold of embodiment
1, wherein the binding affinity and/or avidity for said target, and
a biological activity of the multimeric scaffold are improved.
[0520] 4. The multimeric scaffold of embodiment 1, 2 or 3, wherein
each beta strand of at least one of the FnIII scaffolds has at
least 50% homology to the cognate beta strand domain in any of SEQ
ID NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG.
16). [0521] 5. The multimeric scaffold of embodiment 1, 2 or 3,
wherein each beta strand of at least one of the FnIII scaffolds has
at least 60% homology to the cognate beta strand domain in any of
SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0522] 6. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 70% homology to the cognate beta strand domain in any
of SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0523] 7. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 80% homology to the cognate beta strand domain in any
of SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0524] 8. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 90% homology to the cognate beta strand domain in any
of SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0525] 9. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 95% homology to the cognate beta strand domain in any
of SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16) 1-15, 30-48, or 63. [0526] 10. The multimeric scaffold of
embodiment 1, 2 or 3, wherein each beta strand of at least one of
the FnIII scaffolds has at least 98% homology to the cognate beta
strand domain in any of SEQ ID NOs: 1-15, 30-48, or 63. [0527] 11.
The multimeric scaffold of embodiment 1, 2 or 3, wherein each beta
strand of at least one of the FnIII scaffolds has at least 60%
identity to the cognate beta strand domain in any of SEQ ID
NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16).
[0528] 12. The multimeric scaffold of embodiment 1, 2 or 3, wherein
each beta strand of at least one of the FnIII scaffolds has at
least 70% identity to the cognate beta strand domain in any of SEQ
ID NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG.
16). [0529] 13. The multimeric scaffold of embodiment 1, 2 or 3,
wherein each beta strand of at least one of the FnIII scaffolds has
at least 80% identity to the cognate beta strand domain in any of
SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0530] 14. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 90% identity to the cognate beta strand domain in any
of SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0531] 15. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 95% identity to the cognate beta strand domain in any
of SEQ ID NOs:1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0532] 16. The multimeric scaffold of embodiment 1, 2 or
3, wherein each beta strand of at least one of the FnIII scaffolds
has at least 98% identity to the cognate beta strand domain in any
of SEQ ID NOs: 1-34, 54, 69, or the sequences presented in Table 16
(FIG. 16). [0533] 17. The multimeric scaffold of any one of the
preceeding embodiments, wherein for at least one FnIII scaffold the
A beta strand domain comprises SEQ ID NO:41, 42, 61, 62, 76, or 77,
the B beta strand comprises SEQ ID NO:43, 63, or 78, the C beta
strand comprises SEQ ID NO:44, 64, or 79, the D beta strand
comprises SEQ ID NO:46, 65, or 80, the E beta strand comprises SEQ
ID NO:47, 66, or 81, the F beta strand comprises SEQ ID NO:48, 67,
or 82, and the G beta strand comprises SEQ ID NO:52, 68, or 83.
[0534] 18. The multimeric scaffold of any one of the preceding
embodiments, wherein for at least two of the FnIII scaffolds the A
beta strand comprises SEQ ID NO: 41, 42, 61, 62, 76, or 77, the B
beta strand comprises SEQ ID NO:43, 63, or 78, the C beta strand
comprises SEQ ID NO:44, 64, or 79, the D beta strand comprises SEQ
ID NO:46, 65, or 80, the E beta strand comprises SEQ ID NO:47, 66,
or 81, the F beta strand comprises SEQ ID NO:48, 67, or 82, and the
G beta strand comprises SEQ ID NO:52, 68, or 83. [0535] 19. The
multimeric scaffold of embodiment 17 or 18, wherein the AB loop
comprises SEQ ID NO:35, 55, or 70, the CD loop comprises SEQ ID
NO:37, 57, or 72, and the EF loop comprises SEQ ID NO:39, 59, or
74. [0536] 20. The multimeric scaffold of embodiment 17 or 18,
wherein the BC loop comprises SEQ ID NO:36, 56, or 71, the DE loop
comprises SEQ ID NO:38, 58, or 73 and the FG loop comprises SEQ ID
NO:39, 59, or 73. [0537] 21. The multimeric scaffold of any one of
the preceing embodiments, wherein for at least one FnIII scaffold
the A beta strand domain comprises SEQ ID NO:41 or 42, the B beta
strand comprises SEQ ID NO:43, the C beta strand comprises SEQ ID
NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the E beta
strand comprises SEQ ID NO:47, the F beta strand comprises SEQ ID
NO:49 or 51, and the G beta strand comprises SEQ ID NO:52 or 53.
[0538] 22. The multimeric scaffold of any one of the preceding
embodiments, wherein for at least two of the FnIII scaffolds the A
beta strand comprises SEQ ID NO: 41 or 42, the B beta strand
comprises SEQ ID NO:43, the C beta strand comprises SEQ ID NO:45,
or 131, the D beta strand comprises SEQ ID NO:46, the E beta strand
comprises SEQ ID NO:47, the F beta strand comprises SEQ ID NO:49 or
51, and the G beta strand comprises SEQ ID NO:52 or 53. [0539] 23.
The multimeric scaffold of any one of embodiments 1-16, 21, or 22,
wherein at least one of the FnIII scaffolds comprise the amino acid
sequence:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKET FTT,
wherein X.sub.AB, X.sub.BC X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein n=2-26. [0540] 24. The
multimeric scaffold of any one of embodiments 1-16, 21, 22 or 23,
wherein at least two of the FnIII scaffolds comprise the amino acid
sequence:
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKET FTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein n=2-26. [0541] 25. The
multimeric scaffold of any one of embodiments 21-24, wherein the AB
loop comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37,
and the EF loop comprises SEQ ID NO:39. [0542] 26. The multimeric
scaffold of any one of embodiments 21-24, wherein the BC loop
comprises SEQ ID NO: 36, the DE loop comprises SEQ ID NO:38, and
the FG loop comprises SEQ ID NO: 40. [0543] 27. The multimeric
scaffold of any one of embodiments 21-25, wherein for
(X.sub.FG).sub.nn=1, 2, 3, 4, 5, 6, 7, 8, or 9. [0544] 28. The
multimeric scaffold of any one of embodiments 1-19, 21, 22, 23, 24,
25, or 27, wherein the BC loop of at least one of the FnIII
scaffolds comprises the sequence: S-X-a-X-b-X-X-X-G, wherein X
represents any amino acid, wherein (a) represents proline or
alanine and wherein (b) represents alanine or glycine. [0545] 29.
The multimeric scaffold of any one of embodiments 1-19, 21, 22, 23,
24, 25, or 27, wherein the BC loop of at least one of the FnIII
scaffolds comprises the sequence: S-P-c-X-X-X-X-X-X-T-G, wherein X
represents any amino acid and wherein (c) represents proline,
serine or glycine. [0546] 30. The multimeric scaffold of any one of
embodiments 1-19, 21, 22, 23, 24, 25, or 27, wherein the BC loop of
at least one of the FnIII scaffolds comprises the sequence:
A-d-P-X-X-X-e-f-X-I-X-G, wherein X represents any amino acid,
wherein (d) represents proline, glutamate or lysine, wherein (e)
represents asparagine or glycine, and wherein (f) represents serine
or glycine. [0547] 31. The multimeric scaffold of any one of
embodiments 1-19, 21, 22, 23, 24, 25, or 28-30, wherein the FG loop
of at least one of the FnIII scaffolds comprises the sequence:
X-a-X-X-G-X-X-X-b, wherein X represents any amino acid, wherein (a)
represents asparagine, threonine, or lysine, and wherein (b)
represents serine or alanine. [0548] 32. The multimeric scaffold of
any one of embodiments 1-19, 21, 22, 23, 24, 25, or 28-30, wherein
the FG loop of at least one of the FnIII scaffolds comprises the
sequence: X-a-X-X-X-X-b-N-P-A, wherein X represents any amino acid,
wherein (a) represents asparagine, threonine or lysine and wherein
(b) represents serine or glycine. [0549] 33. The multimeric
scaffold of any one of embodiments 1-19, 21, 22, 23, 24, 25, or
28-30, wherein the FG loop of at least one of the FnIII scaffolds
comprises 11 amino acids having a sequence of
X-a-X-X-G-X-X-S-N-P-A, wherein X represents any amino acid, and
wherein (a) represents asparagine, threonine or lysine. [0550] 34.
The multimeric scaffold of any one of embodiments 1-19, 21, 22, 23,
24, 25, or 27-33, wherein the DE loop of at least one of the FnIII
scaffolds comprises the sequence: X-X-X-X-X-X, wherein X represents
any amino acid. [0551] 35. The multimeric scaffold of any one of
embodiments 1-18, 20, 21, 22, 23, 24, or 26, wherein the AB loop of
at least one of the FnIII scaffolds comprises the sequence:
K-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid,
histidine, tyrosine, isoleucine, valine, leucine, phenylalanine,
threonine, alanine, proline, or serine, and wherein (a) represents
serine, threonine, alanine, or glycine. [0552] 36. The multimeric
scaffold of any one of embodiments 1-18, 20, 21, 22, 23, 24, or 26,
wherein the AB loop of at least one of the FnIII scaffolds
comprises the sequence: K-X-X-X-X-X-X-X-a, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine, and
wherein (a) represents serine, threonine, alanine, or glycine.
[0553] 37. The multimeric scaffold of any one of embodiments 1-18,
20, 21, 22, 23, 24, 26, or 35-36, wherein the CD loop of at least
one of the FnIII scaffolds comprises 7, 8, or 9 residues wherein
each residue in the CD loop is randomized and wherein each residue
may be asparagine, aspartic acid, histidine, tyrosine, isoleucine,
valine, leucine, phenylalanine, threonine, alanine, proline, or
serine. [0554] 38. The multimeric scaffold of any one of
embodiments 1-18, 20, 21, 22, 23, 24, 26, or 35-37, wherein the EF
loop of at least one of the FnIII scaffolds comprises 8 residues
having the sequence X-b-L-X-P-X-c-X, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine,
wherein (b) represents asparagine, lysine, arginine, aspartic acid,
glutamic acid, or glycine, and wherein (c) represents isoleucine,
threonine, serine, valine, alanine, or glycine. [0555] 39. The
multimeric scaffold of any one of the preceding embodiments,
wherein the multimeric scaffold comprises at least three FnIII
scaffolds. [0556] 40. The multimeric scaffold of any one of the
preceding embodiments, wherein the multimeric scaffold comprises at
least four FnIII scaffolds. [0557] 41. The multimeric scaffold of
any one of the preceding embodiments, wherein the multimeric
scaffold comprises at least five FnIII scaffolds. [0558] 42. The
multimeric scaffold of any one of the preceding embodiments,
wherein the multimeric scaffold comprises at least six FnIII
scaffolds. [0559] 43. The multimeric scaffold of any one of the
preceding embodiments, wherein the multimeric scaffold comprises at
least seven FnIII scaffolds. [0560] 44. The multimeric scaffold of
any one of the preceding embodiments, wherein the multimeric
scaffold comprises at least eight FnIII scaffolds. [0561] 45. The
multimeric scaffold of any one of the preceding embodiments,
wherein the multimeric scaffold comprises more than eight FnIII
scaffolds. [0562] 46. The multimeric scaffold of any one of the
preceding embodiments, wherein at least one of the FnIII scaffolds
is fused to a heterologous moiety. [0563] 47. The multimeric
scaffold of embodiment 46, wherein the heterologous moiety is
selected from the group consisting of: polyethylene glycol (PEG), a
cytotoxic agent, a radionuclide, imaging agent, biotin, human serum
albumin (HSA) or an FcRn binding portion thereof, an Fc region of
an antibody, a light chain constant region of an antibody, an
albumin binding domain, an IgG molecule, transferrin, a binding
peptide, a non-FnIII scaffold, an epitope tag, a nucleic acid, a
recombinant polypeptide polymer, or a cytokine. [0564] 48. The
multimeric scaffold of any one of the preceding embodiments,
wherein the target is a cell-surface antigen, a soluble antigen, an
immobilized antigen, an immunosilent antigen, an intracellular
antigen, an intranuclear antigen, a self antigen, a non-self
antigen, a cancer antigen, a bacterial antigen, or a viral antigen.
[0565] 49. The multimeric scaffold of any one of the preceding
embodiments, wherein the multimeric scaffold is a receptor agonist.
[0566] 50. The multimeric scaffold of any one of the preceding
embodiments, wherein the multimeric scaffold binds the target with
a K.sub.D of less than 500 .mu.M. [0567] 51. The multimeric
scaffold of any one of the preceding embodiments, wherein the
multimeric scaffold binds the target with a K
.sub.D of less than 100 .mu.M. [0568] 52. The multimeric scaffold
of any one of the preceding embodiments, wherein two or more FnIII
scaffolds bind the same target at the same epitope. [0569] 53. The
multimeric scaffold of any one of the preceding embodiments,
wherein two or more FnIII scaffolds are identical. [0570] 54. The
multimeric scaffold of any one of the preceding embodiments,
wherein two or more FnIII scaffolds are not identical. [0571] 55.
The multimeric scaffold of any one of the preceding embodiments,
wherein the multimeric scaffold binds at least two different
non-overlapping epitopes on the same target. [0572] 56. The
multimeric scaffold of any one of the preceding embodiments,
wherein two or more FnIII scaffolds bind the same target at
non-overlapping epitopes. [0573] 57. The multimeric scaffold of any
one of embodiments 1-52, wherein the FnIII scaffolds bind to the
same epitope on two or more copies of a target molecule on a cell
surface. [0574] 58. The multimeric scaffold of any one of
embodiments 1-51, or 54, wherein two or more FnIII scaffolds bind
different targets. [0575] 59. The multimeric scaffold of any one of
embodiments 1-51, or 54, wherein the multimeric scaffold binds at
least two different targets. [0576] 60. The multimeric scaffold of
any one of the preceding embodiments, wherein at least two of the
FnIII scaffolds are connected in tandem by a peptide linker. [0577]
61. The multimeric scaffold of any one of the preceding
embodiments, wherein the linker comprises 1 to about 1000 amino
acids. [0578] 62. The multimeric scaffold of any one of the
preceding embodiments, wherein the linker comprises 1 to about 50
amino acids. [0579] 63. The multimeric scaffold of any one of the
preceding embodiments, wherein the linker comprises 1 to 25 amino
acids. [0580] 64. The multimeric scaffold of any one of the
preceding embodiments, wherein the linker comprises 1 to 15 amino
acids. [0581] 65. The multimeric scaffold of any one of the
preceding embodiments, wherein the linker comprises 1 to 5 amino
acids [0582] 66. The multimeric scaffold of any one of the
preceding embodiments, wherein the linker is a flexible peptide
linker comprising at least 50% glycine residues. [0583] 67. The
multimeric scaffold of any one of the preceding embodiments,
wherein the linker sequence comprises one more sequence of the
group consisting of: (G-G-G-S).sub.x, (G-G-G-G-S).sub.x,
(G-G-G-G-S-A).sub.x, (G-A).sub.x, (G-G-G-T-P-T).sub.x, and
(G-G-G-G-S-G-T-G-S-A-M-A-S).sub.x where x is a positive integer.
[0584] 68. The multimeric scaffold of any one of the preceding
embodiments, wherein the linker is a functional moiety. [0585] 69.
The multimeric scaffold of any one of the preceding embodiments,
wherein at least one FnIII scaffold is operably linked to an IgG
domain or a full length IgG light or heavy chain. [0586] 70. The
multimeric scaffold of embodiment 69, wherein the IgG domain is
selected from the group consisting of: [0587] I. an Fc region;
[0588] II. a CH1 region; [0589] III. a CH2 region; [0590] IV. a CH3
region; [0591] V. a hinge region; [0592] VI. a Ckappa region;
[0593] VII. a Clambda region; [0594] VIII. a CH1-hinge-CH2-CH3
region; and [0595] IX. a variable region. [0596] 71. An isolated
nucleic acid molecule encoding the multimeric scaffold of any one
of the preceeding embodiments. [0597] 72. An expression vector
operably linked to the nucleic acid of embodiment 71. [0598] 73. A
host cell comprising the vector of embodiment 72. [0599] 74. A
method of producing a multimeric scaffold comprising culturing the
host cell of embodiment 73 under conditions in which the multimeric
scaffold encoded by the nucleic acid molecule is expressed. [0600]
75. The method of embodiment 74, wherein the expressed multimeric
scaffold is secreted into the culture media. [0601] 76. The method
of embodiment 75, further comprising obtaining the protein from the
culture media. [0602] 77. A composition comprising the multimeric
scaffold of any one of embodiments 1-70 in a pharmaceutically
acceptable excipient. [0603] 78. A method for treating or
inhibiting growth of cancer in a patient comprising administering
an effective amount of the composition of embodiment 77. [0604] 79.
The method of embodiment 78, wherein the cancer is selected from
the group consisting of: squamous cell cancer, small-cell lung
cancer, non-small cell lung cancer, non-Hodgkin's lymphoma,
blastoma, gastrointestinal cancer, renal cancer, ovarian cancer,
liver cancer, stomach cancer, bladder cancer, hepatoma, breast
cancer, colon cancer, colorectal cancer, pancreatic cancer,
endometrial carcinoma, salivary gland carcinoma, kidney cancer,
liver cancer, prostate cancer, vulval cancer, thyroid cancer,
hepatic carcinoma, head and neck cancer, lung cancer,
adenocarcinoma, renal cell carcinoma, or hepatocellular carcinoma.
[0605] 80. A method for treating an autoimmune disorder, an
inflammatory disorder, or a respiratory infection in a patient
comprising administering an effective amount of the composition of
embodiment 77. [0606] 81. The method of embodiment 80, wherein the
respiratory infection is caused by a virus or bacteria. [0607] 82.
The method of embodiment 81, wherein the virus is respiratory
syncytial virus, parainfluenza virus or human metapneumovirus.
[0608] 83. The method of embodiment 80, wherein the inflammatory
disorder is asthma, chronic inflammation resulting from chronic
viral or bacterial infections, chronic obstructive pulmonary
disease; encephalitis, inflammatory bowel disease, inflammatory
osteolysis, pulmonary fibrosis, septic shock, undifferentiated
arthropathy, or undifferentiated spondyloarthropathy. [0609] 84.
The method of embodiment 80, wherein the autoimmune disorder is
age-related macular degeneration, allograft rejection, ankylosing
spondylitis, antiphospholipid syndrome, arthritis, atherosclerosis,
anaphylactic shock, autoimmune Addison's disease, alopecia greata,
autoimmune diseases of the adrenal gland, autoimmune hemolytic
anemia, autoimmune hepatitis, autoimmune oophoritis, autoimmune
orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous
pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic
fatigue immune dysfunction syndrome, chronic inflammatory
demyelinating polyneuropathy, chronic inflammation, Churg-Strauss
syndrome, cicatrical pemphigoid, cold agglutinin disease, corneal
and other tissue transplantation, CREST syndrome, Crohn's disease,
cystic fibrosis, diabetic retinopathies, discoid lupus,
endocarditis, endotoxic shock, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, hemangiomas, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenia purpura, IgA
neuropathy, juvenile arthritis, lichen planus, lupus erythematosus,
Meniere's disease, mixed connective tissue disease, multiple
sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia
gravis, neovascular glaucoma, organ ischemia, pemphigus vulgaris,
peritonitis, pernicious anemia, polyarteritis nodosa,
polychrondritis, polyglandular syndromes, polymyalgia rheumatica,
polymyositis and dermatomyositis, primary agammaglobulinemia,
primary biliary cirrhosis, psoriasis, psoriatic arthritis,
Raynauld's phenomenon, Reiter's syndrome, reperfusion injury,
retrolental fibroplasia, rheumatoid arthritis, sarcoidosis,
scleroderma, sepsis, septicemia, Sjogren's syndrome, spinal cord
injury, stiff-man syndrome, systemic lupus erythematosus, takayasu
arteritis, temporal arteritis/giant cell arteritis, thyroid
hyperplasias, ulcerative colitis, uveitis, vasculitides such as
dermatitis herpetiformis vasculitis, vitiligo, and Wegener's
granulomatosis. [0610] 85. A library of diverse fibronectin type
III (FnIII) scaffolds comprising: [0611] I. seven beta strand
domains designated A, B, C, D, E, F, and G; [0612] II. linked to
six loop regions, wherein a loop region connects each beta strand
and is designated AB, BC, CD, DE, EF, and FG; [0613] wherein each
beta strand has at least 50% homology to the cognate beta strand of
a FnIII domain of interest (FOI) and at least one loop is a
non-naturally occurring variant of the cognate loop in the FOI, and
wherein the FG loop is at least one amino acid shorter than the
cognate FG loop in the FOI. [0614] 86. A library of diverse
fibronectin type III (FnIII) scaffolds comprising: [0615] I. seven
beta strand domains designated A, B, C, D, E, F, and G; [0616] II.
linked to six loop regions, wherein a loop region connects each
beta strand and is designated AB, BC, CD, DE, EF, and FG; [0617]
wherein each beta strand domain has at least 50% homology to the
cognate beta strand of a FnIII domain of interest (FOI) and at
least one loop is a non-naturally occurring variant of the cognate
loop in the FOI, and wherein the FG loop consists of no more than 9
amino acids. [0618] 87. The library of embodiment 85 or 86, wherein
each beta strand has at least 50% homology to SEQ ID NOs:1-34, 54,
69, or the sequences presented in Table 16 (FIG. 16). [0619] 88.
The library of embodiment 85 or 86, wherein each beta strand has at
least 60% homology to any of SEQ ID NOs:1-34, 54, 69, or the
sequences presented in Table 16 (FIG. 16). [0620] 89. The library
of embodiment 85 or 86, wherein each beta strand has at least 70%
homology to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0621] 90. The library of
embodiment 85 or 86, wherein each beta strand has at least 80%
homology to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0622] 91. The library of
embodiment 85 or 86, wherein each beta strand has at least 90%
homology to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0623] 92. The library of
embodiment 85 or 86, wherein each beta strand of the native FnIII
domain has at least 95% homology to any of SEQ ID NOs:1-34, 54, 69,
or the sequences presented in Table 16 (FIG. 16). [0624] 93. The
library of embodiment 85 or 86, wherein each beta strand has at
least 98% homology to any of SEQ ID NOs:1-34, 54, 69, or the
sequences presented in Table 16 (FIG. 16). [0625] 94. The library
of embodiment 85 or 86, wherein each beta strand has at least 60%
identity to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0626] 95. The library of
embodiment 85 or 86, wherein each beta strand has at least 70%
identity to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0627] 96. The library of
embodiment 85 or 86, wherein each beta strand has at least 80%
identity to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0628] 97. The library of
embodiment 85 or 86, wherein each beta strand has at least 90%
identity to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0629] 98. The library of
embodiment 85 or 86, wherein each beta strand has at least 95%
identity to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0630] 99. The library of
embodiment 85 or 86, wherein each beta strand has at least 98%
identity to any of SEQ ID NOs:1-34, 54, 69, or the sequences
presented in Table 16 (FIG. 16). [0631] 100. The library of
embodiment 85 or 86, wherein the A beta strand domain comprises SEQ
ID NO: 41, 42, 61, 62, 76, or 77, the B beta strand comprises SEQ
ID NO:43, 63, or 78, the C beta strand comprises SEQ ID NO:44, 64,
or 79, the D beta strand comprises SEQ ID NO:46, 65, or 80, the E
beta strand comprises SEQ ID NO:47, 66, or 81, the F beta strand
comprises SEQ ID NO:48, 67, or 82, and the G beta strand comprises
SEQ ID NO:52, 68, or 83. [0632] 101. The library of embodiment 100,
wherein the AB loop comprises SEQ ID NO:35, 55, or 70, the CD loop
comprises SEQ ID NO:37, 57, or 72, and the EF loop comprises SEQ ID
NO:39, 59, or 74. [0633] 102. The library of embodiment 100,
wherein the BC loop comprises SEQ ID NO:36, 56, or 71, the DE loop
comprises SEQ ID NO:38, 58, or 73 and the FG loop comprises SEQ ID
NO: 40, 60 or 75. [0634] 103. The library of embodiment 85 or 86,
wherein the A beta strand domain comprises SEQ ID NO: 41 or 42, the
B beta strand comprises SEQ ID NO:43, the C beta strand comprises
SEQ ID NO:45, or 131, the D beta strand comprises SEQ ID NO:46, the
E beta strand comprises SEQ ID NO:47, the F beta strand comprises
SEQ ID NO:49 or 51, and the G beta strand comprises SEQ ID NO:52 or
53. [this embodiment will list SEQ ID NOs for Tn3 beta strands]
[0635] 104. The library of embodiment 85 or 86, wherein the FnIII
scaffolds comprise the amino acid sequence: [0636]
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKET FTT,
wherein X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and
X.sub.FG represent the amino acid residues present in the AB, BC,
CD, DE, EF, and FG loops, respectively, wherein X.sub.1 represents
amino acid residue A or T, and wherein n=3-26 and m=1-9. [0637]
105. The library of embodiment 103 or 104, wherein the AB loop
comprises SEQ ID NO:35, the CD loop comprises SEQ ID NO:37, and the
EF loop comprises SEQ ID NO:39. [0638] 106. The library of
embodiment 103 or 104, wherein the BC loop comprises BC loop
comprises SEQ ID NO:36, the DE loop comprises SEQ ID NO:38, and the
FG loop comprises SEQ ID NO:40. [0639] 107. The library of any one
of embodiments 85, 86, 100, 101, 103, or 105, wherein the amino
acid sequence of the BC loop of the FnIII scaffold comprises the
sequence of: S-X-a-X-b-X-X-X-G, wherein X represents any amino
acid, wherein (a) represents proline or alanine and wherein (b)
represents alanine or glycine. [0640] 108. The library of any one
of embodiments 85-, 86, 100, 101, 103, or 105, wherein the amino
acid sequence of the BC loop of the FnIII scaffold comprises the
sequence of: S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino
acid and wherein (c) represents proline, serine or glycine. [0641]
109. The library of any one of embodiments 85, 86, 100, 101, 103,
or 105, wherein the amino acid sequence of the BC loop of the FnIII
scaffold comprises the sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X
represents any amino acid, wherein (d) represents proline,
glutamate or lysine, wherein (e) represents asparagine or glycine,
and wherein (f) represents serine or glycine. [0642] 110. The
library of any one of embodiments 85, 86, 100, 101, 103, or 105,
wherein the amino acid sequence of the FG loop of the FnIII
scaffold comprises the sequence: X-X-X-X-X-X-X-X-X, wherein X
represents any amino acid. [0643] 111. The library of any one of
embodiments 85, 86, 100, 101, 103, or 105, wherein the amino acid
sequence of the FG loop of the FnIII scaffold comprises the
sequence: X-a-X-X-G-X-X-X-b, wherein X represents any amino acid,
wherein (a) represents asparagine, threonine, or lysine, and
wherein (b) represents serine or alanine.
[0644] 112. The library of any one of embodiments 85, 86, 100, 101,
103, or 105, wherein the amino acid sequence of the DE loop of the
FnIII scaffold comprises the sequence: X-X-X-X-X-X, wherein X
represents any amino acid. [0645] 113. The library of any one of
embodiments 85, 86, 100, 100, 103, or 106, wherein the amino acid
sequence of the AB loop of the FnIII scaffold comprises the
sequence: K-X-X-X-X-X-a, wherein X represents asparagine, aspartic
acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine, and wherein
(a) represents serine, threonine, alanine, or glycine. [0646] 114.
The library of any one of embodiments 85-, 86, 100, 100, 103, or
106, wherein the amino acid sequence of sad AB loop of the FnIII
scaffold comprises the sequence: K-X-X-X-X-X-X-X-a, wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein (a) represents serine, threonine,
alanine, or glycine. [0647] 115. The library of any one of
embodiments 85-, 86, 100, 100, 103, or 106, wherein the amino acid
sequence of the CD loop of the FnIII scaffold comprises 7, 8, or 9
residues wherein each residue in the CD loop is randomized and
wherein each residue may be asparagine, aspartic acid, histidine,
tyrosine, isoleucine, valine, leucine, phenylalanine, threonine,
alanine, proline, or serine. [0648] 116. The library of any one of
embodiments 85-, 86, 100, 100, 103, or 106, wherein the amino acid
sequence of the EF loop of the FnIII scaffold comprises the
sequence: X-b-L-X-P-X-c-X, wherein X represents asparagine,
aspartic acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine, wherein (b)
represents asparagine, lysine, arginine, aspartic acid, glutamic
acid, or glycine, and wherein (c) represents isoleucine, threonine,
serine, valine, alanine, or glycine. [0649] 117. The library of any
one of embodiments 85-116, wherein the library is displayed on the
surface of a ribosome, bacteriophage, virus, bacteria, yeast, or
mammalian cell. [0650] 118. A method for identifying a fibronectin
type III (FnIII) scaffold from the library of any one of
embodiments 85-117, wherein the FnIII scaffold has increased
protein stability as compared to an the FOI, and binds a target,
comprising: [0651] I. contacting the target ligand with the library
of any one of embodiments 85-117 under conditions suitable for
forming a scaffold:target ligand complex; [0652] II. obtaining from
the complex, the scaffold that binds the target ligand; [0653] III.
determining if the stability of the scaffold obtained in step (II)
is greater than that of the FOI. [0654] 119. The method of
embodiment 118, further comprising randomizing at least one loop of
the scaffold obtained in step (II) and repeating steps (I) and (II)
using the further randomized scaffold. [0655] 120. A method for
obtaining a fibronectin type III (FnIII) scaffold variant having
increased stability as compared to an FnIII scaffold of interest
(FOI), comprising: engineering a variant of the FOI, wherein the FG
loop of the variant comprises the deletion of at least 1 amino
acid, wherein the variant exhibits an increased stability as
compared to the FOI. [0656] 121. The method of embodiment 120,
wherein the length and sequence of the FG loop is determined prior
to engineering by aligning the amino acid sequence of the FOI with
the amino acid sequence of at least one native FnIII domain. [0657]
122. The method of embodiment 120, wherein the length and sequence
of the FG loop is determined prior to engineering by modeling the
three dimensional structure of at least one native FnIII domain on
the amino acid sequence of the FOI. [0658] 123. The method of any
one of embodiments 118-122, wherein the protein stability is
measured by melting temperature, differential scanning calorimetry
(DSC), circular dichroism (CD), polyacrylamide gel electrophoresis
(PAGE), protease resistance, isothermal calorimetry (ITC), nuclear
magnetic resonance (NMR), internal fluorescence, and/or biological
activity. [0659] 124. The method of any one of embodiments 118-123,
wherein the stability is increased by at least 10% in Cm as
compared to the FOI. [0660] 125. The method of any one of
embodiments 118-124, wherein the stability is increased by at least
20% in Cm as compared to the FOI. [0661] 126. The method of any one
of embodiments 118-125, wherein the stability is measured by urea
denaturation. [0662] 127. The method of any one of embodiments
118-125, wherein the stability is measured by guanidine
denaturation. [0663] 128. The method of any one of embodiments
118-127, wherein the scaffold exhibits a decrease of at least 10%
in protease sensitivity as compared to the FOI. [0664] 129. The
method of any one of embodiments 118-128, wherein the scaffold
exhibits an increased melting temperature as compared to the FOI.
[0665] 130. The recombinant scaffold of embodiment 118-129, wherein
the melting temperature is increased by at least 2.degree. C. as
compared to the FOI. [0666] 131. The method of any one of
embodiments 118-130, wherein the FOI comprises any of SEQ ID
NOs:1-34, 54, 69, or the sequences presented in Table 16 (FIG. 16).
[0667] 132. The method of any one of embodiments 118-131, wherein
the FOI comprises SEQ ID NO: 1. [0668] 133. The method of any one
of embodiments 118-131, wherein the FOI SEQ ID NO: 54 or 69. [0669]
134. A fibronectin type III scaffold having increased protein
stability produced by the method of any one of embodiments 118-133,
wherein the scaffold exhibits an increased stability of: (a) of at
least 10% in Cm as measured in a urea denaturation experiment; (b)
of at least 10% in Cm as measured in a guanidine denaturation
experiment; (c) of at least 10% in protease sensitivity; or (d)
increased melting temperature, as compared to that of the FOI.
TABLE-US-00001 [0669] TABLE 1 Sequences and SEQ ID Nos of molecular
components to assemble representative scaffolds on the invention:
SEQ Name/Brief Description Sequence ID NO Tn3
IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSL 1
ICRRGDMSSNPAKETFTT (cys residues of disulfide bond are underlined)
SS3
IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSAGNLKPDTEYCVSL 2
ISRRGDMSSNPAKECFTT (cys residues of disulfide bond are underlined)
Tn3 + SS3
IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYCVS- L
3 ICRRGDMSSNPAKECFTT (cys residues of disulfide bonds are
underlined) 3rd FnIII of tenascin C
RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPD 4
(w/N-term aa) TEYEVSLISRRGDMSSNPAKETFTT (underlined A beta strand
residues may be removed) 10th FnIII of
LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITV 5
fibronectin YAVTGRGDSPASSKPISINYRT 3rd FnIII of
PTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIY 6
fibronectin AVEENQESTPVVIQQET 6th FnIII of
PYNTEVTETTIVITWTPAPRIGFKLGVRPSQGGEAPREVTSDSGSIVVSGLTPGVEYVYTIQVLR 7
fibronectin DGQERDAPIVNKVVT FnIII from growth
PPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSV 8
hormone R PVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLP FnIII from .beta.
PPSLNVTKDGDSYSLRWETMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPST 9
common R RYWARVRVRTSRTGYNGIWSEWSEARSWDTE FnIII from IL-5R
PPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESKIVTILHKGFSA
10 SVRTILQNDHSLLASSWASAELHA 29.sup.th FnIII from
LSVTDVTTSSLRLNWEAPPGAFDSFLLRFGVPSPSTLEPHPRPLLQRELMVPGTRHSAVLRDLRS
11 Tenascin XB GTLYSLTLYGLRGPHKADSIQGTART 31.sup.st FnIII from
LRALNLTEGFAVLHWKPPQNPVDTYDIQVTAPGAPPLQAETPGSAVDYPLHDLVLHTNYTATVRG
12 Tenascin XB LRGPNLTSPASITFTT 32.sup.nd FnIII from
LEAKEVTPRTALLTWTEPPVRPAGYLLSFHTPGGQTQEILLPGGITSHQLLGLFPSTSYNARLQA
13 Tenascin XB MWGQSLLPPVSTSFTT Truncated 3rd FnIII
IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSL
14 of tenascin C ISRRGDMSSNPAKETFTT FnIII-growth hormone R
PKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFN
15 SSFTSIWIPYCIKLTSNGGTVDEKCFSV FnIII from PTPR-F
PSGFPQNLHVTGLTTSTTELAWDPPVLAERNGRIISYTVVFRDINSQQELQNITTDTRFTLTGLK
16 PDTTYDIKVRAWTSKGSGPLSPSIQSRTMPVE FnIII from PTPR-F
PKPPIDLVVTETTATSVTLTWDSGNSEPVTYYGIQYRAAGTEGPFQEVDGVATTRYSIGGLSPFS
17 EYAFRVLAVNSIGRGPPSEAVRARTGE FnIII from collagen
LSPPRNLRISNVGSNSARLTWDPTSRQINGYRIVYNNADGTEINEVEVDPTIIFPLKGLTPLTEY
18 type XIV TIAIFSIYDEGQSEPLTGVFTT 3rd FnIII of tenascin
IEKVKDVTDTTALITWFKPLAEIDGIQLTYGIKDVPGDRTTINLTEDEVQYSIGNLKPDTEYEVS
19 C-charge variant SLISRRGDMSSNPAKQTFTT Archaeoglobus fulgidus
PAISNVRVSDVTNSSATIRWDVSLAANNRVLFSTNSDLSSPQWSAWDNSTDSPMITLSGLSAGTA
20 DSM 4304 NCBI Acc. #: YYFSVYSFRPDNASLYSNSSIMSFTT NC_000917
Staphylothermus marinus
SEPQNLKATAGNNNITLTWDPPIDDGGCRIVEYRIYRGTNNNNLEYYASVNGSTTTFIDKNIVYS
21 F1 NCBI Acc. #: QTYYYKVSAVNNIVEGPKSNTASATPTSS NC_009033
Sulfolobus acidocaldarius
PPPKPVIRFAQAGNNSISLSWYDTNTSGYYIQWWSSIDNNKSTINVGNVSSYLFINLTNGVTYYF
22 DSM 639 NCBI Acc. #: RIIPYNQAGNGTSSDIISLTPGAV NC_007181 1st
FnIII Sulfolobus acidocaldarius
PDSPSVKVIVGDRNATVIWSKPYNGGFPILGYYLTVKTDNSSYTINVGNVSKYTLTNLTPEVLYE
23 DSM 639 NCBI Acc. #: VMVVAYNKLGNSSPGIVNFVALTT NC_007181 2nd
FnIII Sulfolobus acidocaldarius
LTTASISVSVYKKVNGVLISWNKTENTTYYNLLISDKKGKIIVNITTTNTSYFAYIPYGIYNVTI
24 DSM 639 NCBI Acc. #: RATNQVGTNSTSFPIVFYIPPFI NC_007181 3rd FnIII
Sulfolobus acidocaldarius
PLVKFSIGNNSILNLKWNNVTGATFYLVYVNTTLIANVTTDSYSLNLTPGFHVIRVVAANPIYNS
25 DSM 639 NCBI Acc. #: SPASLGILIQQHSVTSSIT NC_007181 4th FnIII
Sulfolobus solfataricus
PLPPKITSYSAGNESVTLGWNPVRLSSGYEIIYWNNMGFNSSINVGNVTSYTVTGLKDGITYYFE
26 P2 NCBI Acc. #: VLAYNSIGYSSPSSIIALTPASV NC_002754 1st FnIII
Sulfolobus solfataricus
PNPPQLVSVKYGNDNVTLNWLPPTFSGGYLLLGYYVIVKNENSMVSSHFVNSTSLTISNLTPNVT
27 P2 NCBI Acc. #: YNVFIYAVNKLGNSSPLVLTVVPITKA NC_002754 2nd FnIII
Sulfolobus solfataricus
PITKASVFAFITKLGNGILVNWTTSFPANTLELYNPNGNLISQIAAIKGNSSYLFRVPQGNYTLV
28 P2 NCBI Acc. #: IIASNSAGVSKYVYQVVYYL NC_002754 3rd FnIII
Sulfolobus solfataricus
PPASPQVLSIGFGNNLYISWNNEANVITYLVYVNNSLVYEGPSNSIVTNISNGTYLVKVIGVNPA
29 P2 NCBI Acc. #: GSSSPGIAVIHYTGDYVT NC_002754 4th FnIII
Sulfolobus tokodaii str.
PPKPQIASIASGNETITVKWYDTNASGYYITWSNFSQKVTINGNVTSYTIKHLKDGVTYYIQIVP
30 7 NCBI Acc. #: YNSLGNGTPSDIISATPSSV NC_003106 1st FnIII
Sulfolobus tokodaii str.
PNPPIIKVKIGNLNATLTWYDTFNGGYPIEGYYLYVNGKGINVGNITSYVLTNLTAGELYTIELI
31 7 NCBI Acc. #: AYNKIGNSSISSVSFIAASKA NC_003106 2nd FnIII
Sulfolobus tokodaii str.
ASKANLTVTVYKKINGFLVSWNSTSKAKYILTVSKENVVLLNVSTTNTSYFVKPFGVYNISLEAV
32 7 NCBI Acc. #: NIVGITKYAFILIYYIQ NC_003106 3rd FnIII Sulfolobus
tokodaii str.
PASPTVNWSITLNTVSLNWSKVSGAEYYLIYDNGKLITNTTNTAFTFNLTIGQNEIEVYAANAYY
33 7 NCBI Acc. #: KSAPYIINDVRNYIVV NC_003106 4th FnIII 14.sup.th
FnIII of
ARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLY
34 fibronectin TLNDNARSSPVVIDAST 3.sup.rd FnIII of tenascin KDVTDTT
35 C, AB loop 3.sup.rd FnIII of tenascin FKPLAEIDG 36 C, BC loop
3.sup.rd FnIII of tenascin KDVPGDR 37 C, CD loop 3.sup.rd FnIII of
tenascin TEDENQ 38 C, DE loop 3.sup.rd FnIII of tenascin GNLKPDTE
39 C, EF loop 3.sup.rd FnIII of tenascin RRGDMSSNPA 40 C, FG loop
3.sup.rd FnIII of tenascin RLDAPSQIEV 41 C, beta strand A 3.sup.rd
FnIII of tenascin IEV 42 C, beta strand A N-terminal truncation
3.sup.rd FnIII of tenascin ALITW 43 C, beta strand B 3.sup.rd FnIII
of tenascin IELTYGI 44 C, beta strand C 3.sup.rd FnIII of tenascin
CELTYGI 45 C, beta strand C (Tn3) 3.sup.rd FnIII of tenascin TTIDL
46 C, beta strand D 3.sup.rd FnIII of tenascin YSI 47 C, beta
strand E 3.sup.rd FnIII of tenascin YEVSLIS 48 C, beta strand F
3.sup.rd FnIII of tenascin YEVSLIC 49 C, beta strand F (Tn3)
3.sup.rd FnIII of tenascin YCVSLIS 50 C, beta strand F (SS3)
3.sup.rd FnIII of tenascin YCVSLIC 51 C, beta strand F (Tn3 + SS3)
3.sup.rd FnIII of tenascin KETFTT 52 C, beta strand G 3.sup.rd
FnIII of tenascin KECFTT 53 C, beta strand G (SS3 & Tn3 + SS3)
WT 10FnIII of fibronectin
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPG
54 (w/N-term aa) VDYTITVYAVTGRGDSPASSKPISINYRT (underlined A beta
strand residues may be removed) WT 10FnIII of fibronectin, VAATPTS
55 AB loop WT 10FnIII of fibronectin, DAPAVTVRY 56 BC loop WT
10FnIII of fibronectin, TGGNSPV 57 CD loop WT 10FnIII of
fibronectin, PGSKST 58 DE loop WT 10FnIII of fibronectin, SGLKPGVD
59 EF loop WT 10FnIII of fibronectin, VTGRGDSPASSKPI 60 FG loop WT
10FnIII of fibronectin, VSDVPRDLEV 61 beta strand A WT 10FnIII of
fibronectin, LEV 62 beta strand A N-terminal truncation WT 10FnIII
of fibronectin, LLISW 63
beta strand B WT 10FnIII of fibronectin, YRITYGE 64 beta strand C
WT 10FnIII of fibronectin, GEFTV 65 beta strand D WT 10FnIII of
fibronectin, ATI 66 beta strand E WT 10FnIII of fibronectin,
YTITVYA 67 beta strand F WT 10FnIII of fibronectin, SINYRT 68 beta
strand G WT 14FnIII of fibronectin,
VSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTD
69 (w/N-term aa) YKIYLYTLNDNARSSPVVIDAST (underlined A beta strand
residues may be removed) WT 14FnIII of fibronectin, TDATETT 70 AB
loop WT 14FnIII of fibronectin, RTKTETITG 71 BC loop WT 14FnIII of
fibronectin, ANGQTP 72 CD loop WT 14FnIII of fibronectin, KPDVRS 73
DE loop WT 14FnIII of fibronectin, TGLQPGTD 74 EF loop WT 14FnIII
of fibronectin, LNDNARSSPV 75 FG loop WT 14FnIII of fibronectin,
SPPRRARV 76 Beta strand A WT 14FnIII of fibronectin, ARV 77 Beta
strand A N-terminal truncation WT 14FnIII of fibronectin, ITISW 78
Beta strand B WT 14FnIII of fibronectin, FQVDAVP 79 Beta strand C
WT 14FnIII of fibronectin, IQRTI 80 Beta strand D WT 14FnIII of
fibronectin, YTI 81 Beta strand E WT 14FnIII of fibronectin,
YKIYLYT 82 Beta strand F WT 14FnIII of fibronectin, VIDAST 83 Beta
strand G Fc region with hinge
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
84
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CH1-hinge-Fc region
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
85
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGK Kappa light chain
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
86 YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Lambda light chain
QPKAAPSVLTFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY
87 AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC Linker region 1
GGGGSGGGGSGGGGSA 88 Linker region 2 GGGGSGGGGSGTGSAMASGGGGSA 89
Linker region from C1 RLDAPGQ 90 (G-G-G-G-S) units are in bold;
natural tenascin C sequence underlined Linker region from RLDAPGQ
91 C2 and C8 (G-G-G-G-S) units are in bold; natural tenascin C
sequence underlined Linker region from C3 RLDAPGQ 92 (G-G-G-G-S)
units are in bold; natural tenascin C sequence underlined Linker
region from C4 RLDAPGQ 93 (G-G-G-G-S) units are in bold; natural
tenascin C sequence underlined Linker region from C5 RLDAPGQ 94
natural tenascin C sequence underlined Linker region from C6
RLDAPGQ 95 (G-G-G-G-S) units are in bold; natural tenascin C
sequence underlined Linker region from C7 RLDAPGQ 96 (G-G-G-G-S)
units are in bold; natural tenascin C sequence underlined
Tenascin-consensus FnIII
LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGT
256 (w/N-term aa) EYTVSIYGVKGGHRSNPLSAEFTT (underlined A beta
strand residues may be removed) Tenascin-consensus FnIII SEVTEDS
257 AB loop Tenascin-consensus FnIII TAPDAAFDS 258 BC loop
Tenascin-consensus FnIII SEKVGEA 259 CD loop Tenascin-consensus
FnIII PGSERS 260 DE loop Tenascin-consensus FnIII TGLKPGTE 261 EF
loop Tenascin-consensus FnIII VKGGHRSNPL 262 FG loop
Tenascin-consensus FnIII LPAPKNLVV 263 Beta strand A
Tenascin-consensus FnIII LVV 264 Beta strand A N-terminal
truncation Tenascin-consensus FnIII LRLSW 265 Beta strand B
Tenascin-consensus FnIII FLIQYQE 266 Beta strand C
Tenascin-consensus FnIII INLTV 267 Beta strand D Tenascin-consensus
FnIII YDL 268 Beta strand E Tenascin-consensus FnIII YTVSIYG 269
Beta strand F Tenascin-consensus FnIII SAEFTT 270 Beta strand G
EXAMPLES
[0670] The invention is now described with reference to the
following examples. These examples are illustrative only and the
invention should in no way be construed as being limited to these
examples but rather should be construed to encompass any and all
variations which become evident as a result of the teachings
provided herein.
Example 1
Design of Various Multivalent Tn3 Formats
[0671] Multivalent formats of the Tn3 scaffold have been designed.
The multivalent formats contain one or more Tn3 modules fused to
themselves, fused to other protein motifs that can oligomerize, or
fused to themselves and to other protein motifs that can
oligomerize are shown in FIG. 1. In each case, the resulting
molecular entity contains at least 2 Tn3 modules. The polypeptide
linkers connecting the Tn3 modules to each other or to other
protein motifs can be structured or unstructured and with or
without a function. Three exemplary classes of multivalent Tn3
scaffold proteins are specifically provided: (i) linear (L)
multivalent proteins containing Tn3 modules fused to each other via
a polypeptide linker; (ii) antibody-like (Ig) multivalent proteins
containing one or more linearly fused Tn3 modules fused to the
light and heavy chains of an antibody or antibody fragment and
(iii) Fc-containing multivalent proteins containing one or more
linearly fused Tn3 modules fused to an antibody Fc region (FIG.
1).
Example 2
Expression and Purification of Multivalent TRAIL R2-specific
Tn3-containing Proteins
[0672] A series of eight multivalent Tn3-module containing scaffold
proteins (also referred to as "Tn3 proteins" or "Tn3 scaffolds")
with binding specificity for human TRAIL R2 were prepared. Examples
were prepared from each of the three multivalent formats described
in Example 1, and all of these proteins presented 2 or more of the
TRAIL R2-binding Tn3 module A1 (clone 1E11, G6 or 1C12). For
several TRAIL R2-specific multivalent Tn3 protein, a corresponding
control Tn3 protein (clone DE a Tn3 domain specific for the
Synagis.RTM. antibody) that did not bind TRAIL R2 was also
generated, differing only in the sequence and binding specificity
of the component Tn3 modules. Tn3 clone D1 is a Tn3 protein wherein
the BC, DE, and FG loops of a 1E11 clone are replaced with
alternative loops with sequences corresponding to SEQ ID NO: 99,
38, and 107, respectively (see TABLE 4). Sequence identity numbers
of the multivalent Tn3 protein constructs that were expressed are
shown in TABLE 2, and all the possible constructs are represented
schematically in TABLE 3 and FIG. 2. The loop sequences for the
clones are provided in TABLE 4.
TABLE-US-00002 TABLE 2 Names, formats, valencies, and specificities
of expressed Tn3- containing proteins Name Format Number of (clone)
type SEQ ID NO Tn3 modules Specificity A1(1E11) Monomer 134 1 TRAIL
R2 A2(1E11) L 139 2 TRAIL R2 A3(1E11) L 140 4 TRAIL R2 A4(1E11) L
141 6 TRAIL R2 A5(1E11) L 142 8 TRAIL R2 A5(G6) L 145 8 TRAIL R2
A6(1E11) Fc 151 2 TRAIL R2 A7(1E11) Fc 164 4 TRAIL R2 A8(1E11) Fc
165 8 TRAIL R2 A9(1C12) Ig 154 (HC), 4 TRAIL R2 154 (LC) A9(1E11)
Ig 158 (HC), 4 TRAIL R2 159 (LC) B1(D1) Monomer 180 1 non TRAIL
R2-binding control of A1 B2(D1) L not expressed 2 non TRAIL
R2-binding control of A2 B3(D1) L 146 4 non TRAIL R2-binding
control of A3 B4(D1) L 147 6 non TRAIL R2-binding control of A4
B5(D1) L 148 8 non TRAIL R2-binding control of A5 B6(D1) Fc 181 2
non TRAIL R2-binding control of A6 B7(D1) Fc not expressed 4 non
TRAIL R2-binding control of A7 B8(D1) Fc not expressed 8 non TRAIL
R2-binding control of A8 B9(D1) Ig 182 (HC), 4 non TRAIL 183 (LC)
R2-binding control of A9 L = linear Tn3 fusions, Fc = Fc-Tn3
fusions, Ig = antibody-like Tn3 fusions
TABLE-US-00003 TABLE 3 Schematic Representation of Tn3 Scaffold
Constructs Construct Components Tn3 Module (Tn3)
IEV(X.sub.AB).sub.nALITW(X.sub.BC).sub.nCELX.sub.1YGI(X.sub.CD).sub.nTTID-
L(X.sub.DE).sub.nYSI(X.sub.EF).sub.nYEVSLIC(X.sub.FG).sub.nKETFTT
X.sub.AB, X.sub.BC, X.sub.CD, X.sub.DE, X.sub.EF, and X.sub.FG
represent the amino acid residues present in the AB, BC, CD, DE,
EF, and FG loops, respectively where n = 2-26, X.sub.1 represents
amino acid residue A or T. Gly-Ser linker GGGGS module,
(G.sub.4S).sub.n The (G.sub.4S)n module wherein n = 1 is shown
above where n = 1-7 Poly-Histidine Tag HHHHHHHH ( ) An optional
component of the constructs detailed below-useful for purification
Name Construct Overview A1 or B1 A(Tn3)GGGTLG A2 or B2
S(G.sub.4S).sub.1A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.2GTL A3
or B3
S(G.sub.4S).sub.1A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.3A(Tn-
3)(G.sub.4S).sub.2GTLG A4 or B4
S(G.sub.4S).sub.1A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.3A(Tn-
3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.2GTGSAMAS(G.sub.4S).sub.1A(Tn3)
(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.2GTLG A5 or B5
S(G.sub.4S).sub.1A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.3A(Tn-
3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.2GTGSAMAS(G.sub.4S).sub.1A(Tn3)
(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.-
4S).sub.2GTLG A6 or B6
(Tn3)GAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV-
KFNWYDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK A7 or B7
AMAS(G.sub.4S).sub.1A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.2G-
TGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A8 or B8
AMAS(G.sub.4S).sub.1A(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.3A-
(Tn3)(G.sub.4S).sub.3A(Tn3)(G.sub.4S).sub.2GTGAEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
A9 or B9 heavy chain
SQ(Tn3)GGGTPTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
constant region
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
fusion
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN-
GKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A9 or B9
light chain
SQ(Tn3)GGGTPTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
constant region SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVKTSFNRGEC
fusion M13 or 79 A(Tn3)GGGTLG C1
A(Tn3)A(G.sub.4S).sub.1RLDAPGQ(Tn3)GGGTLG C2
A(Tn3)(G.sub.4S).sub.3RLDAPGQ(Tn3)GGGTLG C3
A(Tn3)(G.sub.4S).sub.3RLDAPGQ(Tn3)GGGTLG C4
A(Tn3)(G.sub.4S).sub.7RLDAPGQ(Tn3)GGGTLG C5
A(Tn3)TRLDAPGQ(Tn3)GGGTLG C6
A(Tn3)(G.sub.4S).sub.1RLDAPGQ(Tn3)GGGTLG C7
A(Tn3)(G.sub.4S).sub.2RLDAPGQ(Tn3)GGGTLG C8
A(Tn3)(G.sub.4S).sub.3RLDAPGQ(Tn3)GGGTLG
TABLE-US-00004 TABLE 4 Loop Sequences of Tn3 Clones Used in These
Studies AB Loop BC Loop CD Loop DE Loop EF Loop FG Loop Clone (SEQ
ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO)
1E11.dagger. KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE
FDPYGAKSNPA (NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 106)
D1 KDVTDTT SPGERIWMFTG KDVPGDR TEDENQ GNLKPDTE PNYERISNPA (NO: 35)
(NO: 99) (NO: 37) (NO: 38) (NO: 39) (NO: 107) G6.dagger. KDVTDTT
AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYGMRSKPA (NO: 35) (NO: 97)
(NO: 37) (NO: 102) (NO: 39) (NO: 108) 1C12.dagger. KDVTDTT
AKPEKWDGSIYG KDVPGDR NSRHTA GNLKPDTE FTPYGAKSNPA (NO: 35) (NO: 98)
(NO: 37) (NO: 103) (NO: 39) (NO: 109) M13 KDVTDTT HDAFGYDFG KDVPGDR
PDHFHN GNLKPDTE ANDHGFDSNPA (NO: 35) (NO: 100) (NO: 37) (NO: 104)
(NO: 39) (NO: 110) 79 KDVTDTT IPPHNADSSIIG KDVPGDR YDVAFD GNLKPDTE
DTFYGFDSNPA (NO: 35) (NO: 101) (NO: 37) (NO: 105) (NO: 39) (NO:
111) G3.dagger. KDVTDTT AKPEKWDGPPLW KDVPGDR NSRHTA GNLKPDTE
FTPYGAKSNPA (NO: 35) (NO: 168) (NO: 37) (NO: 103) (NO: 39) (NO:
109) C4.dagger. KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE
FDPYNKRNVPA (NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 169)
F4.dagger. KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE FDPYGLKSRPA
(NO: 35) (NO: 97) (NO: 37) (NO: 102) (NO: 39) (NO: 170)
F4mod1.dagger. KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHTA GNLKPDTE
FDPYGLKSRPA (NO: 35) (NO: 9897 (NO: 37) (NO: 102) (NO: 39) (NO:
170) F4mod12 KDVTDTT AKPWVDPPPLWG KDVPGDR QQKHNQ GNLKPDTE
FDPYGLKSRPA (NO: 35) (NO: 97) (NO: 37) (NO: 179) (NO: 39) (NO: 170)
.dagger.Clones comprising a C beta strand having the sequence
CELAYGI (SEQ ID NO: 131), all other clones comprise a C beta strand
having the sequence CELTYGI (SEQ ID NO: 45)
[0673] Preparation of Expression Constructs:
[0674] Enzymes used were from New England Biolabs (Ipswich, Mass.),
DNA purification kits were from Qiagen (Germantown, Md.), and DNA
primers were from IDT (Coralville, Iowa). Preparation of expression
constructs encoding 2 or more linearly fused Tn3 modules was as
follows. The DNA encoding a TRAIL R2-specific Tn3 module (e.g.,
1E11, SEQ ID NO: 134; G6, SEQ ID NO: 138; etc.) was amplified by
PCR with the primers "Tn3 gly4serl module forward" (SEQ ID NO: 112)
and "Tn3 gly4ser2 module reverse" (SEQ ID NO: 113) (TABLE 5).
[0675] After cleanup of the PCR product, the amplified DNA was
divided in two, with one half digested with BpmI, and the other
half digested with AcuI. The digested samples were purified using a
PCR cleanup kit and ligated with T4 DNA ligase to make a DNA
product encoding two Tn3 modules (A2). This material was purified
by agarose gel electrophoresis and again split into two. Digestion
with NcoI and KpnI followed by ligation into NcoI/KpnI digested
pSec-oppA(L25M) (described in WO 2009/058379 A2, Example 18)
yielded the bacterial expression construct for protein A2. Ligation
of undigested product into pCR 2.1-TOPO vector (Invitrogen,
Carlsbad, Calif.) provided genetic material for generation of
higher order fusions. To make a DNA fragment encoding four Tn3
modules (A3), the TOPO cloned A2 DNA was PCR amplified with primers
"module amp forward" (SEQ ID NO: 114) and "module amp reverse" (SEQ
ID NO: 115) (TABLE 5), purified, and split in two for digestion
with AcuI or BpmI. The rest of the process for making the A3
expression construct was the same as that used for making the A2
construct, wherein the DNA encoding A3 was assembled from A2
building blocks. Again, concurrent cloning of assembled A3 DNA into
pCR 2.1-TOPO provided genetic material for generation of higher
order fusions.
[0676] For preparation of A4 and A5 bacterial expression
constructs, an adapter module was introduced at the 3' end of the
multi-Tn3 coding sequence within the A3 expression construct. To do
this, the A3 expression vector was first digested with KpnI and
EcoRI, and the excised fragment was replaced with a duplex cassette
containing the oligonucleotides "insert BamHI in pSec forward" (SEQ
ID NO: 116) and "insert BamHI in pSec reverse" (SEQ ID NO: 117)
(TABLE 5). PCR amplification of A2 and A3 sequences from the
corresponding pCR 2.1 TOPO constructs was performed with the
primers "module insert BamHI forward" (SEQ ID NO: 118) and "module
amp reverse" (SEQ ID NO: 115) (TABLE 5). Amplified products were
double digested with BamHI/KpnI, and cloned into similarly digested
A3 expression construct.
[0677] Proteins A6-A9 were expressed by transient transfection of
293F cells, as described in Example 16 of WO 2009/058379 A2.
Briefly, expression vectors were generated by PCR amplifying the
Tn3 module (or modules) from the bacterial expression constructs,
and cloning these into in house vectors encoding the Fc region, the
kappa light chain constant region and/or the CHI-hinge-CH2-CH3
heavy chain constant regions for expression of Fc fusion or
antibody proteins. For protein A9, a Tn3 module replaces the
antibody variable regions in the human IgG1 heavy chain and kappa
light chain. The primers that add compatible NheI and KasI sites
for making Fc fusions of the tandem constructs are shown in TABLE
5.
TABLE-US-00005 TABLE 5 Primer Sequences Used in the Construction of
Multivalent Tn3 Proteins Sequence Name Sequence SEQ ID NO Tn3
gly4ser1 module GGCGCTAGGCTGAGTAGGTCCTGGAGTGCGGCCATGGCCAGCGG 112
forward GGGCGGAGGGAGTGCCATTGAAGTGAAAGATGTGACCGATACC Tn3 gly4ser2
module CCTCAGCCGATCACCACCTGAAGGCTACGCAGGTACCGCTACCG 113 reverse
CCACCTCCGCTCCCACCGCCACCGGTGGTAAAGGTTTC Module amp forward
GGCGCTAGGCTGAGTAGGTCCTGGAGTGCGG 114 Module amp reverse
CCTCAGCCGATCACCACCTGAAGGCTACGCAGG 115 Module insert
GGGATCCGCTACGGGCCACTCGATCGAGGTCCGTGCTGATCGAG 116 BamHI in pSec
CGATCGGTACCCTGGGCCATCATCATCATCATCACCACCACTGA forward G Module
insert AATTCTCAGTGGTGGTGATGATGATGATGATGGCCCAGGGTACC 117 BamHI in
pSec GATCGCTCGATCAGCACGGACCTCGATCGAGTGGCCCGTAGCGG reverse ATCCCGTAC
Module insert GGCGCTAGGCTGAGTAGGTCCTGGGGATCCGCCATGGCCAGC 118 BamHI
forward Module insert NheI
GGCGCTAGGCTGAGTAGGTCCTGGCTAGCTGCCATGGCCAGC 119 forward Module
insert KasI CCTCAGCCGATCACCACCTGAAGGCGGCGCCGGTACC 120 reverse
[0678] Expression and Purification of Proteins:
[0679] Monovalent or linear Tn3 proteins were expressed in
BL21(DE3) E. coli (EMD/Novagen, Gibbstown, N.J.) and the His-tagged
proteins were purified from the culture media using Ni NTA
Superflow resin (Qiagen). Surprisingly, despite large differences
in the molecular weights, all of these constructs expressed at
medium to high levels in E. coli and were efficiently secreted into
the media (TABLE 6 and FIG. 3).
[0680] To express Fc fusion and antibody-like proteins (A6-A9),
293F cells were transiently transfected with the appropriate
expression constructs. Harvests of supernatant were performed on
days 6 and 10 and the protein was purified by protein A affinity
chromatography.
[0681] All purified proteins were analysed by SDS-PAGE on NuPage
Novex 4-12% bis tris gels in MES buffer without reducing agent, and
were visualized using SimplyBlue SafeStain (Invitrogen, Carlsbad,
Calif.). Size exclusion chromatography was also used to analyze
purified proteins, and where necessary, aggregated material was
removed on either a Superdex 75 10/300GL or Superdex 200 10/300GL
column (GE Healthcare, Piscataway, N.J.), to a final level below
10% of total protein. An Acrodisc unit with a Mustang E membrane
(Pall Corporation, Port Washington, N.Y.) was used as indicated by
the manufacturer to remove endotoxin from bacterially expressed
protein preparations.
TABLE-US-00006 TABLE 6 Yield After Purification of Representative
Multivalent Tn3 Protein Formats Protein (Clone) Yield (mg/L) A1
(1E11) 400 A2 (1E11) 300 A3 (1E11) 135 A4 (1E11) 90 A5 (1E11)
40
Example 3
TRAIL R2Binding Affinity for Mono- and Polyvalent Tn3 Proteins
[0682] To measure the effect of Tn3 valency on binding affinity for
a series of TRAIL R2-specific Tn3 proteins, a competition ELISA
experiment was performed. A 96-well NUNC MaxiSorp plate (Thermo
Fisher, Rochester, N.Y.), was coated with A9(1C12) (SEQ ID NO:
154+SEQ ID NO: 145) a TRAIL R2 specific scaffold in an
antibody-like format, in PBS at 2 .mu.g/ml overnight at 4.degree.
C. Plates were blocked with PBS 0.1% Tween 20+10 mg/ml BSA.
Dilutions of A1 (1E11 monomer), and linear format A2 (1E11
bivalent) or A3 (1E11 tetravalent) multimeric scaffolds were
incubated on the coated plate with 0.75 nM of biotinylated TRAIL
R2-Fc for two hours at room temperature in PBS 0.1% Tween 20+1
mg/ml BSA, washed. Bound biotinylated TRAIL R2Fc was detected with
streptavidin HRP, TMB, and neutralized with acid. Absorbance was
read at 450 nm. Data is shown in FIG. 4. Binding affinities
(IC.sub.50) are shown in TABLE 7 and were calculated as the
concentration of competing protein required to reduce maximal
binding of biotinylated TRAIL R2-Fc by 50%.
[0683] The IC.sub.50 values for A2 and A3 were at least 30-fold
lower than those of the monomer A1 and are at the limit of this
assay (i.e., approx. equal to the concentration of biotinylated
TRAIL R2-Fc) Binding of biotinylated TRAIL R2-Fc to immobilized
TRAIL R2-specific Tn3 was displaced by the TRAIL R2 binding
constructs.
[0684] Relative to the monomeric A1 protein, the bi- and
tetravalent A2 and A3 proteins bound TRAIL R2-Fc with 30-40-fold
higher affinity, which is an indication that the multiple Tn3
modules retain their binding activity and contribute to higher
affinity through an avidity effect. The true difference in affinity
between mono- and bi- or tetravalent Tn3 proteins may be greater
than 30-40-fold given the IC.sub.50 values for A2 and A3 were
approximately equal to the concentration of biotinylated TRAIL
R2-Fc used in the assay (0.75 nM).
TABLE-US-00007 TABLE 7 IC.sub.50 Values for the Inhibition of
Binding of TRAIL R2-Fc to immobilized TRAIL R2 Binding A9(1C12) Tn3
Protein Clone Valency IC.sub.50 (nM) A1 (1E11) 1 16 A2 (1E11) 2 0.5
A3 (1E11) 4 0.4
Example 4
Flow Cytometry for Confirmation of Cell Binding
[0685] Flow cytometry was used to confirm the specificity of
binding of a multivalent TRAIL R2-specific Tn3 protein to
endogenous TRAIL R2 expressed on the cell surface of H2122 cells.
Adherent H2122 cells (a non-small cell lung cancer adenocarcinoma
cell line), were detached from tissue culture flasks using Accutase
(Innovative Cell Technologies, San Diego, Calif.). Cells were
rinsed with complete medium (RPMI 1640 medium supplemented with 10%
FBS) and resuspended in PBS/2% FBS at approximately
2.times.10.sup.6 cells/mL. Tn3 protein A9(1E11) (SEQ ID NO: 158+SEQ
ID NO: 159), a tetravalent antibody-like format multimeric
scaffold, or the format-matched control Tn3 protein B9 (clone D1),
were prepared at 40 nM concentrations in PBS/2% FBS.
[0686] Cells were plated on 96 well U-bottom plates at 75 .mu.l per
well, and protein samples were added at 25 .mu.l per well (to a
final concentration of 10 nM). The plate was incubated at 4.degree.
C. for approximately 1 hour, then washed 3 times with PBS/2% FBS.
Anti-human IgG Alexa Fluor 488 conjugated secondary antibody added
was added (100 .mu.l/well), and the plate was incubated at
4.degree. C. for approximately 30 minutes and washed as described
above. Cells were resuspended in 100 .mu.l of PBS/2% FBS, and flow
cytometry analysis was performed using a BD LSR II cytometer (BD
Biosciences, San Jose, Calif.). A shift (increase) in fluorescently
labeled H2122 cells when incubated with the TRAIL R2 specific Tn3
protein relative to control confirmed that the TRAIL R2 specific
Tn3 protein could bind to cellular TRAIL R2 (FIG. 5).
Example 5
Effect of Valency and Format on Apoptosis of H2122 Cells by TRAIL
R2-specific Tn3 Proteins
[0687] Apoptotic cell death can be induced in cancer cells lines by
crosslinking of cell surface TRAIL R2. This effect can be
determined in cell assays that measure the number of viable cells.
To this end, lung carcinoma cell lines H2122 cells were plated in
96 well plates at a density of 10,000 cells/well in 75 .mu.l of
complete medium (RPMI 1640 medium supplemented with 10% FBS).
Following overnight incubation at 37.degree. C., media was
supplemented with 25 .mu.l of additional media containing a serial
dilution of TRAIL R2-specific (clone 1E11) or negative control
(clone D1) Tn3 proteins. All treatments were performed in duplicate
wells. Commercially available TRAIL ligand (Chemicon/Millipore,
Billerica, Mass.) was used as a positive control for TRAIL
receptor-induced cell death. After 72 hrs, the CellTiter-Glo kit
from Promega (Madison, Wis.) was used according to the
manufacturer's instructions to assay ATP levels, which is a measure
of the number of viable cells in the culture. Assay luminescence
was measured on an Envision Plate reader (PerkinElmer, Waltham,
Mass.). Inhibition of cell viability was determined by dividing the
luminescence values for treated cells by the average luminescence
for untreated viable cells. Dose response plots of inhibition vs
compound concentration were generated, and cell killing potency
(EC.sub.50) was determined as the concentration of protein required
to inhibit 50% of the cell viability.
[0688] To test the effect of valency on the proapoptotic activity
of multivalent TRAIL R2-specific Tn3 proteins, H2122 cells were
treated with the monovalent Tn3 protein A1 (clone 1E11), and the
series of linearly fused Tn3 proteins A2-A5 (each clone 1E11) which
contain 2, 4, 6 or 8 Tn3 modules. While the mono- and bivalent Tn3
proteins showed no or negligible killing activity, proteins
containing 4, 6 and 8 Tn3 modules potently inhibited H2122 cell
viability, with potency increasing as a function of valency (FIG.
6A; TABLE 8). Protein A3 (tetravalent) had a similar potency to
TRAIL, the natural TRAIL R2 ligand, while proteins A4 (hexavalent)
and A5 (octavalent) were 1-2 logs more potent. It is clear from
this assay that for a given molecular format, cell killing improves
with higher valency, up to a point where the assay can no longer
discriminate.
TABLE-US-00008 TABLE 8 EC.sub.50 Values for Killing of H2122 by
Multivalent Constructs Clone EC.sub.50 (nM) Maximum Inhibition % A3
(1E11) 0.013 91 A4 (1E11) 0.0009 97 A5 (1E11) 0.0006 97 human TRAIL
0.027 98
[0689] To demonstrate that inhibition of cell viability is
dependent on TRAIL R2 binding, 100 pM of protein A5 (clone G6)
(i.e., 167.times. the EC.sub.50) was incubated with H2122 cells in
the presence of soluble TRAIL R2-Fc protein. Dose dependent
repression of cell killing by soluble TRAIL R2-Fc is an indication
that cell killing is dependent on protein A5 binding to cell
surface TRAIL R2 (FIG. 6B). Similar results were seen with protein
A5 comprising clone 1E11 loops (data not shown).
[0690] In addition to the number of binding modules, the activity
of multivalent Tn3 proteins may also be affected by the molecular
format used to present the individual binding units. To test the
effect of molecular format on activity, H2122 cells were treated
with different TRAIL R2-specific Tn3 proteins presenting the same
number of Tn3 binding modules. The ability of the tetravalent
proteins A3, A7 and A9 (each clone 1E11) to induce killing of H2122
cells was tested in the cell viability assay, as was the pair of
octavalent Tn3 proteins A5 and A8 (each clone 1E11). Inactive mono-
and bivalent proteins were included as negative controls, and TRAIL
as a positive control (FIG. 7; TABLE 9 and TABLE 10). In FIG. 7A,
for the three constructs tested with a valency of four, it is
apparent that A3 (linear format) and A7 (Fc-fusion format) are
similar in their cell killing activity and are more potent in
killing H2122 cells than A9 (antibody-like fusion format). This
clearly shows that the spatial orientation of Tn3 modules can have
a considerable effect on bioactivity, wherein A3 is approximately
150-fold more potent than A9 protein in inhibiting 112122 cell
viability (TABLE 9). FIG. 7B shows that both formats of octavalent
TRAIL R2-binding Tn3 proteins, A5 (linear) and A8 (Fc-fusion), have
similar efficacy in inhibiting the viability of H2122 cells. The
EC.sub.50 data for these constructs is shown in TABLE 9. The
ability to fine tune affinity, valency, and spatial orientation
affords great flexibility in terms of the ability to precisely
engineer a desired therapeutic outcome.
TABLE-US-00009 TABLE 9 EC.sub.50 Values for Killing of H2122 by
Multivalent Constructs with a Valency of Four Clone EC.sub.50 (nM)
Maximum Inhibition % A9 (1E11) 1.98 80 A7 (1E11) 0.02 88 A3 (1E11)
0.013 91 human TRAIL 0.027 98
TABLE-US-00010 TABLE 10 EC.sub.50 Values for Killing of H2122 by
Multivalent Constructs with a Valency of Eight Maximum Clone
EC.sub.50 (nM) Inhibition % A5 (1E11) 0.0006 97 A8 (1E11) 0.0002 98
human TRAIL 0.027 98
Example 6
Dose Dependent Cell Killing in the Cell Lines Colo205 and
Jurkat
[0691] To demonstrate that multivalent TRAIL R2-specific Tn3
proteins could kill cancer cell lines other than H2122, other TRAIL
R2 expressing cell lines were also tested. The colorectal
adenocarcinoma cell line Colo205 (FIG. 8A) and Jurkat T cell
leukemia line (FIG. 8B) were tested for their ability to be killed
by proteins A3 (tetravalent, linear format) (SEQ ID NO: 143) and A5
(octavalent, linear format) (SEQ ID NO: 145) (each clone G6). Each
cell line was incubated with A3, A5, the positive control TRAIL, or
a negative control protein B5 (SEQ ID NO: 148) which does not bind
TRAIL R2, and the cell viability assay was performed as described
for 142122. In each of these cell lines, A5 shows extremely potent
inhibition of cell viability. The lower valency A3 protein also
induces cell killing, albeit with lower potency than A5. Thus, the
higher valency construct shows greater activity. As expected, TRAIL
could also inhibit cell viability, but not octavalent negative
control protein B5, which does not bind TRAIL R2.
TABLE-US-00011 TABLE 11 EC.sub.50 Values for Killing of Colo205 by
Linear Tandem Constructs Clone EC.sub.50 (nM) Maximum Inhibition %
A3 (G6) 0.04 97 A5 (G6) 0.0005 100 human TRAIL 0.08 100
TABLE-US-00012 TABLE 12 EC.sub.50 Values for Killing of Jurkat
cells by Linear Tandem Constructs Maximum Clone EC.sub.50 (nM)
Inhibition % A3 (G6) 0.05 83 A5 (G6) 0.0001 100 human TRAIL 0.009
99 Cells were analyzed by the CellTiter-Glo assay as in Example
5.
Example 7
Design, Expression, and Activity of Mouse CD40L-Specific Bivalent
Tandem Scaffolds
[0692] Bivalent murine CD40L-specific Tn3 proteins (TABLE 13) were
prepared by fusing a pair of identical Tn3 modules. M13 is a Tn3
protein that specifically binds Murine CD40L. The M13 sequence
corresponds to the sequence of Tn3 wherein the sequences of the BC,
DE, and FG loops are replaced with alternative loops with sequences
corresponding to SEQ ID NOs: 100, 104, and 110, respectively (see
TABLE 4). Linkers containing 1 (Construct Cl(M13)), 3 (Construct
C2(M13)), 5 (Construct C3(M13)), or 7 (Construct C4(M13)) copies of
the Gly.sub.4Ser (GS) unit were used resulting in total linker
lengths between 13 and 43 amino acids (see FIG. 9A and TABLE
3).
TABLE-US-00013 TABLE 13 Names, valencies, and specificities of
expressed Tn3-containing proteins Number of Linker Name (clone) Tn3
modules length Specificity M13 (M13) 1 N/A Murine CD40L C1 (M13) 2
13 Murine CD40L C2 (M13) 2 23 Murine CD40L C3 (M13) 2 33 Murine
CD40L C4 (M13) 2 43 Murine CD40L
[0693] Briefly, the expression constructs were generated as
follows: Fragment A was generated by PCR amplification of Murine
CD40L binder pSec-M13 cloned in the pSec-oppA(L25M) vector
described in Example 1 with a primer specific for the pSec vector
upstream of the Tn3 gene and primer "1-3 GS linker reverse" (SEQ ID
NO: 123) (see TABLE 14 for sequences of Tn3 specific primers used).
Fragments B1 GS and B3GS were generated by PCR amplification of the
same template with primers "1 GS linker" (SEQ ID NO: 121) or "3
Glinker" (SEQ ID NO: 122), respectively, and a primer specific for
the pSec vector downstream of the Tn3 gene. Upon gel-purification
of the fragments, Fragment A and B1GS or Fragment A and B3GS were
mixed, and the tandem constructs were generated by overlap PCR in a
PCR reaction with the two pSec vector specific primers. The
products were digested with NcoI and KpnI and cloned back into the
pSec-oppA(L25M) vector as described in Example 1, yielding the two
constructs: C1(M13) and C2(M13). In order to generate the 5 and 7
GS linker constructs, linker inserts generated by PCR amplification
of the oligonucleotides "5 GSLinker" (SEQ ID NO: 124) and "7
GSLinker" (SEQ ID NO: 125), respectively, with primers "GS L Amp
forward" (SEQ ID NO: 126) and "GS L Amp reverse" (SEQ ID NO: 127)
were digested with PstI and XmaI and cloned into a vector fragment
generated by cutting pSecM13-1GS-M13 with PstI and XmaI yielding
the constructs C3(M13) and C4(M13).
TABLE-US-00014 TABLE 14 Primer sequences used in the construction
of Tandem bivalent MuCD40L specific constructs Sequence Name
Sequence SEQ ID NO 1 GSLinker
AAAGAAACCTTTACCACTGCAGGTGGCGGAGGTTCACGCTTG 121
GATGCCCCCGGGCAGATTGAAGTGAAAGATGTGACCGAT 3 GSLinker
AAAGAAACCTTTACCACTGCAGGTGGCGGAGGTTCAGGTGGC 122
GGAGGTTCAGGTGGCGGAGGTTCACGCTTGGATGCCCCCGGG
CAGATTGAAGTGAAAGATGTGACCGAT 1-3 GSlinker reverse
CTGCAGTGGTAAAGGTTTCTTTCG 123 5 GSLinker
AAAGAAACCTTTACCACTGCAGGTGGCGGGGGTAGCGGTGGC 124
GGAGGTTCTGGTGGCGGGGGTAGCGGTGGCGGAGGTTCTGGT
GGCGGGGGTAGCCGCTTGGATGCCCCCGGGCA 7 GSLinker
AAAGAAACCTTTACCACTGCAGGTGGCGGGGGTAGCGGTGGC 125
GGAGGTTCTGGTGGCGGGGGTAGCGGTGGCGGAGGTTCTGGT
GGCGGGGGTAGCGGTGGCGGAGGTTCTGGTGGCGGGGGTAGC CGCTTGGATGCCCCCGGGCA GS
L Amp forward AAAGAAACCTTTACCCACTGCAGGT 126 GS L Amp reverse
TTCAATCTGCCCGGGGGCATCCAA 127
[0694] Monovalent and bivalent tandem constructs comprising
identical Tn3 scaffolds were recombinantly expressed and purified
from E. coli as described in Example 2. FIG. 9B depicts an SDS-PAGE
analysis of the purified protein preps under reducing and
non-reducing conditions.
[0695] In order to test the binding efficiencies of the bivalent
tandem M13-M13 constructs and compare them to the monovalent M13
scaffold, their competitive inhibition of Murine CD40L binding to
Murine CD40 receptor immobilized on a biosensor chip was
tested.
[0696] Briefly, a fragment of the Murine CD40 receptor in the form
of a chimeric fusion with the Fc region of IgG1 was immobilized
onto a GLC chip (Bio-Rad) at a density of about 3000 response
units. For competition binding assays, 3-fold serial dilutions of
monovalent M13 or the M13 tandem bivalent constructs with different
linker length were incubated for 20 min with a fixed concentration
of E. coli produced recombinant Murine CD40L (0.5 .mu.g/ml) in PBS
containing 0.1% (v/v) Tween-20 and 0.5 mg/mL BSA. These samples
were then injected over the GLC chip at a flow rate of 30 .mu.L/min
for 300 seconds and the level of bound CD40L was recorded at a
fixed time point within the sensorgram and compared to the
corresponding level of bound protein in the absence of any
competitor. After each binding measurement, residual CD40L was
desorbed from the chip surface by injecting 10 mM glycine-HCl (pH
2.0). Non-specific binding effects were corrected by subtracting
sensorgrams from interspots of the chip. IC.sub.50 values
corresponding to the concentrations of Tn3 constructs required to
displace 50% of murine CD40L were calculated using GraphPad
Prism.
[0697] As shown in FIG. 9C, the half maximal inhibitory
concentration (IC.sub.50) for the M13 monomer was 71 nM while the
IC.sub.50 for the bivalent tandem construct C1 (M13) was 29 nM.
Similar IC.sub.50 values of 5 or 6 nM were obtained for the
bivalent constructs containing longer linkers (constructs C2(M13),
C3(M13) and C4(M13), respectively). Due to the concentration of
CD40L used in the assay, this is at the lower limit of IC.sub.50s
that can be observed in this assay. The bivalent constructs all had
a lower IC.sub.50 value compared to the monovalent construct,
indicating enhanced binding activity of the bivalent tandem
constructs compared to a single M13 Tn3 module. The linker length
in these bivalent constructs exhibits some effect on assay potency,
with the shortest linker length construct having intermediate
potency, while those constructs with linkers of 23 or more amino
acids are equivalent in this assay.
[0698] To test the activity of the bivalent tandem Tn3 constructs
in a cell based activity and compare them to the monovalent M13
scaffold, inhibition of Murine CD40L-induced CD86 expression on
B-cells was tested. As a control, the commercially available
anti-murine CD40L specific antibody (MR1) was tested in
parallel.
[0699] The assay utilizes PBMC prepared from blood from healthy
volunteers. Briefly, freshly drawn blood was collected in BD
Vacutainer.RTM. CPT.TM. Cell Preparation Tube with heparin. After
centrifugation, the cell layer containing PBMCs was collected and
washed twice with PBS and once with RPMI 1640 medium. The cells
were resuspended in complete RPMI 1640 medium (supplemented with
10% heat-inactivated fetal bovine serum, 1% P/S) at a concentration
of 5.times.10.sup.6 cells/ml.
[0700] The murine CD40L-expressing Th2 cell line D10.G4.1 was
washed and resuspended in complete RPMI 160 medium at a
concentration of 1.times.10.sup.6 cells/ml.
[0701] M13, M13-M13 tandem bivalent constructs C.sub.1-C.sub.4, or
MR1 antibody (BioLegend Cat. No: 106508) were serially diluted
(1:3) in complete RPMI 1640 medium. A 50 .mu.l sample of each
dilution was added to wells in a 96 well U bottom tissue culture
plate. Each well then received 50 .mu.l of D10.G4.1 cells
(5.times.10.sup.4), and after mixing, plates were incubated at
37.degree. C. for 1 hr. 100 .mu.l of resuspended PBMC
(5.times.10.sup.5 cells) were then added to each well and incubated
at 37.degree. C. for 20-24 hrs.
[0702] PBMC were collected and stained with APC-anti-human CD86 (BD
bioscience, Cat #555660) and FITC-anti-human CD19 (BD bioscience,
Cat #555412) in FACS buffer (PBS pH 7.4, 1% BSA, 0.1% sodium azide)
at 4.degree. C. for 30 min in the dark. After two washes in FACS
buffer, samples were then analyzed by FACS LSRII (Becton
Dickinson). CD86 expression on CD19 gated B cells was evaluated.
The analysis of CD86 expression as a function of test protein was
performed using GraphPad Prism software.
[0703] As shown in FIG. 9D, the bivalent M13-M13 tandem constructs
all inhibited CD86 expression with an IC.sub.50 of 100 to 200 pM,
comparable to the IC.sub.50 of the MR1 antibody (100 pM) and about
3 logs more potent than the M13 monovalent scaffold itself. In
contrast to the biochemical assay, no effect of linker length was
observed in this cell based assay, and bivalent constructs with
linkers ranging from 13 to 43 amino acids in length all show
equivalent enhanced potency relative to the monovalent protein.
Example 8
Expression of Bi-Specific Tandem Scaffolds
[0704] To generate bispecific Tn3 constructs with specificity for
TRAIL R2 and Human CD40L (HuCD40L), two Tn3 modules, one with
specificity for TRAIL R2 (clone 1E11) and one with specificity for
human CD40L (clone 79), were fused together with variable length
linkers separating the two modules (TABLE3 and TABLE 15). The
sequence of the clone 79 protein (SEQ ID NO: 184) corresponds to
the sequence of a Tn3 module wherein the BC, DE, and EF loops have
been replaced with alternative loops corresponding to SEQ ID NOs:
101, 105, and 111, respectively. Expression constructs for the
tandem bispecific scaffolds containing linkers with 1 and 3
Gly.sub.4Ser (GS) repeats (constructs C6 and C8, respectively) were
generated as described in Example 7 except that plasmids carrying
the Tn3 variants A1 and 79 were used initially as PCR templates.
Construct C5 (containing a short linker derived from the natural
sequence linking the second and third FnIII domains in human
tenascin C, which may be considered part of the A beta strand of
the third FnIII domain although it is not required for scaffold
binding) and construct C7 were generated in a similar way to C6 and
C8, using the additional primers listed in TABLE 17, except that "0
GSlinker reverse" was used in place of "1-3 GSLinker reverse" for
C5.
TABLE-US-00015 TABLE 15 Names, valencies, and specificities of
expressed Tn3-containing proteins Number of Tn3 Linker Name modules
length Specificity A1 (1E11) 1 N/A TRAIL R2 79 (79) 1 N/A HuCD40L
C5 (1E11 & 79) 2 8 TRAIL R2 + HuCD40L C6 (1E11 & 79) 2 13
TRAIL R2 + HuCD40L C7 (1E11 & 79) 2 18 TRAIL R2 + HuCD40L C8
(1E11 & 79) 2 23 TRAIL R2 + HuCD40L
TABLE-US-00016 TABLE 17 Additional Primer sequences used in the
construction of bispecific tandem constructs Sequence Name Sequence
SEQ ID NO 0 GSLinker AAAGAAACCTTTACCACCACGCGTTTGGATGCCCCCGGGCAGATTG
128 AAGTGAAAGATGTGACCGAT 0 GSlinker reverse CGTGGTGGTAAAGGTTTCTTTCG
129 2 GSLinker AAAGAAACCTTTACCACTGCAGGTGGCGGAGGTTCAGGTGGCGGAG 130
GTTCACGCTTGGATGCCCCCGGGCAGATTGAAGTGAAAGATGTGAC CGAT
[0705] Monovalent as well as tandem bispecific Tn3 scaffolds were
recombinantly expressed in E. coli media as described in Example 2.
Expression levels of the soluble constructs were analyzed using
SDS-PAGE. FIG. 10 demonstrates acceptable expression levels for the
constructs tested.
Example 9
Specific Binding of BiSpecific Tandem Scaffolds
[0706] To measure the binding of bispecific Tn3 constructs to CD40L
and TRAIL R2, a capture ELISA assay was employed. Briefly,
8.times.His-tagged protein constructs: A1, 79, C5, C6, C7 or C8
(see TABLE 15 for details) were captured from E. coli media onto
anti-His antibody coated wells as follows. A 96-well MaxiSorb plate
was coated with Qiagen anti-His antibody at 2 .mu.g/ml overnight.
The coated plate was blocked with PBS containing 0.1% v/v Tween-20
and 4% w/v skim milk powder (PBST 4% milk) for 1.5 hours. The
coated plate was washed with PBST and diluted bacterial media
(diluted 30-fold) containing soluble expressed proteins was added
and plates were incubated at room temperature for 2 hours. After
washing with PBST, wells containing the captured constructs were
incubated for 1.5 hours with varying concentrations of either
biotinylated TRAIL R2 (FIG. 11A) or a complex generated by
preincubation of E. coli produced His-tagged HuCD40L with
biotinylated anti-His antibody (FIG. 11B). After washing with PBST,
bound TRAIL R2 or HuCD40L/anti-H is antibody complex was detected
with streptavidin-horseradish peroxidase (RPN1231V; GE Healthcare;
1000.times.working dilution) for 20 min., washing with PBST, and
detecting colorimetrically by addition of TMB substrate (Pierce).
The absorbance was read at 450 nm.
[0707] Binding of the bispecific tandem TRAIL R2-HuCD40L-specific
scaffolds to TRAIL R2, and binding of the bispecific tandem TRAIL
R2-HuCD40L-specific scaffolds to HuCD40L are depicted in FIG. 11A
and FIG. 11B, respectively. Bispecific tandem scaffolds, designated
C5 to C8, comprising a TRAIL R2 specific Tn3 domain fused to a
HuCD40L specific Tn3 domain bound TRAIL R2 and HuCD40L; however,
the monomeric/monospecific Tn3 constructs A1 and 79 bound either
TRAIL R2 or HuCD40L according to their known specificities but not
both targets.
[0708] Simultaneous binding of tandem TRAIL R2-HuCD40L-specific
constructs to TRAIL R2 and HuCD40L was determined using an
AlphaScreen.TM. assay. Dilutions of E. coli media containing
proteins A1, 79, C5, C6, C7 and C8 were incubated with 10 nM TRAIL
R2-Fc fusion protein, 50 nM biotinylated HuCD40L (produced in E.
coli), streptavidin AlphaScreen donor beads (0.02 mg/ml) and
Protein A AlphaScreen acceptor beads (0.02 mg/ml) in PBS+0.01%
Tween+0.1% BSA. Samples were incubated 1 h in the dark prior to
reading in a PerkinElmer Envision reader. The donor bead population
was excited with a laser at 680 nm causing the release of singlet
oxygen. Singlet oxygen has a limited lifetime allowing it to travel
up to 200 nm by diffusion before falling back to ground state.
Singlet oxygen excites the acceptor beads causing light emission
between 520-620 nm which is measured by the Envision reader. Only
when donor and acceptor beads are in proximity is a signal
generated. Thus, an increase in signal is observed when the two
bead types are brought together by molecules interacting with the
two targets simultaneously. In the absence of binding to either
target no signal should be detected.
[0709] As shown in FIG. 12, the tandem bispecific constructs
simultaneously bound TRAIL R2 and HuCD40L generating a strong
AlphaScreen signal; however, the monovalent Tn3 scaffolds, A1 and
79, did not generate a signal indicating they could not bring donor
and acceptor beads in proximity by simultaneously binding both
targets.
Example 10
Increased Stability of Tn3 Scaffolds Having 9 Amino Acid Length FG
Loop
[0710] To measure the effect of FG loop length on Tn3 stability,
unfolding of six
[0711] HuCD40L-specific Tn3 scaffolds by guanidine hydrochloride
(GuHCl) at pH 7.0 was assessed by intrinsic tryptophan
fluorescence. These Tn3 monomeric scaffolds contained FG loop
lengths of 9, 10 or 11 amino acids. Samples of 0.05 mg/mL Tn3
scaffold containing different concentrations of guanidine
hydrochloride were prepared in 50 mM sodium phosphate pH 7.0.
Fluorescence emission spectra were acquired on a Horiba Fluoromax-4
spectrofluorometer at an excitation wavelength of 280 nm. Relative
fluorescence emission intensity at 360 nm was plotted as a function
of GuHCl concentration for each protein. Each scaffold contained
unique BC, DE, and FG loop sequences. Clones A3 (SEQ ID NO:185;
note that the A3 monomeric scaffold in this example is distinct
from the construct designated A3 as provided in Table 3), 71 (SEQ
ID NO: 186), 79 (SEQ ID NO: 184), 127 (SEQ ID NO: 187), 252 (SEQ ID
NO: 188), and 230 (SEQ ID NO: 189) were more than 50% unfolded in
3.0M GuHCl at pH 7.0, which is the GuHCl concentration required to
effect 50% unfolding (C.sub.m) of parental Tn3. C.sub.m values for
clones A3, 79, 127, 252, and 230 were 2.2M, 2.7M, 2.4M, 2.7M, 2.4M,
respectively. The FG loop lengths for these clones is 11, 11, 11,
10 and 11 amino acids respectively, while the FG loop length for
parental Tn3 is 10 amino acids. Surprisingly, clone 71, the only
variant having an FG loop length of 9 amino acids, exhibited a Cm
of 4.2M, a significantly higher stability than parental Tn3
scaffold or the other five variants tested. Results are shown in
FIG. 13.
[0712] To determine whether the enhanced stability of Tn3 clone 71
was intrinsic to its sequence, or a consequence of the shortened FG
loops length, this clone and two additional monomeric Tn3 scaffold
proteins, (A6 (SEQ ID NO: 190; note that the A6 monomeric scaffold
in this example is distinct from the construct designated A6 as
provided in Table 3) and P1C01 (SEQ ID NO: 191)), with an FG loop
length of 9 amino acids (but different BC, DE and FG loop
sequences) were analyzed by differential scanning calorimetry (DSC)
and compared to the parental Tn3 scaffold which contains an FG loop
that is 10 amino acids long. Tn3 protein samples at 1 mg/mL in PBS
pH 7.2 were analyzed. In all cases, the midpoint of thermal
unfolding was higher for clones with the 9 residue FG loops as
compared to parental (WT) Tn3, which has a 10 residue FG loop.
Thermal unfolding was reversible, or partially reversible (clone
A6) as evidenced by superimposable thermograms when the same sample
was cooled and reheated. As shown in FIG. 14, the melting
temperature (T.sub.m) for parental Tn3 was 72.1.degree. C., for
P1C01 the Tm was 75.2.degree. C., for A6 the T. was 77.5.degree.
C., and for 71 the T. was 74.4.degree. C.
[0713] These findings were corroborated by testing the same Tn3
protein variants in a guanidine hydrochloride stability experiment.
Unfolding of parental (WT) Tn3, P1C01, A6, and 71 by guanidine
hydrochloride (GuHCl) at pH 7.0 was assessed by intrinsic
tryptophan fluorescence as described above. As shown in FIG. 15, in
agreement with the DSC data in FIG. 14, Tn3 clones A6, 71, and
P1C01 all have midpoints of unfolding at significantly higher GuHCl
concentrations than parental (WT) Tn3 scaffold, indicating the
stability of Tn3 proteins having FG loops that are 9 amino acids in
length, i.e. shorter than that in the parental Tn3 scaffold, is
enhanced.
Example 11
Stability Analysis of FG Loop Length
[0714] As described above, preliminary analysis indicated that Tn3
molecules having an FG loop length of 9 residues are significantly
more stable than those having longer FG loops. In these studies, we
conducted stability analysis on a set of random Tn3s to assess the
effect of FG loop length on thermal stability.
[0715] A Tn3 library was subcloned into the pSEC expression vector.
This library codes for Tn3s with BC, DE, and FG loops of varying
sequence as well as varying but defined length. The FG loop, which
is the focus of these studies, can be 9, 10, or 11 residues long.
The BC loop may be 9, 11, or 12 residues long. The DE loop in this
library has a fixed length of 6 residues. The subcloned library was
used to transform DH5.alpha. competent cells, from which a plasmid
pool was purified and used to transform BL21(DE3) cells. BL21
colonies were sequenced to identify 96 clones which coded for
full-length Tn3s. The final 96 clones were grown in a 96 deep-well
plate at a 500 .mu.l scale using standard Magic Media expression
(37.degree. C. shaking for 24 hours post-innoculation) and analyzed
on SDS-PAGE. 29 random clones having moderate-to-high expression
levels were scaled up to 50 mL scale expression and purified using
standard immobilized metal affinity chromatography. Identities of
all proteins were confirmed by mass spectrometry.
[0716] The random clones were analyzed for stability by DSC.
Briefly, DSC measurements were conducted on a VP-Capillary DSC
(MicroCal). Proteins were exchanged into PBS (pH 7.2) through
extensive dialysis, and adjusted to a concentration of 0.25-0.5
mg/ml for DSC analysis. Samples were scanned from 20-95.degree. C.
at a scan rate of 90.degree. C./hour, with no repeat scan. The
results are shown in TABLE 18.
TABLE-US-00017 TABLE 18 Comparison of T.sub.m values of Tn3s with
FG9 vs FG10/11 FG9 T.sub.m(.degree. C.) FG10/11 T.sub.m(.degree.
C.) A1 64.8 E12 (FG10) 65.0 A3 71.8 F5 (FG10) 60.0 B2 70.0 G1
(FG11) 64.3 B4 69.4 G4 (FG11) 67.6 C5 66.6 G8 (FG11) 64.2 C7 66.0
H6 (FG11) 70.3 C8 64.1 H7 (FG11) 71.7 C11 59.5 H8 (FG10) 61.9 D1
73.7 H9 (FG10) 59.5 D8 72.1 H10 (FG11) 67.6 D10 65.6 H11 (FG11)
63.7 D11 65.6 H12 (FG11) 65.6 D12 66.4 E1 75.0 E3 66.0 E9 75.3 E11
61.9 n = 17 n = 12 Mean 67.9 Mean 65.1
[0717] In this study, the thermal stability of Tn3s with loop
length FG9 or FG10 and 11 was compared. The trend shows that Tn3
domains having an FG loop of length 9 are more thermostable than
those with loop length FG10 or 11. A control, the wild-type Tn3
domain (with an FG loop of 10 residues) had a Tm of 72.degree. C.
when run in parallel with the above samples. The range of T.sub.m
values seen with each loop length indicates that other factors also
play a role in determining Tn3 domain thermostability.
Example 12
Generation and Characterization of a Trispecific Tn3
[0718] In these experiments, a Tn3 molecule having binding
specificity for three different targets was generated and
characterized. D1, the Tn3 domain specific for the Synagis.RTM.
antibody, was linked to 1E11, a Tn3 domain specific for TRAIL
receptor 2, and 79, a Tn3 domain specific for CD40L, respectively
(FIG. 17A). The construct was expressed in BL21(DE3) E. coli cells
and purified using standard methods (see FIG. 17B).
[0719] To confirm that the trispecific constructs were capable of
binding pairs of all three targets simultaneously, both AlphaScreen
and ELISA experiments were conducted. For AlphaScreen experiments,
trispecific Tn3, subsets of two of the three total target molecules
(one biotinylated and the other containing an antibody Fc region),
Protein A donor beads, and streptavidin acceptor beads were
combined in a 384-well white Optiplate, as described above.
AlphaScreen signal can only be observed when the streptavidin donor
bead and Protein A acceptor bead are within proximity of each other
(200 nm of each other), which in this assay is accomplished through
bridging by the trispecific molecule. The ability of D1-1E11-79 to
simultaneously bind huCD40L and TRAIL R2-Fc (FIG. 18A), and to
simultaneously bind huCD40L and Synagis.RTM. (FIG. 18B) was
confirmed by AlphaScreen as follows: in a 384-well white Optiplate,
the following components were combined in a total volume of 30
.mu.l: 20 mM purified D1-1E11-79, 50 mM biotinylated-huCD40L, (0,
1, 2.5, 5, 10, or 42 nM) TrailR2-Fc (FIG. 18A) or (0, 1, 2.5, 5,
10, or 42 nM) Synagis.RTM. (FIG. 18B), 5 .mu.l each of 1/50
dilutions of AlphaScreen Protein A acceptor beads and streptavidin
donor beads. After 1 hour incubation in the dark, the plate was
read on an Envision plate reader in AlphaScreen mode.
[0720] Because Synagis.RTM. and TRAIL R2-Fc both contain an Fc
domain, the AlphaScreen assay could not be used to demonstrate
simultaneous binding of these molecules to the trispecific
construct. In place of this, an ELISA experiment was conducted.
MaxiSorp plates were coated with TRAIL R2-Fc (100 .mu.l at 1
.mu.g/ml), blocked with 4% milk, then followed by addition of
varying concentrations of the trispecific construct. Biotinylated
Synagis.RTM., the second target ligand, was added and detected by
the addition of HRP-streptavidin (FIG. 19). of D1-1E11-79 was also
shown to be capable of binding both TRAIL R2-Fc and Synagis.RTM.
simultaneously, as indicated by the ELISA results in FIG. 19.
Therefore we can conclude that this construct can bind all three
pairs of its targets simultaneously.
Example 13
Lead Isolation
[0721] The first step in developing an agonist Tn3 is to isolate a
Tn3 monomer that can bind to TRAIL R2 and when linked into a
multivalent format can bind two or more TRAIL R2 extracellular
domains in a way that engages the apoptotic pathway. Since not all
binders may act as agonists, we decided to first isolate a panel of
binders and then screen for agonism in a secondary in vitro cell
killing assay. We first panned a large phage displayed library of
Tn3's with variation in the BC, DE, and FG loops on recombinant
TRAIL R2-Fc to isolate an initial panel of binders. The Tn3
scaffold chosen as the basis for this library was not a native
3.sup.rd FnIII domain from tenascin C but a version that had an
engineered disulfide to improve stability. An in house Tn3/gene 3
fused phage display library was constructed containing
randomization in the BC, DE, and FG loops. Multiple binders were
found by a phage ELISA in which TRAIL R2 was directly coated on a
plate and binding of 1:3 diluted phage in PBS+0.1% Tween 20 (PBST)
1% milk was detected by anti-M13-peroxidase conjugated antibody (GE
Healthcare Biosciences, Piscataway, N.J.). A majority of the
binders had an undesirable free cysteine in one of the loops and
were not chosen for further study. A subset of the clones lacking
an unpaired cysteine were cloned into expression vectors generating
either an Fc fusion or antibody-like construct (FIG. 1) and tested
in the tumor cell line H2122 for cell killing (data not shown).
Although the Fc fusion format failed to kill cells regardless of
its fused Tn3, the antibody-like format did elicit a response for
more than one binder.
Example 14
Affinity Maturation
[0722] Clone 1C12 (SEQ ID NO: 132) (see FIG. 20) showed the best
cell killing in the initial screening assays and was therefore
chosen for affinity maturation. Affinity maturation was performed
by saturation mutagenesis of portions of the loops using either
Kunkel mutagenesis or PCR with oligonucleotides containing
randomization, assembly, and ligation into the phage display
vector. Round one and three consisted of saturation mutagenesis in
parts of the BC and FG loops respectively and round 2 combined
saturation mutagenesis of parts of all three loops separately,
panning, gene shuffling, and then panning of the shuffled mutants
to obtain the highest affinity output clone. Pools of affinity
matured clones were recovered after panning by PCR directly from
the phage or by prepping the single stranded DNA using a Qiagen kit
(Qiagen, Valencia, Calif.) and then PCR. PCR products were digested
NcoI to KpnI (New England Biolabs, Ipswich, Mass.) and cloned into
our in house expression vector pSEC. The clones were expressed in
MagicMedia (Invitrogen, Carlsbad, Calif.) and run on a gel to
verify that expression did not differ greatly between clones.
Improved clones were identified by a competition ELISA in which
plates were coated with tetravalent, antibody-like 1C12 (SEQ ID
NOs: 154 and 155), and the inhibition in binding of 0.75 nM TRAIL
R2 biotin in the presence of dilutions of Tn3 in MagicMedia was
measured using streptavidin-horseradish peroxidase (GE Healthcare
Biosciences, Piscataway, N.J.). TMB (KPL, Gaithersburg, Md.) was
added and neutralized with acid. Absorbance was read at 450 nm.
[0723] Affinity measurements were performed on the ProteOn XPR36
protein interaction array system (Bio-Rad, Hercules, Calif.) with
GLC sensor chip at 25.degree. C. ProteOn phosphate buffered saline
with 0.005% Tween 20, pH 7.4 (PBS/Tween) was used as running
buffer. TRAIL R2 was immobilized on the chip and a two-fold, 12
point serial dilution of the Tn3 binders (1C12 (SEQ ID NO: 132),
1E11 (SEQ ID NO: 134), G3 (SEQ ID NO: 133), C4 (SEQ ID NO: 135),
and G6 (SEQ ID NO: 138)) were prepared in PBS/Tween/0.5 mg/ml BSA,
pH 7.4 at starting concentrations ranging from 36 .mu.M to 700 nM.
Samples of each concentration were injected into the six analyte
channels at a flow rate of 30 .mu.l/min. for 300 seconds. The
K.sub.d was determined by using the equilibrium analysis setting
within the ProteOn software. The sequences of the best clones from
each round are shown in FIG. 20. The total improvement in affinity
after three rounds of affinity maturation was almost two orders of
magnitude with the best clones having affinities in the 40-50 nM
range (TABLE 19).
TABLE-US-00018 TABLE 19 Equilibrium binding constants of monomeric
best clones from affinity maturation of lead clone 1C12 as measured
by Surface Plasmon Resonance (SPR). Round Clone K.sub.d(nM) Fold
Improvement Lead isolation 1C12 4130 .+-. 281 Affinity maturation 1
G3 422 .+-. 45 10 Affinity maturation 2 1E11 103 .+-. 9 40 Affinity
maturation 3 C4 50 .+-. 2 83 Affinity maturation 3 G6 43 .+-. 2
96
Example 15
Effect of Tn3 Affinity on Potency in Antibody-Like Format
[0724] In order to assess the effect of affinity of the individual
TN3 subunit on potency, all of the clones in TABLE 19 were
reformatted into the antibody-like construct depicted in FIG. 1. To
express the antibody-like proteins, 293F cells were transiently
transfected with the appropriate expression constructs. Harvests of
supernatant were performed on days 6 and 10 and the protein was
purified by protein A affinity chromatography. All purified
proteins were analyzed by SDS-PAGE on NuPage Novex 4-12% bis tris
gels in MES buffer without reducing agent, and were visualized
using SimplyBlue SafeStain (Invitrogen, Carlsbad, Calif.).
[0725] Size exclusion chromatography was also used to analyze
purified proteins, and where necessary, aggregated material was
removed on either a Superdex 75 10/300GL or Superdex 200 10/300GL
column (GE Healthcare, Piscataway, N.J.), to a final level below
10% of total protein. An Acrodisc unit with a Mustang E membrane
(Pall Corporation, Port Washington, N.Y.) was used as indicated by
the manufacturer to remove endotoxin from bacterially expressed
protein preparations.
[0726] H2122 cells were then tested for sensitivity to the
agonistic antibody-like constructs using a CellTiter-Glo cell
viability assay. In this assay, luminescence is directly
proportional to the levels of ATP within a given well of a 96 well
plate, which in turn is directly proportional to the amount of
metabolically active viable cells. For the H2122 cell line, cells
were plated in 96 well plates at a density of 10,000 cells/well in
75 .mu.l of complete medium (RPMI 1640 medium supplemented with 10%
FBS). Following overnight incubation at 37.degree. C., media was
supplemented with 25 .mu.l of additional media containing a serial
dilution of TRAIL R2-specific or negative control proteins. All
treatments were performed in duplicate wells. Commercially
available TRAIL ligand (Chemicon/Millipore, Billerica, Mass.) was
used as a positive control for TRAIL receptor-induced cell
death.
[0727] After 72 hours, the CellTiter-Glo kit was used according to
the manufacturer's instructions. Assay luminescence was measured on
an Envision Plate reader (PerkinElmer, Waltham, Mass.). Inhibition
of cell viability was determined by dividing the luminescence
values for treated cells by the average luminescence for untreated
viable cells.
[0728] Two variables determine potency: the concentration at which
a construct inhibits the viability of cells by 50% (EC.sub.50) and
the maximum inhibition of cell viability. FIG. 21 shows that as a
general trend, greater affinity of the Tn3 monomer leads to a lower
EC.sub.50 of the antibody-like constructs as G6 has a lower
EC.sub.50 than 1E11 and 1E11 has a lower EC.sub.50 than 1C12.
Example 16
Pharmacokinetics of Linear Tn3's
[0729] To determine the half life of the linear Tn3 tandems as a
function of the number of Tn3 modules per tandem, the G6 monomer
(SEQ ID NO: 138), G6 tandem 4 (SEQ ID NO: 143), G6 tandem 6 (SEQ ID
NO: 192), and G6 tandem 8 (SEQ ID NO: 145) were injected into a
mouse and serum concentration of the Tn3s was monitored by an
ELISA. The route of administration was intraperitoneal (IP)
injection. The experimental design is shown in TABLE 20. Mice were
bled 150 .mu.l per time point. Tn3's were detected in serum by an
ELISA in which in house produced TRAIL R2 coated plates were
incubated with serum diluted in PBST 1% milk. Initial ELISAs were
performed to determine for a given time point the correct dilution
range in order for the signal to be within the dynamic range of the
assay. Bound Tn3 was detected with a 1 in 1,000 dilution of
polyclonal anti-Tn3 serum from rabbit in PBST 1% milk (Covance,
Princeton, N.J.) followed by a 1 in 10,000 dilution in PBST 1% milk
of donkey anti-rabbit HRP (Jackson ImmunoResearch, West Grove,
Pa.). For each construct, a standard curve was made. Statistical
analysis was performed using an in house statistical program.
[0730] The term "maximum plasma concentration" ("C.sub.max") refers
to the highest observed concentration of tandem Tn3 in plasma
following administration of the test material to the patient.
[0731] The term "T.sub.max" refers to the time to maximum plasma
concentration C.sub.max.
[0732] The term "area under the curve" ("AUC") is the area under
the curve in a plot of the concentration of tandem Tn3 in plasma
against time. AUC can be a measure of the integral of the
instantaneous plasma concentrations (C.sub.p) during a time
interval and has the units of mass*time/volume. However, AUC is
usually given for the time interval zero to infinity. Thus, as used
herein "AUC.sub.inf" refers to an AUC from over an infinite time
period.
[0733] The term "biological half-life" ("T.sub.1/2") is defined as
the time required for the plasmatic concentration of tandem Tn3 to
reach half of its original value.
[0734] The term "CL/F" refers to the apparent total body clearance
calculated as Dose/AUC.sub.inf.
[0735] Tn3 biological half-life (T.sub.1/2) increases with
increasing number of tandem Tn3's per linear molecule. Adding seven
Tn3's to make a tandem 8 from a monomer increased the half life by
almost 50%. Increases in valency did not affect the T. However,
increases in valency from 1 to 8 resulted in approximately ten-fold
and 7-fold increases in C.sub.max and AUC.sub.inf, respectively.
Furthermore, when valency increase from 1 to 8, an approximately
7-fold decrease in clearance (CL/F) was observed.
TABLE-US-00019 TABLE 20 Experimental design of anti-TRAIL R2 linear
tandem pharmacokinetic assay. Group # Test Dose Route Volume Time
points # Mice 1 G6 10 mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr), (3)
(3) (3) monomer (30 min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr) 2 G6 10
mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr), (3) (3) (3) tandem 4 (30
min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr) 3 G6 10 mg/kg IP 10 ml/kg (15
min, 1 hr, 16 hr), (3) (3) (3) tandem 6 (30 min, 4 hr, 24 hr) (2
hr, 6 hr, 48 hr) 4 G6 10 mg/kg IP 10 ml/kg (15 min, 1 hr, 16 hr),
(3) (3) (3) tandem 8 (30 min, 4 hr, 24 hr) (2 hr, 6 hr, 48 hr)
Total 36
TABLE-US-00020 TABLE 21 Pharmacokinetic properties of Tandem Tn3's
Pharmacokinetic Parameters Test C.sub.max T.sub.max AUC.sub.inf
T.sub.1/2 CL/F Material (.mu.g/mL) (hr) (hr .mu.g/mL) (hr)
(mL/hr/kg) G6 monomer 3.65 1 9.31 1.22 1070 G6 tandem 4 8.07 1 23.2
1.46 431 G6 tandem 6 24.6 1 36.5 1.69 274 G6 tandem 8 38.6 1 64.2
1.76 156
Example 17
Engineered Enhancement of Cyno Cross-reactivity
[0736] For pre-clinical toxicity testing in cynomolgus monkeys
(Macaca fascicularis), it is desirable to develop an anti-TRAIL
R2-Tn3 that cross reacts with cynomolgus TRAIL R2 (cyno TRAIL R2).
Our initial affinity matured lead clones had poor cross reactivity
with cyno TRAIL R2, although the homology to human TRAIL R2 is 88%.
The cross reactivity was enhanced by making a library based upon
clone F4 (SEQ ID NO: 137), which was the clone with the best cyno
cross reactivity among the clones that resulted from affinity
maturation.
[0737] Two libraries were made by saturation mutagenesis: one with
diversity in the FG loop alone and one with diversity in the BC and
FG loops. A low error rate mutagenic PCR was also used to allow for
mutations outside the loops that may be beneficial for enhanced
cyno TRAIL R2 binding. Four rounds of phage panning were done on in
house produced cyno TRAIL R2, and outputs were cloned into the pSEC
expression vector. For screening of initial hits in an ELISA
format, Tn3's were secreted into MagicMedia (Invitrogen, Carlsbad,
Calif.) and were captured from supernatant using an anti-his tag
antibody (R and D Systems, Minneapolis, Minn.).
[0738] Binding of either human or cyno TRAIL R2-Fc in solution to
captured Tn3 was detected by anti-human-Fc-HRP. Clones that had
significant binding to cyno TRAIL R2-Fc and did not appear to lose
binding to human TRAIL R2-Fc were selected for a subsequent
screening ELISA in which either human or cyno TRAIL R2-Fc was
coated on a plate and Tn3 supernatants were titrated and then
detected with anti-his tag HRP. Because the level of variation in
expression levels from clone to clone was low, and also because
avidity from having divalent TRAIL R2-Fc in solution could not mask
differences in Tn3 affinity, this ELISA allowed for affinity
discrimination. It was found that one mutation, a mutation from D
to G two amino acids before the DE loop, was present in all
engineered cyno cross reactive clones (FIG. 23A). This D to G
mutation was engineered into the original F4 to make a clone named
F4 mod 1 (SEQ ID NO: 193) and the cross reactivity for cyno was
greatly improved without sacrificing binding to human TRAIL R2
(FIG. 23B). In this ELISA, inhibition of binding of 0.75 nM of
human or cyno TRAIL R2-Fc to F4 mod 1 coated plates by purified F4
or F4 mod 1 was measured.
[0739] It is desired that the binding of a cyno cross reactive
enhanced clone to cyno TRAIL-R2-Fc be within tenfold of its binding
to human TRAIL R2-Fc. Also, it is desired that the binding of a
cyno cross reactive enhanced clone to cyno TRAIL-R2-Fc be within
tenfold of the binding of F4 to human TRAIL R2-Fc. The IC.sub.50
for F4 mod 1 binding to cyno TRAIL R2 differs by less than three
fold from the IC.sub.50 for F4 mod 1 binding to human TRAIL R2. In
addition, the IC.sub.50 for F4 mod 1 binding to human TRAIL R2 is
six-fold stronger than the IC.sub.50 for F4 binding to human TRAIL
R2. Accordingly, F4 mod 1 meets the intended cross reactivity
requirements.
Example 18
Germline Engineering of Enhanced Cyno Cross Reactive Clone
[0740] Clone F4 mod 1 was further engineered to eliminate non
essential mutations from germline in order to reduce possible
immunogenicity risk. A panel of twelve different modifications was
made to determine if there was an effect from a given mutation on
the binding to both human and cyno TRAIL R2. FIG. 24A shows a
comparison of the final clone F4 mod 12 (SEQ ID NO: 194), which
incorporates all tested germline mutations that do not affect
binding, to other constructs, namely the Tn3 germline, the original
F4 parent, and clone F4 mod 1 (initial enhanced cyno cross reactive
engineered).
[0741] The amino acid sequence of F4 mod 12 starts with the native
Tn3 sequence SQ, ends with L, has a reversion of the framework 2
mutation from A to T, and has a reversion of the final two amino
acids of the DE loop from TA to NQ. FIG. 24B shows that F4, F4 mod
1, and F4 mod 12 all are within six-fold of each other in their
binding to human TRAIL R2. It also shows that F4 mod 1 and F4 mod
12 are within twofold of each other in their binding to cyno TRAIL
R2.
[0742] F4 mod 12 was reformatted into a tandem 6 (SEQ ID NO: 167)
and tandem 8 (SEQ ID NO: 166) construct and tested to confirm that
there is not loss in potency relative to G6 tandem 6 (SEQ ID NO:
144) and tandem 8 (SEQ ID NO: 145). FIG. 24C and FIG. 24D show no
loss in potency for the germline engineered, enhanced cyno cross
reactive F4 mod 12 tandems in comparison to the G6 tandems in the
Colo205 cell line.
Example 19
Activity of G6 Tandem 8 in TRAIL Resistant Cell Lines
[0743] Multiple cell lines are resistant to killing by TRAIL. Thus,
we evaluated whether the enhanced potency of G6 tandem 8 constructs
relative to TRAIL in TRAIL sensitive cell lines will translate into
potency of G6 tandem 8 in TRAIL resistant cell lines. Sensitivity
to Apo2L/TRAIL in several cancer cell lines was determined with the
CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison,
Wis.). Briefly, cells were plated in 96-well plates, allowed to
adhere overnight and then treated with various concentrations of
recombinant human Apo2L/TRAIL and TRAIL mimetic G6 Tandem 8 in
medium containing 10% FBS. After a period of 48-72 hrs, cell
viability was determined following manufacturer's protocols. FIG.
25 shows that for the TRAIL resistant cell line HT29 G6 tandem 8
shows potent cell killing activity while TRAIL does not. TABLE 22
shows that G6 tandem 8 has cell killing activity in many, but not
all of the TRAIL resistant cell lines tested.
TABLE-US-00021 TABLE 22 Activity of G6 tandem 8 and TRAIL in TRAIL
resistant cell lines. G6 TRAIL G6 TRAIL Tandem 8 % Max Tandem 8
IC50 [nM] IC50 [nM] Kill % Max Kill Resistant T84 >8.3 0.247
14.44 71.53 to TRAIL LoVo >8.3 0.005 45.99 74.22 but CaCo-2
>8.3 0.044 18.23 54.84 sensitive HT29 >8.3 0.01 28.00 85.40
to TRAIL HPAF-II >8.3 0.0 6 45.33 91.33 mimetics Hep3B >8.3
0.023 13.35 70.15 SKHEP-1 >8.3 0.055 19.48 80.19 HepG2 >8.3
0.040 33.31 84.00 Resistant SW620 >8.3 >10 -5.71 4.65 to
TRAIL SW837 >8.3 >10 19.98 25.32 and Hs766T >8.3 >10
20.99 47.86 TRAIL NCI-H522 >8.3 >10 32.69 31.38 mimetics
CI-H23 >8.3 >10 22.08 39.59 BT-549 >8.3 >10 4.49 27.99
SNB-7 >8.3 >10 8.9 4.7 786-0 >8.3 >10 -0.12 7.19 SN
-387 >8.3 >10 -0.63 33.1 SN -475 >8.3 >10 0.49 20.88 SN
-393 >8.3 >10 1.50 0.46 indicates data missing or illegible
when filed
Example 20
Immunogenicity Study of TRAIL R2 Binding Monomers
[0744] Immunogenicity is a potential issue for any therapeutic
protein even if it is human in origin. Immunogenic responses can
limit efficacy through neutralizing antibodies that can lead to
inflammation. One of the most important factors in the development
of an immune response is the presence of epitopes that can
stimulate CD4+ T cell proliferation. In the EpiScreen test
(Antitope, Cambridge, UK), CD8+ T cell depleted Peripheral Blood
Mononuclear Cells (PBMCs) are incubated with test proteins and CD4+
T cell proliferation and IL-2 secretion are monitored (see, Baker
& Jones, Curr. Opin. Drug Discovery Dev. 10:219-227, 2007;
Jaber & Baker, J. Pharma. Biomed. Anal. 43:1256-1261, 2007;
Jones et al., J. Thrombosis and Haemostasis 3:991-1000, 2005; Jones
et al., J. Interferon Cytokine Res. 24:560-72, 2004). The PBMCs are
isolated from a pool of donors which represent the HLA-DR allotypes
expressed in the world's population.
[0745] The Tn3 monomers shown in FIG. 26 were expressed (with a
GGGGHHHHHHHH linker-His tag), purified, and verified to be
monomeric by SEC, and filtered for endotoxin removal as described
above. All non-wild type clones tested were from the engineering
round to enhance cyno cross reactivity (FIG. 23A). However, these
clones had mutations to germline that have been shown not to affect
binding in the F4 mod 1 background. These clones were tested in an
ELISA to verify that the germlining mutations did not affect
binding. In both the T cell proliferation assay and the IL-2
secretion assay, a stimulation index (SI) of greater than two had
been previously established as a positive response for a given
donor. The mean SI, or average of the SI of the positive responding
population, is indicative of the strength of the response. A
control protein known to induce a strong response, keyhole limpet
haemocyanin (KLH), was included in both assays.
[0746] TABLE 23 shows the mean SI for all test proteins, which was
significantly lower than for KLH and was not much higher than the
cutoff of 2 for a positive mean SI. In addition, the frequency of
response for the test proteins was very low (ten percent or less
for all tested proteins except for the control which had a response
in excess of 90%). Previous studies by Antitope have revealed that
an EpiScreen response of less than 10% is indicative of low
clinical immunogenicity risk. Thus, our observation that all Tn3s
tested have 10% or less frequency of response indicates a low risk
of clinical immunogenicity.
TABLE-US-00022 TABLE 23 Results of Antitope EpiScreen
immunogenicity assay. Tested Tn3s are ranked from 1 (most
immunogenic) to 4 (least immunogenic). Frequency (%) Mean SI of
Response Sample Prolif IL-2 Prolif IL-2 Ranking F4mod12 2.82 2.30 4
4 4= 00322S-A07 2.91 2.06 8 8 2 00322S-G09 2.88 2.26 10 10 1
00322V-A10 2.67 2.33 8 6 3= 00322V-F11 3.14 2.37 6 6 3= wild type
2.05 2.00 6 4 4= KLH 6.51 3.98 96 92 N/A
Example 21
Aggregation State of Unpurified and Purified G6 Tandem 8 Tn3's
[0747] It is known in the art that proteins containing multiple
cysteines, e.g., a protein made up of tandem repeats that contains
an internal disulfide bond, often does not exhibit proper disulfide
pairing. Scrambling of disulfides can reduce or eliminate
expression into media. If the protein does express into media, it
may be a mixture of improperly folded protein with intermolecular
as well as mismatched intramolecular disulfide pairs leading to
aggregation. Our SEC data revealed that the majority of the tandem
proteins in the bacterial expression media were in a monomeric,
properly folded state. After Ni-NTA purification of the Hi-tagged
G6 tandem 8 protein, approximately 15% of the protein was
aggregated. The observed aggregation was reduced to 4% (FIG. 27A)
by reduction with 2 mM DTT, indicating that most of the aggregation
was disulfide mediated. Most of the aggregates were removed by SEC
purification (FIG. 27B), as described above.
Example 22
Determination of TRAIL Mimetics, G6TN6 and G6TN8, Tumor Growth
Inhibition of in Colo205 Colorectal Cancer Xenograft Models
[0748] The anti-tumor activity of TRAIL Tn3 mimetics, G6 tandem 6
(G6TN6) (SEQ ID NO: 144) and G6 tandem 8 (G6TN8) (SEQ ID NO: 145),
were evaluated in Colo205, a human colorectal carcinoma xenograft
model. Colo205 cells were maintained as a semiadhesive monolayer
culture at 37.degree. C. under 5% CO.sub.2 in Roswell Park Memorial
Institute (RPMI) 1640 medium that contained 10% fetal bovine serum
(FBS). Cells harvested by trypsinization were resuspended to a
final concentration of 3.times.10.sup.7 cells/mL in Hank's balanced
salt solution (HBSS). Athymic female nude mice were each injected
subcutaneously (SC) in the right flank with 3.times.10.sup.6
Colo205 cells. The study was initiated when tumors reached an
average of .about.177 mm.sup.3. The study design is summarized in
TABLE 24. TRAIL was diluted from stock solution with 20 mM Tris-HCl
300 mM Arginine-HCl pH 7 and administered intravenously (IV) at
dose indicated in TABLE 24, daily for a total of 5 doses according
to body weight (10 mL/kg). G6 tandem 6 (G6TN6) and G6 tandem 8
(G6TN8) were each diluted from a stock solution with PBS and
administered intravenously (IV) at doses indicated in TABLE 24,
daily for a total of 5 doses according to body weight (10 mL/kg).
Tumor volumes and body weight measurements were recorded. Tumor
measurements were made using an electronic caliper and tumor volume
(mm.sup.3) was calculated using the formula tumor volume=[length
(mm).times.width (mm).sup.2]/2. Tumor growth inhibition (TGI) was
calculated as percent TGI=(1-T/C).times.100, where T=final tumor
volumes from a treated group after the last dose, and C=final tumor
volumes from the control group after the last dose.
[0749] During the dosing phase (DP) (FIG. 28), 3 mg/kg and 30 mg/kg
of G6TN6 resulted in significant TGI of 92% (p<0.0001) and 93%
(p<0.0001), respectively (TABLE 25). Similarly, after equimolar
adjustment for final concentration, 2.25 mg/kg and 25.5 mg/kg of
G6TN8 resulted in significant TGI of 93% (p<0.0001) and 94%
(p<0.0001), respectively (TABLE 25). 30 mg/kg of TRAIL resulted
in TGI of 60% (p<0.001), (TABLE 25).
[0750] By day 34 of the regrowth phase (RP) (FIG. 28, while 3 mg/kg
G6TN6 did not result in any CR (complete regression), 2.25 mg/kg
G6TN8 resulted in a 90% CR. At a higher dose of 30 mg/kg G6TN6 50%
CR was achieved. On the other hand, 25.5 mg/kg G6TN8 resulted in
100% CR (TABLE 26). Results from both doses suggest that G6TN8
resulted in greater efficacy in comparison to G6TN6. However, both
showed efficacy at certain doses. More importantly, both constructs
significantly outperformed TRAIL which did not result in any PR or
CR.
[0751] As shown in FIG. 29, no body weight loss was observed for
both G6TN6 and G6TN8 at all doses during the dosing and regrowth
phase of the study.
TABLE-US-00023 TABLE 24 Study design for Trail and TRAIL mimetics
(G6TN6 and G6TN8) in Colo205 tumor xenograft model Dose Test Dose
Volume Dose Group Material (mg/kg) (mL/kg) Route Schedule 1
Untreated NA NA NA NA 2 PBS NA 10 IV QDX5 3 Trail 30 mg/kg 10 IV
QDX5 4 G6TN6 30 mg/kg 10 IV QDX5 5 G6TN6 3 mg/kg 10 IV QDX5 6 G6TN8
25.5 mg/kg 10 IV QDX5 7 G6TN8 2.25 mg/kg 10 IV QDX5
TABLE-US-00024 TABLE 25 Effect of TRAIL and TRAIL mimetics (G6TN6
and G6TN8) on TGI during dosing phase of the study. P Value
(compared to Treatment group % TGI untreated control) Trail 30
mg/kg 60 P < 0.001 G6TN6 30 mg/kg 93 P < 0.0001 G6TN6 3 mg/kg
92 P < 0.0001 G6TN8 25.5 mg/kg 94 P < 0.0001 G6TN8 2.25 mg/kg
93 P < 0.0001
TABLE-US-00025 TABLE 26 Effect of TRAIL and TRAIL mimetics (G6TN6
and G6TN8) on TGI during regrowth phase by day 34 of the study.
Treatment group PR.sup.a (%) CR.sup.b (%) Trail -- -- G6TN6 30
mg/kg 50 50 G6TN6 3 mg/kg 100 -- G6TN8 25.5 mg/kg -- 100 G6TN8 2.25
mg/kg 10 90 .sup.apercent partial regression (PR; percentage of
mice in group where tumor volume is less than 50% of volume at time
of staging for two successive measurements) .sup.bpercent complete
regression (CR; percentage of mice in group where no palpable tumor
detectable for two successive measurements)
Example 23
Binding Additional Targets
[0752] FnIII scaffolds that bind to particular targets may be
generated by the methods described herein and/or known in the art
(see for Example WO 2009/058379). Alternatively, the scaffolds
described herein are subjected to "loop grafting" in which the loop
sequences of a scaffold of known binding specificity are grafted to
the beta strand sequences of the desired scaffold (e.g., the beta
strand sequences of a Tn3 scaffold or the sequences presented in
FIG. 16). TABLE 27 provides a non-limiting example of loop
sequences for grafting to desired beta strands, for example those
provided in TABLE 1.
TABLE-US-00026 TABLE 27 Loop Sequences for Loop Grafting AB Loop BC
Loop CD Loop DE Loop EF Loop FG Loop Target (SEQ ID NO) (SEQ ID NO)
(SEQ ID NO) (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) .alpha.v.beta.3
Integrin VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTLRGDWSEDSKPI
See: US7,7556,925 (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO:
210) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTVRGDWYEYSKPI
(NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 211) VAATPTS
DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDWTEHSKPI (NO: 55) (NO:
56) (NO: 57) (NO: 58) (NO: 59) (NO: 212) VAATPTS DAPAVTVRY TGGNSPV
SGLKPGVD SGLKPGVD VTARGDWVEGSKPI (NO: 55) (NO: 56) (NO: 57) (NO:
58) (NO: 59) (NO: 213) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD
VTPRGDWTEGSKPI (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO:
214) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTPRGDWIEFSKPI
(NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 215 VAATPTS
DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDWNEGSKPI (NO: 55) (NO:
56) (NO: 57) (NO: 58) (NO: 59) (NO: 216) VAATPTS DAPAVTVRY TGGNSPV
SGLKPGVD SGLKPGVD VTFRGDWIELSKPI (NO: 55) (NO: 56) (NO: 57) (NO:
58) (NO: 59) (NO: 217) Estrogen Receptor VAATPWTWV DAPAVTVRY
TGGNSPV SGLKPGVD SGLKPGVD VTGRGDSPASSKPI See: US7,598,352 LRETS
(NO: 56) (NO: 57) (NO: 58) (NO: 59) (NO: 60) (NO: 218) VAATPWVLI
DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD VTGRGDSPASSKPI TRSTS (NO: 56)
(NO: 57) (NO: 58) (NO: 59) (NO: 60) (NO: 219) VAATPTS DAPWYQGRY
TGGNSPV SGLKPGVD SGLKPGVD VTGRLRAQLVSKPI (NO: 55) (NO: 220) (NO:
57) (NO: 58) (NO: 59) (NO: 221) VAATPTS DAPRTKQY TGGNSPV SGLKPGVD
SGLKPGVD VTGRLRDLLQSKPI (NO: 55) (NO: 222) (NO: 57) (NO: 58) (NO:
59) (NO: 223) VAATPTS DAPAVTVRY TGGNSPV SGLKPGVD SGLKPGVD
VRGLVRFRVVNSSL (NO: 55) (NO: 56) (NO: 57) (NO: 58) (NO: 59)
CMWARSKPI (NO: 224) VEGFR-2 VAATPTS RHPHFPTRY TGGNSPV PLQPPL
SGLKPGVD VTKERNGRELFTPI See: US7858739 (NO: 55) (NO: 225) (NO: 57)
(NO: 226) (NO: 59) (NO: 227) VAATPTS RHPHFPTRY TGGNSPV PLQPPT
SGLKPGVD VTDGRNGRLLSIPI (NO: 55) (NO: 225) (NO: 57) (NO: 228) (NO:
59) (NO: 229) VAATPTS RHPHFPTRY TGGNSPV PLQPPT SGLKPGVD
VTMGLYGHELLTPP (NO: 55) (NO: 225) (NO: 57) (NO: 228) (NO: 59) I
(NO: 230) VAATPTS RHPHFPTRY TGGNSPV PLQPPT SGLKPGVD VTDGENGQFLLVPI
(NO: 55) (NO: 225) (NO: 57) (NO: 228) (NO: 59) (NO: 231) EGFR
VAATPTS HERDGSRQY TGGNSPV PGGVRT SGLKPGVD VTDYFNPTTHEYIY See:
WO2010/060095 (NO: 55) (NO: 232) (NO: 57) (NO: 233) (NO: 59) QTTPI
(NO: 234) VAATPTS WAPVDRYQY TGGNSPV PRDVYT SGLKPGVD VTDYKPHADGPHTY
(NO: 55) (NO: 235) (NO: 57) (NO: 236) (NO: 59) HESPI (NO: 237)
VAATPTS TQGSTHYQY TGGNSPV PGMVYT SGLKPGVD VTDYFDRSTHEYKY (NO: 55)
(NO: 238) (NO: 57) (NO: 239) (NO: 59) RTTPI (NO: 240) VAATPTS
YWEGLPYQY TGGNSPV PRDVNT SGLKPGVD VTDWYNPDTHEYIY (NO: 55) (NO: 241)
(NO: 57) (NO: 242) (NO: 59) HTIPI (NO: 243) IGF-IR VAATPTS
SPYLRVARY TGGNSPV PSSART SGLKPGVD VTPSNIIGRHYGPI See: WO2008/066752
(NO: 55) (NO: 244) (NO: 57) (NO: 245) (NO: 59) (NO: 246) VAATPTS
VNDPQRNRY TGGNSPV PAYYPT SGLKPGVD VTYSHIKYLYHKPI (NO: 55) (NO: 247)
(NO: 57) (NO: 248) (NO: 59) (NO: 249) VAATPTS SDSLKVSRY TGGNSPV
PKQYHT SGLKPGVD VTPSNIIGRHYGPI (NO: 55) (NO: 250) (NO: 57) (NO:
251) (NO: 59) (NO: 252) VAATPTS SAPLKVARY TGGNSPV PKNVYT SGLKPGVD
VTKMRDYRPI (NO: 55) (NO: 253) (NO: 57) (NO: 254) (NO: 59) (NO:
255)
[0753] The foregoing examples illustrate various aspects of the
invention and practice of the methods of the invention. The
examples are not intended to provide an exhaustive description of
the many different embodiments of the invention. Thus, although the
forgoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
those of ordinary skill in the art will realize readily that many
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims.
[0754] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
Sequence CWU 1
1
270183PRTArtificialDisulfide-bonded variant 1Ile Glu Val Lys Asp
Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe 1 5 10 15 Lys Pro Leu
Ala Glu Ile Asp Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp
Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn 35 40
45 Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser
50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys
Glu Thr 65 70 75 80 Phe Thr Thr 283PRTArtificialDisulfide-bonded
variant 2Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Phe 1 5 10 15 Lys Pro Leu Ala Glu Ile Asp Gly Ile Glu Leu Thr
Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp
Leu Thr Glu Asp Glu Asn 35 40 45 Gln Tyr Ser Ile Gly Asn Leu Lys
Pro Asp Thr Glu Tyr Cys Val Ser 50 55 60 Leu Ile Ser Arg Arg Gly
Asp Met Ser Ser Asn Pro Ala Lys Glu Cys 65 70 75 80 Phe Thr Thr
383PRTArtificialDisulfide-bonded variant 3Ile Glu Val Lys Asp Val
Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe 1 5 10 15 Lys Pro Leu Ala
Glu Ile Asp Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn 35 40 45
Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Cys Val Ser 50
55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu
Cys 65 70 75 80 Phe Thr Thr 490PRTHomo sapiens 4Arg Leu Asp Ala Pro
Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu
Ile Thr Trp Phe Lys Pro Leu Ala Glu Ile Asp Gly Ile 20 25 30 Glu
Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile 35 40
45 Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro
50 55 60 Asp Thr Glu Tyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp
Met Ser 65 70 75 80 Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr 85 90
587PRTHomo sapiens 5Leu 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 Arg Thr 85 682PRTHomo sapiens 6Pro Thr Val Asp Gln Val
Asp Asp Thr Ser Ile Val Val Arg Trp Ser 1 5 10 15 Arg Pro Gln Ala
Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 20 25 30 Val Glu
Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 35 40 45
Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 50
55 60 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln
Gln 65 70 75 80 Glu Thr 780PRTHomo sapiens 7Pro Tyr Asn Thr Glu Val
Thr Glu Thr Thr Ile Val Ile Thr Trp Thr 1 5 10 15 Pro Ala Pro Arg
Ile Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly 20 25 30 Gly Glu
Ala Pro Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val 35 40 45
Ser Gly Leu Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu 50
55 60 Arg Asp Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val
Thr 65 70 75 80 8102PRTHomo sapiens 8Pro Pro Ile Ala Leu Asn Trp
Thr Leu Leu Asn Val Ser Leu Thr Gly 1 5 10 15 Ile His Ala Asp Ile
Gln Val Arg Trp Glu Ala Pro Arg Asn Ala Asp 20 25 30 Ile Gln Lys
Gly Trp Met Val Leu Glu Tyr Glu Leu Gln Tyr Lys Glu 35 40 45 Val
Asn Glu Thr Lys Trp Lys Met Met Asp Pro Ile Leu Thr Thr Ser 50 55
60 Val Pro Val Tyr Ser Leu Lys Val Asp Lys Glu Tyr Glu Val Arg Val
65 70 75 80 Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr Gly Glu Phe Ser
Glu Val 85 90 95 Leu Tyr Val Thr Leu Pro 100 996PRTHomo sapiens
9Pro Pro Ser Leu Asn Val Thr Lys Asp Gly Asp Ser Tyr Ser Leu Arg 1
5 10 15 Trp Glu Thr Met Lys Met Arg Tyr Glu His Ile Asp His Thr Phe
Glu 20 25 30 Ile Gln Tyr Arg Lys Asp Thr Ala Thr Trp Lys Asp Ser
Lys Thr Glu 35 40 45 Thr Leu Gln Asn Ala His Ser Met Ala Leu Pro
Ala Leu Glu Pro Ser 50 55 60 Thr Arg Tyr Trp Ala Arg Val Arg Val
Arg Thr Ser Arg Thr Gly Tyr 65 70 75 80 Asn Gly Ile Trp Ser Glu Trp
Ser Glu Ala Arg Ser Trp Asp Thr Glu 85 90 95 1089PRTHomo sapiens
10Pro Pro Val Asn Phe Thr Ile Lys Val Thr Gly Leu Ala Gln Val Leu 1
5 10 15 Leu Gln Trp Lys Pro Asn Pro Asp Gln Glu Gln Arg Asn Val Asn
Leu 20 25 30 Glu Tyr Gln Val Lys Ile Asn Ala Pro Lys Glu Asp Asp
Tyr Glu Thr 35 40 45 Arg Ile Thr Glu Ser Lys Leu Val Thr Ile Leu
His Lys Gly Phe Ser 50 55 60 Ala Ser Val Arg Thr Ile Leu Gln Asn
Asp His Ser Leu Leu Ala Ser 65 70 75 80 Ser Trp Ala Ser Ala Glu Leu
His Ala 85 1191PRTHomo sapiens 11Leu Ser Val Thr Asp Val Thr Thr
Ser Ser Leu Arg Leu Asn Trp Glu 1 5 10 15 Ala Pro Pro Gly Ala Phe
Asp Ser Phe Leu Leu Arg Phe Gly Val Pro 20 25 30 Ser Pro Ser Thr
Leu Glu Pro His Pro Arg Pro Leu Leu Gln Arg Glu 35 40 45 Leu Met
Val Pro Gly Thr Arg His Ser Ala Val Leu Arg Asp Leu Arg 50 55 60
Ser Gly Thr Leu Tyr Ser Leu Thr Leu Tyr Gly Leu Arg Gly Pro His 65
70 75 80 Lys Ala Asp Ser Ile Gln Gly Thr Ala Arg Thr 85 90
1281PRTHomo sapiens 12Leu Arg Ala Leu Asn Leu Thr Glu Gly Phe Ala
Val Leu His Trp Lys 1 5 10 15 Pro Pro Gln Asn Pro Val Asp Thr Tyr
Asp Ile Gln Val Thr Ala Pro 20 25 30 Gly Ala Pro Pro Leu Gln Ala
Glu Thr Pro Gly Ser Ala Val Asp Tyr 35 40 45 Pro Leu His Asp Leu
Val Leu His Thr Asn Tyr Thr Ala Thr Val Arg 50 55 60 Gly Leu Arg
Gly Pro Asn Leu Thr Ser Pro Ala Ser Ile Thr Phe Thr 65 70 75 80 Thr
1381PRTHomo sapiens 13Leu Glu Ala Lys Glu Val Thr Pro Arg Thr Ala
Leu Leu Thr Trp Thr 1 5 10 15 Glu Pro Pro Val Arg Pro Ala Gly Tyr
Leu Leu Ser Phe His Thr Pro 20 25 30 Gly Gly Gln Thr Gln Glu Ile
Leu Leu Pro Gly Gly Ile Thr Ser His 35 40 45 Gln Leu Leu Gly Leu
Phe Pro Ser Thr Ser Tyr Asn Ala Arg Leu Gln 50 55 60 Ala Met Trp
Gly Gln Ser Leu Leu Pro Pro Val Ser Thr Ser Phe Thr 65 70 75 80 Thr
1483PRTHomo sapiens 14Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala
Leu Ile Thr Trp Phe 1 5 10 15 Lys Pro Leu Ala Glu Ile Asp Gly Ile
Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Thr Glu Asp Glu Asn 35 40 45 Gln Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Ser
Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe
Thr Thr 1593PRTHomo sapiens 15Pro Lys Phe Thr Lys Cys Arg Ser Pro
Glu Arg Glu Thr Phe Ser Cys 1 5 10 15 His Trp Thr Asp Glu Val His
His Gly Thr Lys Asn Leu Gly Pro Ile 20 25 30 Gln Leu Phe Tyr Thr
Arg Arg Asn Thr Gln Glu Trp Thr Gln Glu Trp 35 40 45 Lys Glu Cys
Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys Tyr Phe 50 55 60 Asn
Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys Leu Thr 65 70
75 80 Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser Val 85 90
1697PRTHomo sapiens 16Pro Ser Gly Phe Pro Gln Asn Leu His Val Thr
Gly Leu Thr Thr Ser 1 5 10 15 Thr Thr Glu Leu Ala Trp Asp Pro Pro
Val Leu Ala Glu Arg Asn Gly 20 25 30 Arg Ile Ile Ser Tyr Thr Val
Val Phe Arg Asp Ile Asn Ser Gln Gln 35 40 45 Glu Leu Gln Asn Ile
Thr Thr Asp Thr Arg Phe Thr Leu Thr Gly Leu 50 55 60 Lys Pro Asp
Thr Thr Tyr Asp Ile Lys Val Arg Ala Trp Thr Ser Lys 65 70 75 80 Gly
Ser Gly Pro Leu Ser Pro Ser Ile Gln Ser Arg Thr Met Pro Val 85 90
95 Glu 1792PRTHomo sapiens 17Pro Lys Pro Pro Ile Asp Leu Val Val
Thr Glu Thr Thr Ala Thr Ser 1 5 10 15 Val Thr Leu Thr Trp Asp Ser
Gly Asn Ser Glu Pro Val Thr Tyr Tyr 20 25 30 Gly Ile Gln Tyr Arg
Ala Ala Gly Thr Glu Gly Pro Phe Gln Glu Val 35 40 45 Asp Gly Val
Ala Thr Thr Arg Tyr Ser Ile Gly Gly Leu Ser Pro Phe 50 55 60 Ser
Glu Tyr Ala Phe Arg Val Leu Ala Val Asn Ser Ile Gly Arg Gly 65 70
75 80 Pro Pro Ser Glu Ala Val Arg Ala Arg Thr Gly Glu 85 90
1887PRTHomo sapiens 18Leu Ser Pro Pro Arg Asn Leu Arg Ile Ser Asn
Val Gly Ser Asn Ser 1 5 10 15 Ala Arg Leu Thr Trp Asp Pro Thr Ser
Arg Gln Ile Asn Gly Tyr Arg 20 25 30 Ile Val Tyr Asn Asn Ala Asp
Gly Thr Glu Ile Asn Glu Val Glu Val 35 40 45 Asp Pro Ile Thr Thr
Phe Pro Leu Lys Gly Leu Thr Pro Leu Thr Glu 50 55 60 Tyr Thr Ile
Ala Ile Phe Ser Ile Tyr Asp Glu Gly Gln Ser Glu Pro 65 70 75 80 Leu
Thr Gly Val Phe Thr Thr 85 1983PRTArtificialCharge variant 19Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe 1 5 10
15 Lys Pro Leu Ala Glu Ile Asp Gly Ile Gln Leu Thr Tyr Gly Ile Lys
20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asn Leu Thr Glu Asp
Glu Asn 35 40 45 Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu
Tyr Glu Val Ser 50 55 60 Leu Ile Ser Arg Arg Gly Asp Met Ser Ser
Asn Pro Ala Lys Gln Thr 65 70 75 80 Phe Thr Thr
2091PRTArchaeoglobus fulgidus DSM 4304 20Pro Ala Ile Ser Asn Val
Arg Val Ser Asp Val Thr Asn Ser Ser Ala 1 5 10 15 Thr Ile Arg Trp
Asp Val Ser Leu Ala Ala Asn Asn Arg Val Leu Phe 20 25 30 Ser Thr
Asn Ser Asp Leu Ser Ser Pro Gln Trp Ser Ala Trp Asp Asn 35 40 45
Ser Thr Asp Ser Pro Met Ile Thr Leu Ser Gly Leu Ser Ala Gly Thr 50
55 60 Ala Tyr Tyr Phe Ser Val Tyr Ser Phe Arg Pro Asp Asn Ala Ser
Leu 65 70 75 80 Tyr Ser Asn Ser Ser Ile Met Ser Phe Thr Thr 85 90
2194PRTStaphylothermus marinus F1 21Ser Glu Pro Gln Asn Leu Lys Ala
Thr Ala Gly Asn Asn Asn Ile Thr 1 5 10 15 Leu Thr Trp Asp Pro Pro
Ile Asp Asp Gly Gly Cys Arg Ile Val Glu 20 25 30 Tyr Arg Ile Tyr
Arg Gly Thr Asn Asn Asn Asn Leu Glu Tyr Tyr Ala 35 40 45 Ser Val
Asn Gly Ser Thr Thr Thr Phe Ile Asp Lys Asn Ile Val Tyr 50 55 60
Ser Gln Thr Tyr Tyr Tyr Lys Val Ser Ala Val Asn Asn Ile Val Glu 65
70 75 80 Gly Pro Lys Ser Asn Thr Ala Ser Ala Thr Pro Thr Ser Ser 85
90 2289PRTSulfolobus acidocaldarius DSM 639 22Pro Pro Pro Lys Pro
Val Ile Arg Phe Ala Gln Ala Gly Asn Asn Ser 1 5 10 15 Ile Ser Leu
Ser Trp Tyr Asp Thr Asn Thr Ser Gly Tyr Tyr Ile Gln 20 25 30 Trp
Trp Ser Ser Ile Asp Asn Asn Lys Ser Thr Ile Asn Val Gly Asn 35 40
45 Val Ser Ser Tyr Leu Phe Ile Asn Leu Thr Asn Gly Val Thr Tyr Tyr
50 55 60 Phe Arg Ile Ile Pro Tyr Asn Gln Ala Gly Asn Gly Thr Ser
Ser Asp 65 70 75 80 Ile Ile Ser Leu Thr Pro Gly Ala Val 85
2389PRTSulfolobus acidocaldarius DSM 639 23Pro Asp Ser Pro Ser Val
Lys Val Ile Val Gly Asp Arg Asn Ala Thr 1 5 10 15 Val Ile Trp Ser
Lys Pro Tyr Asn Gly Gly Phe Pro Ile Leu Gly Tyr 20 25 30 Tyr Leu
Thr Val Lys Thr Asp Asn Ser Ser Tyr Thr Ile Asn Val Gly 35 40 45
Asn Val Ser Lys Tyr Thr Leu Thr Asn Leu Thr Pro Glu Val Leu Tyr 50
55 60 Glu Val Met Val Val Ala Tyr Asn Lys Leu Gly Asn Ser Ser Pro
Gly 65 70 75 80 Ile Val Asn Phe Val Ala Leu Thr Thr 85
2487PRTSulfolobus acidocaldarius DSM 639 24Leu Thr Thr Ala Ser Ile
Ser Val Ser Val Tyr Lys Lys Val Asn Gly 1 5 10 15 Val Leu Ile Ser
Trp Asn Lys Thr Glu Asn Thr Thr Tyr Asn Leu Leu 20 25 30 Ile Ser
Asp Lys Lys Gly Lys Ile Ile Val Asn Ile Thr Thr Thr Asn 35 40 45
Thr Ser Tyr Phe Ala Tyr Ile Pro Tyr Gly Ile Tyr Asn Val Thr Ile 50
55 60 Arg Ala Thr Asn Gln Val Gly Thr Asn Ser Thr Ser Phe Pro Ile
Val 65 70 75 80 Phe Tyr Ile Pro Pro Phe Ile 85 2584PRTSulfolobus
acidocaldarius DSM 639 25Pro Leu Val Lys Phe Ser Ile Gly Asn Asn
Ser Ile Leu Asn Leu Lys 1 5 10 15 Trp Asn Asn Val Thr Gly Ala Thr
Phe Tyr Leu Val Tyr Val Asn Thr 20 25 30 Thr Leu Ile Ala Asn Val
Thr Thr Asp Ser Tyr Ser Leu Asn Leu Thr 35 40 45 Pro Gly Phe His
Val Ile Arg Val Val Ala Ala Asn Pro Ile Tyr Asn 50 55 60 Ser Ser
Pro Ala Ser Leu Gly Ile Leu Ile Gln Gln His Ser Val Thr 65 70 75 80
Ser Ser Ile Thr 2688PRTSulfolobus solfataricus P2 26Pro Leu Pro Pro
Lys Ile Thr Ser Tyr Ser Ala Gly Asn Glu
Ser Val 1 5 10 15 Thr Leu Gly Trp Asn Pro Val Arg Leu Ser Ser Gly
Tyr Glu Ile Ile 20 25 30 Tyr Trp Asn Asn Met Gly Phe Asn Ser Ser
Ile Asn Val Gly Asn Val 35 40 45 Thr Ser Tyr Thr Val Thr Gly Leu
Lys Asp Gly Ile Thr Tyr Tyr Phe 50 55 60 Glu Val Leu Ala Tyr Asn
Ser Ile Gly Tyr Ser Ser Pro Ser Ser Ile 65 70 75 80 Ile Ala Leu Thr
Pro Ala Ser Val 85 2792PRTSulfolobus solfataricus P2 27Pro Asn Pro
Pro Gln Leu Val Ser Val Lys Tyr Gly Asn Asp Asn Val 1 5 10 15 Thr
Leu Asn Trp Leu Pro Pro Thr Phe Ser Gly Gly Tyr Leu Leu Leu 20 25
30 Gly Tyr Tyr Val Ile Val Lys Asn Glu Asn Ser Met Val Ser Ser His
35 40 45 Phe Val Asn Ser Thr Ser Leu Thr Ile Ser Asn Leu Thr Pro
Asn Val 50 55 60 Thr Tyr Asn Val Phe Ile Tyr Ala Val Asn Lys Leu
Gly Asn Ser Ser 65 70 75 80 Pro Leu Val Leu Thr Val Val Pro Ile Thr
Lys Ala 85 90 2886PRTSulfolobus solfataricus P2 28Pro Ile Thr Lys
Ala Ser Val Phe Ala Phe Ile Thr Lys Leu Gly Asn 1 5 10 15 Gly Ile
Leu Val Asn Trp Thr Thr Ser Phe Pro Ala Asn Ile Thr Leu 20 25 30
Glu Leu Tyr Asn Pro Asn Gly Asn Leu Ile Ser Gln Ile Ala Ala Ile 35
40 45 Lys Gly Asn Ser Ser Tyr Leu Phe Arg Val Pro Gln Gly Asn Tyr
Thr 50 55 60 Leu Val Ile Ile Ala Ser Asn Ser Ala Gly Val Ser Lys
Tyr Val Tyr 65 70 75 80 Gln Val Val Tyr Tyr Leu 85
2983PRTSulfolobus solfataricus P2 29Pro Pro Ala Ser Pro Gln Val Ser
Leu Ile Gly Phe Gly Asn Asn Leu 1 5 10 15 Tyr Ile Ser Trp Asn Asn
Glu Ala Asn Val Ile Thr Tyr Leu Val Tyr 20 25 30 Val Asn Asn Ser
Leu Val Tyr Glu Gly Pro Ser Asn Ser Ile Val Thr 35 40 45 Asn Ile
Ser Asn Gly Thr Tyr Leu Val Lys Val Ile Gly Val Asn Pro 50 55 60
Ala Gly Ser Ser Ser Pro Gly Ile Ala Val Ile His Tyr Thr Gly Asp 65
70 75 80 Tyr Val Thr 3087PRTSulfolobus tokodaii str. 7 30Pro Pro
Lys Pro Gln Ile Ala Ser Ile Ala Ser Gly Asn Glu Thr Ile 1 5 10 15
Thr Val Lys Trp Tyr Asp Thr Asn Ala Ser Gly Tyr Tyr Ile Thr Tyr 20
25 30 Trp Ser Asn Phe Ser Gln Lys Val Thr Ile Asn Val Gly Asn Val
Thr 35 40 45 Ser Tyr Thr Ile Lys His Leu Lys Asp Gly Val Thr Tyr
Tyr Ile Gln 50 55 60 Ile Val Pro Tyr Asn Ser Leu Gly Asn Gly Thr
Pro Ser Asp Ile Ile 65 70 75 80 Ser Ala Thr Pro Ser Ser Val 85
3186PRTSulfolobus tokodaii str. 7 31Pro Asn Pro Pro Ile Ile Lys Val
Lys Ile Gly Asn Leu Asn Ala Thr 1 5 10 15 Leu Thr Trp Tyr Asp Thr
Phe Asn Gly Gly Tyr Pro Ile Glu Gly Tyr 20 25 30 Tyr Leu Tyr Val
Asn Gly Lys Gly Ile Asn Val Gly Asn Ile Thr Ser 35 40 45 Tyr Val
Leu Thr Asn Leu Thr Ala Gly Glu Leu Tyr Thr Ile Glu Leu 50 55 60
Ile Ala Tyr Asn Lys Ile Gly Asn Ser Ser Ile Ser Ser Val Ser Phe 65
70 75 80 Ile Ala Ala Ser Lys Ala 85 3283PRTSulfolobus tokodaii str.
7 32Ala Ser Lys Ala Asn Leu Thr Val Thr Val Tyr Lys Lys Ile Asn Gly
1 5 10 15 Phe Leu Val Ser Trp Asn Ser Thr Ser Lys Ala Lys Tyr Ile
Leu Thr 20 25 30 Val Ser Lys Glu Asn Val Val Leu Leu Asn Val Ser
Thr Thr Asn Thr 35 40 45 Ser Tyr Phe Val Lys Val Pro Phe Gly Val
Tyr Asn Ile Ser Leu Glu 50 55 60 Ala Val Asn Ile Val Gly Ile Thr
Lys Tyr Ala Phe Ile Leu Ile Tyr 65 70 75 80 Tyr Ile Gln
3381PRTSulfolobus tokodaii str. 7 33Pro Ala Ser Pro Thr Val Asn Trp
Ser Ile Thr Leu Asn Thr Val Ser 1 5 10 15 Leu Asn Trp Ser Lys Val
Ser Gly Ala Glu Tyr Tyr Leu Ile Tyr Asp 20 25 30 Asn Gly Lys Leu
Ile Thr Asn Thr Thr Asn Thr Ala Phe Thr Phe Asn 35 40 45 Leu Thr
Ile Gly Gln Asn Glu Ile Glu Val Tyr Ala Ala Asn Ala Tyr 50 55 60
Tyr Lys Ser Ala Pro Tyr Ile Ile Asn Asp Val Arg Asn Tyr Ile Val 65
70 75 80 Val 3482PRTHomo sapiens 34Ala Arg Val Thr Asp Ala Thr Glu
Thr Thr Ile Thr Ile Ser Trp Arg 1 5 10 15 Thr Lys Thr Glu Thr Ile
Thr Gly Phe Gln Val Asp Ala Val Pro Ala 20 25 30 Asn Gly Gln Thr
Pro Ile Gln Arg Thr Ile Lys Pro Asp Val Arg Ser 35 40 45 Tyr Thr
Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile Tyr Leu 50 55 60
Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile Asp Ala 65
70 75 80 Ser Thr 357PRTHomo sapiens 35Lys Asp Val Thr Asp Thr Thr 1
5 369PRTHomo sapiens 36Phe Lys Pro Leu Ala Glu Ile Asp Gly 1 5
377PRTHomo sapiens 37Lys Asp Val Pro Gly Asp Arg 1 5 386PRTHomo
sapiens 38Thr Glu Asp Glu Asn Gln 1 5 398PRTHomo sapiens 39Gly Asn
Leu Lys Pro Asp Thr Glu 1 5 4010PRTHomo sapiens 40Arg Arg Gly Asp
Met Ser Ser Asn Pro Ala 1 5 10 4110PRTHomo sapiens 41Arg Leu Asp
Ala Pro Ser Gln Ile Glu Val 1 5 10 423PRTHomo sapiens 42Ile Glu Val
1 435PRTHomo sapiens 43Ala Leu Ile Thr Trp 1 5 447PRTHomo sapiens
44Ile Glu Leu Thr Tyr Gly Ile 1 5 457PRTArtificialBeta strand C in
disulfide-bonded variant 45Cys Glu Leu Thr Tyr Gly Ile 1 5
465PRTHomo sapiens 46Thr Thr Ile Asp Leu 1 5 473PRTHomo sapiens
47Tyr Ser Ile 1 487PRTHomo sapiens 48Tyr Glu Val Ser Leu Ile Ser 1
5 497PRTArtificialBeta strand F in disulfide-bonded variant 49Tyr
Glu Val Ser Leu Ile Cys 1 5 507PRTArtificialBeta strand F in
disulfide-bonded variant 50Tyr Cys Val Ser Leu Ile Ser 1 5
517PRTArtificialBeta strand F in disulfide-bonded variant 51Tyr Cys
Val Ser Leu Ile Cys 1 5 526PRTHomo sapiens 52Lys Glu Thr Phe Thr
Thr 1 5 536PRTArtificialBeta strand G in disulfide-bonded variants
53Lys Glu Cys Phe Thr Thr 1 5 5494PRTHomo sapiens 54Val 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 557PRTHomo sapiens 55Val Ala Ala Thr Pro Thr Ser
1 5 569PRTHomo sapiens 56Asp Ala Pro Ala Val Thr Val Arg Tyr 1 5
577PRTHomo sapiens 57Thr Gly Gly Asn Ser Pro Val 1 5 586PRTHomo
sapiens 58Pro Gly Ser Lys Ser Thr 1 5 598PRTHomo sapiens 59Ser Gly
Leu Lys Pro Gly Val Asp 1 5 6014PRTHomo sapiens 60Val Thr Gly Arg
Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile 1 5 10 6110PRTHomo sapiens
61Val Ser Asp Val Pro Arg Asp Leu Glu Val 1 5 10 623PRTHomo sapiens
62Leu Glu Val 1 635PRTHomo sapiens 63Leu Leu Ile Ser Trp 1 5
647PRTHomo sapiens 64Tyr Arg Ile Thr Tyr Gly Glu 1 5 655PRTHomo
sapiens 65Gln Glu Phe Thr Val 1 5 663PRTHomo sapiens 66Ala Thr Ile
1 677PRTHomo sapiens 67Tyr Thr Ile Thr Val Tyr Ala 1 5 686PRTHomo
sapiens 68Ser Ile Asn Tyr Arg Thr 1 5 6988PRTHomo sapiens 69Val Ser
Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr 1 5 10 15
Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln 20
25 30 Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr
Ile 35 40 45 Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln
Pro Gly Thr 50 55 60 Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp
Asn Ala Arg Ser Ser 65 70 75 80 Pro Val Val Ile Asp Ala Ser Thr 85
707PRTHomo sapiens 70Thr Asp Ala Thr Glu Thr Thr 1 5 719PRTHomo
sapiens 71Arg Thr Lys Thr Glu Thr Ile Thr Gly 1 5 726PRTHomo
sapiens 72Ala Asn Gly Gln Thr Pro 1 5 736PRTHomo sapiens 73Lys Pro
Asp Val Arg Ser 1 5 748PRTHomo sapiens 74Thr Gly Leu Gln Pro Gly
Thr Asp 1 5 7510PRTHomo sapiens 75Leu Asn Asp Asn Ala Arg Ser Ser
Pro Val 1 5 10 768PRTHomo sapiens 76Ser Pro Pro Arg Arg Ala Arg Val
1 5 773PRTHomo sapiens 77Ala Arg Val 1 784PRTHomo sapiens 78Ile Thr
Ile Trp 1 797PRTHomo sapiens 79Phe Gln Val Asp Ala Val Pro 1 5
805PRTHomo sapiens 80Ile Gln Arg Thr Ile 1 5 813PRTHomo sapiens
81Tyr Thr Ile 1 827PRTHomo sapiens 82Tyr Lys Ile Tyr Leu Tyr Thr 1
5 836PRTHomo sapiens 83Val Ile Asp Ala Ser Thr 1 5 84232PRTHomo
sapiens 84Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu
Ser Leu Ser Pro Gly Lys 225 230 85330PRTHomo sapiens 85Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 86107PRTHomo sapiens 86Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
87104PRTHomo sapiens 87Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90
95 Lys Thr Val Ala Pro Thr Glu Cys 100 8816PRTArtificialLinker
88Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 1
5 10 15 8924PRTArtificialLinker 89Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Thr Gly Ser Ala Met 1 5 10 15 Ala Ser Gly Gly Gly Gly
Ser Ala 20 9013PRTArtificialLinker 90Ala Gly Gly Gly Gly Ser Arg
Leu Asp Ala Pro Gly Gln 1 5 10
9122PRTArtificialLinker 91Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Arg 1 5 10 15 Leu Asp Ala Pro Gly Gln 20
9232PRTArtificialLinker 92Gly 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 Arg Leu Asp Ala Pro Gly Gln 20 25 30 9342PRTArtificialLinker
93Gly 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 Gly Gly Gly Gly Ser Gly
Gly 20 25 30 Gly Gly Ser Arg Leu Asp Ala Pro Gly Gln 35 40
948PRTArtificialLinker 94Thr Arg Leu Asp Ala Pro Gly Gln 1 5
9512PRTArtificialLinker 95Gly Gly Gly Gly Ser Arg Leu Asp Ala Pro
Gly Gln 1 5 10 9617PRTArtificialLinker 96Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Arg Leu Asp Ala Pro Gly 1 5 10 15 Gln
9712PRTArtificialLoop 97Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly 1 5 10 9812PRTArtificialLoop 98Ala Lys Pro Glu Lys Trp Asp Gly
Ser Ile Tyr Gly 1 5 10 9911PRTArtificialLoop 99Ser Pro Gly Glu Arg
Ile Trp Met Phe Thr Gly 1 5 10 1009PRTArtificialLoop 100His Asp Ala
Phe Gly Tyr Asp Phe Gly 1 5 10112PRTArtificialLoop 101Ile Pro Pro
His Asn Ala Asp Ser Ser Ile Ile Gly 1 5 10 1026PRTArtificialLoop
102Gln Gln Lys His Thr Ala 1 5 1036PRTArtificialLoop 103Asn Ser Arg
His Thr Ala 1 5 1046PRTArtificialLoop 104Pro Asp His Phe His Asn 1
5 1056PRTArtificialLoop 105Tyr Asp Val Ala Phe Asp 1 5
10611PRTArtificialLoop 106Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro
Ala 1 5 10 10710PRTArtificialLoop 107Pro Asn Tyr Glu Arg Ile Ser
Asn Pro Ala 1 5 10 10811PRTArtificialLoop 108Phe Asp Pro Tyr Gly
Met Arg Ser Lys Pro Ala 1 5 10 10911PRTArtificialLoop 109Phe Thr
Pro Tyr Gly Ala Lys Ser Asn Pro Ala 1 5 10 11011PRTArtificialLoop
110Ala Asn Asp His Gly Phe Asp Ser Asn Pro Ala 1 5 10
11111PRTArtificialLoop 111Asp Thr Phe Tyr Gly Phe Asp Ser Asn Pro
Ala 1 5 10 11287DNAArtificialPrimer 112ggcgctaggc tgagtaggtc
ctggagtgcg gccatggcca gcgggggcgg agggagtgcc 60attgaagtga aagatgtgac
cgatacc 8711382DNAArtificialPrimer 113cctcagccga tcaccacctg
aaggctacgc aggtaccgct accgccacct ccgctcccac 60cgccaccggt ggtaaaggtt
tc 8211431DNAArtificialPrimer 114ggcgctaggc tgagtaggtc ctggagtgcg g
3111533DNAArtificialPrimer 115cctcagccga tcaccacctg aaggctacgc agg
3311689DNAArtificialPrimer 116gggatccgct acgggccact cgatcgaggt
ccgtgctgat cgagcgatcg gtaccctggg 60ccatcatcat catcatcacc accactgag
8911797DNAArtificialPrimer 117aattctcagt ggtggtgatg atgatgatga
tggcccaggg taccgatcgc tcgatcagca 60cggacctcga tcgagtggcc cgtagcggat
cccgtac 9711842DNAArtificialPrimer 118ggcgctaggc tgagtaggtc
ctggggatcc gccatggcca gc 4211942DNAArtificialPrimer 119ggcgctaggc
tgagtaggtc ctggctagct gccatggcca gc 4212037DNAArtificialPrimer
120cctcagccga tcaccacctg aaggcggcgc cggtacc
3712181DNAArtificialPrimer 121aaagaaacct ttaccactgc aggtggcgga
ggttcacgct tggatgcccc cgggcagatt 60gaagtgaaag atgtgaccga t
81122111DNAArtificialPrimer 122aaagaaacct ttaccactgc aggtggcgga
ggttcaggtg gcggaggttc aggtggcgga 60ggttcacgct tggatgcccc cgggcagatt
gaagtgaaag atgtgaccga t 11112324DNAArtificialPrimer 123ctgcagtggt
aaaggtttct ttcg 24124116DNAArtificialPrimer 124aaagaaacct
ttaccactgc aggtggcggg ggtagcggtg gcggaggttc tggtggcggg 60ggtagcggtg
gcggaggttc tggtggcggg ggtagccgct tggatgcccc cgggca
116125146DNAArtificialPrimer 125aaagaaacct ttaccactgc aggtggcggg
ggtagcggtg gcggaggttc tggtggcggg 60ggtagcggtg gcggaggttc tggtggcggg
ggtagcggtg gcggaggttc tggtggcggg 120ggtagccgct tggatgcccc cgggca
14612624DNAArtificialPrimer 126aaagaaacct ttaccactgc aggt
2412724DNAArtificialPrimer 127ttcaatctgc ccgggggcat ccaa
2412866DNAArtificialPrimer 128aaagaaacct ttaccaccac gcgtttggat
gcccccgggc agattgaagt gaaagatgtg 60accgat
6612923DNAArtificialPrimer 129cgtggtggta aaggtttctt tcg
2313096DNAArtificialPrimer 130aaagaaacct ttaccactgc aggtggcgga
ggttcaggtg gcggaggttc acgcttggat 60gcccccgggc agattgaagt gaaagatgtg
accgat 961317PRTHomo sapiens 131Cys Glu Leu Ala Tyr Gly Ile 1 5
132102PRTHomo sapiens 132Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Glu Lys Trp Asp Gly
Ser Ile Tyr Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn 35 40 45 Ser Arg His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu
Val Ser Leu Ile Cys Phe Thr Pro Tyr Gly Ala Lys Ser Asn 65 70 75 80
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His 85
90 95 His His His His His His 100 133102PRTHomo sapiens 133Ala Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15
Ala Lys Pro Glu Lys Trp Asp Pro Pro Leu Trp Gly Cys Glu Leu Ala 20
25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu
Asn 35 40 45 Ser Arg His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe Thr Pro Tyr
Gly Ala Lys Ser Asn 65 70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr Gly
Gly Gly Thr Leu Gly His His 85 90 95 His His His His His His 100
134102PRTHomo sapiens 134Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln 35 40 45 Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn 65 70 75 80
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His 85
90 95 His His His His His His 100 135102PRTHomo sapiens 135Ala Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15
Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20
25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu
Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr
Asn Lys Arg Asn Val 65 70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr Gly
Gly Gly Thr Leu Gly His His 85 90 95 His His His His His His 100
136102PRTHomo sapiens 136Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln 35 40 45 Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Asn His Arg Ser Leu 65 70 75 80
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His 85
90 95 His His His His His His 100 137102PRTHomo sapiens 137Ala Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15
Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20
25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu
Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr
Gly Leu Lys Ser Arg 65 70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr Gly
Gly Gly Thr Leu Gly His His 85 90 95 His His His His His His 100
138102PRTHomo sapiens 138Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln 35 40 45 Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser Lys 65 70 75 80
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His 85
90 95 His His His His His His 100 139219PRTHomo sapiens 139Ser Gly
Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr 1 5 10 15
Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu 20
25 30 Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp
Arg 35 40 45 Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser
Ile Gly Asn 50 55 60 Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu
Ile Cys Phe Asp Pro 65 70 75 80 Tyr Gly Ala Lys Ser Asn Pro Ala Lys
Glu Thr Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Ala Ile Glu 100 105 110 Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro 115 120 125 Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile 130 135 140 Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His 145 150
155 160 Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu
Val 165 170 175 Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn
Pro Ala Lys 180 185 190 Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 195 200 205 Thr Leu Gly His His His His His His
His His 210 215 140425PRTHomo sapiens 140Ser Gly Gly Gly Gly Ser
Ala Ile Glu Val Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu Ile
Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu 20 25 30 Trp Gly
Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 35 40 45
Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn 50
55 60 Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp
Pro 65 70 75 80 Tyr Gly Ala Lys Ser Asn Pro Ala Lys Glu Thr Phe Thr
Thr Gly Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ala Ile Glu 100 105 110 Val Lys Asp Val Thr Asp Thr Thr Ala
Leu Ile Thr Trp Ala Lys Pro 115 120 125 Trp Val Asp Pro Pro Pro Leu
Trp Gly Cys Glu Leu Ala Tyr Gly Ile 130 135 140 Lys Asp Val Pro Gly
Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His 145 150 155 160 Thr Ala
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 165 170 175
Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro Ala Lys 180
185 190 Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 195 200 205 Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp
Thr Thr Ala 210 215 220 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro
Pro Pro Leu Trp Gly 225 230 235 240 Cys Glu Leu Ala Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr 245 250 255 Ile Asp Leu Gln Gln Lys
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys 260 265 270 Pro Asp Thr Glu
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly 275 280 285 Ala Lys
Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 290 295 300
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys 305
310 315 320 Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro
Trp Val 325 330 335 Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr
Gly Ile Lys Asp 340 345 350 Val Pro Gly Asp Arg Thr Thr Ile Asp Leu
Gln Gln Lys His Thr Ala 355 360 365 Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu Tyr Glu Val Ser Leu 370 375 380 Ile Cys Phe Asp Pro Tyr
Gly Ala Lys Ser Asn Pro Ala Lys Glu Thr 385 390 395 400 Phe Thr Thr
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Thr Leu 405 410 415 Gly
His His His His His His His His 420 425 141639PRTHomo sapiens
141Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr
1 5 10 15 Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu 20 25 30 Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg 35 40 45 Thr Thr Ile Asp Leu Gln Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn 50 55 60 Leu Lys Pro Asp Thr Glu Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro 65 70 75 80 Tyr Gly Ala Lys Ser Asn
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu 100 105 110 Val Lys
Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro 115 120 125
Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile 130
135 140 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys
His 145 150 155 160 Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr Glu Val 165 170 175 Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala
Lys Ser Asn Pro Ala Lys 180 185
190 Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
195 200 205 Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala 210 215 220 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly 225 230 235 240 Cys Glu Leu Ala Tyr Gly Ile Lys Asp
Val Pro Gly Asp Arg Thr Thr 245 250 255 Ile Asp Leu Gln Gln Lys His
Thr Ala Tyr Ser Ile Gly Asn Leu Lys 260 265 270 Pro Asp Thr Glu Tyr
Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly 275 280 285 Ala Lys Ser
Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 290 295 300 Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys 305 310
315 320 Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp
Val 325 330 335 Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly
Ile Lys Asp 340 345 350 Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln
Gln Lys His Thr Ala 355 360 365 Tyr Ser Ile Gly Asn Leu Lys Pro Asp
Thr Glu Tyr Glu Val Ser Leu 370 375 380 Ile Cys Phe Asp Pro Tyr Gly
Ala Lys Ser Asn Pro Ala Lys Glu Thr 385 390 395 400 Phe Thr Thr Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Thr Gly 405 410 415 Ser Ala
Met Ala Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp 420 425 430
Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp 435
440 445 Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp
Val 450 455 460 Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His
Thr Ala Tyr 465 470 475 480 Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu
Tyr Glu Val Ser Leu Ile 485 490 495 Cys Phe Asp Pro Tyr Gly Ala Lys
Ser Asn Pro Ala Lys Glu Thr Phe 500 505 510 Thr Thr Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 515 520 525 Ser Ala Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 530 535 540 Trp Ala
Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 545 550 555
560 Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu
565 570 575 Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr 580 585 590 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr
Gly Ala Lys Ser 595 600 605 Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly
Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gly Thr Leu Gly His
His His His His His His His 625 630 635 142845PRTHomo sapiens
142Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr
1 5 10 15 Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu 20 25 30 Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg 35 40 45 Thr Thr Ile Asp Leu Gln Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn 50 55 60 Leu Lys Pro Asp Thr Glu Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro 65 70 75 80 Tyr Gly Ala Lys Ser Asn
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu 100 105 110 Val Lys
Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro 115 120 125
Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile 130
135 140 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys
His 145 150 155 160 Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr Glu Val 165 170 175 Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala
Lys Ser Asn Pro Ala Lys 180 185 190 Glu Thr Phe Thr Thr Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 195 200 205 Gly Gly Gly Ser Ala Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala 210 215 220 Leu Ile Thr Trp
Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly 225 230 235 240 Cys
Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr 245 250
255 Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys
260 265 270 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly 275 280 285 Ala Lys Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Gly 290 295 300 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Ile Glu Val Lys 305 310 315 320 Asp Val Thr Asp Thr Thr Ala
Leu Ile Thr Trp Ala Lys Pro Trp Val 325 330 335 Asp Pro Pro Pro Leu
Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp 340 345 350 Val Pro Gly
Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala 355 360 365 Tyr
Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu 370 375
380 Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro Ala Lys Glu Thr
385 390 395 400 Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Thr Gly 405 410 415 Ser Ala Met Ala Ser Gly Gly Gly Gly Ser Ala
Ile Glu Val Lys Asp 420 425 430 Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala Lys Pro Trp Val Asp 435 440 445 Pro Pro Pro Leu Trp Gly Cys
Glu Leu Ala Tyr Gly Ile Lys Asp Val 450 455 460 Pro Gly Asp Arg Thr
Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr 465 470 475 480 Ser Ile
Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile 485 490 495
Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro Ala Lys Glu Thr Phe 500
505 510 Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly 515 520 525 Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala
Leu Ile Thr 530 535 540 Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu
Trp Gly Cys Glu Leu 545 550 555 560 Ala Tyr Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Asp Leu 565 570 575 Gln Gln Lys His Thr Ala
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 580 585 590 Glu Tyr Glu Val
Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser 595 600 605 Asn Pro
Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly 610 615 620
Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr 625
630 635 640 Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp
Pro Pro 645 650 655 Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys
Asp Val Pro Gly 660 665 670 Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys
His Thr Ala Tyr Ser Ile 675 680 685 Gly Asn Leu Lys Pro Asp Thr Glu
Tyr Glu Val Ser Leu Ile Cys Phe 690 695 700 Asp Pro Tyr Gly Ala Lys
Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr 705 710 715 720 Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 725 730 735 Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala 740 745
750 Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr
755 760 765 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu
Gln Gln 770 775 780 Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu Tyr 785 790 795 800 Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly Ala Lys Ser Asn Pro 805 810 815 Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Gly Ser Gly Gly Gly Gly 820 825 830 Ser Gly Thr Leu Gly
His His His His His His His His 835 840 845 143425PRTHomo sapiens
143Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr
1 5 10 15 Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu 20 25 30 Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg 35 40 45 Thr Thr Ile Asp Leu Gln Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn 50 55 60 Leu Lys Pro Asp Thr Glu Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro 65 70 75 80 Tyr Gly Met Arg Ser Lys
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu 100 105 110 Val Lys
Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro 115 120 125
Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile 130
135 140 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys
His 145 150 155 160 Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr Glu Val 165 170 175 Ser Leu Ile Cys Phe Asp Pro Tyr Gly Met
Arg Ser Lys Pro Ala Lys 180 185 190 Glu Thr Phe Thr Thr Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 195 200 205 Gly Gly Gly Ser Ala Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala 210 215 220 Leu Ile Thr Trp
Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly 225 230 235 240 Cys
Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr 245 250
255 Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys
260 265 270 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly 275 280 285 Met Arg Ser Lys Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Gly 290 295 300 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Ile Glu Val Lys 305 310 315 320 Asp Val Thr Asp Thr Thr Ala
Leu Ile Thr Trp Ala Lys Pro Trp Val 325 330 335 Asp Pro Pro Pro Leu
Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp 340 345 350 Val Pro Gly
Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala 355 360 365 Tyr
Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu 370 375
380 Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro Ala Lys Glu Thr
385 390 395 400 Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Thr Leu 405 410 415 Gly His His His His His His His His 420 425
144639PRTHomo sapiens 144Ser Gly Gly Gly Gly Ser Ala Ile Glu Val
Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu Ile Thr Trp Ala Lys
Pro Trp Val Asp Pro Pro Pro Leu 20 25 30 Trp Gly Cys Glu Leu Ala
Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 35 40 45 Thr Thr Ile Asp
Leu Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn 50 55 60 Leu Lys
Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro 65 70 75 80
Tyr Gly Met Arg Ser Lys Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly 85
90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile
Glu 100 105 110 Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
Ala Lys Pro 115 120 125 Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala Tyr Gly Ile 130 135 140 Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Gln Gln Lys His 145 150 155 160 Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 165 170 175 Ser Leu Ile Cys
Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro Ala Lys 180 185 190 Glu Thr
Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 195 200 205
Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala 210
215 220 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly 225 230 235 240 Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly
Asp Arg Thr Thr 245 250 255 Ile Asp Leu Gln Gln Lys His Thr Ala Tyr
Ser Ile Gly Asn Leu Lys 260 265 270 Pro Asp Thr Glu Tyr Glu Val Ser
Leu Ile Cys Phe Asp Pro Tyr Gly 275 280 285 Met Arg Ser Lys Pro Ala
Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 290 295 300 Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys 305 310 315 320 Asp
Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val 325 330
335 Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp
340 345 350 Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His
Thr Ala 355 360 365 Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
Glu Val Ser Leu 370 375 380 Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser
Lys Pro Ala Lys Glu Thr 385 390 395 400 Phe Thr Thr Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Thr Gly 405 410 415 Ser Ala Met Ala Ser
Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp 420 425 430 Val Thr Asp
Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp 435 440 445 Pro
Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val 450 455
460 Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr
465 470 475 480 Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val
Ser Leu Ile 485 490 495 Cys Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro
Ala Lys Glu Thr Phe 500 505 510 Thr Thr Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 515 520 525 Ser Ala Ile Glu Val Lys Asp
Val Thr Asp Thr Thr Ala Leu Ile Thr 530 535 540 Trp Ala Lys Pro Trp
Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 545 550 555 560 Ala Tyr
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 565 570 575
Gln Gln Lys His Thr Ala Tyr Ser Ile
Gly Asn Leu Lys Pro Asp Thr 580 585 590 Glu Tyr Glu Val Ser Leu Ile
Cys Phe Asp Pro Tyr Gly Met Arg Ser 595 600 605 Lys Pro Ala Lys Glu
Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser
Gly Thr Leu Gly His His His His His His His His 625 630 635
145845PRTHomo sapiens 145Ser Gly Gly Gly Gly Ser Ala Ile Glu Val
Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu Ile Thr Trp Ala Lys
Pro Trp Val Asp Pro Pro Pro Leu 20 25 30 Trp Gly Cys Glu Leu Ala
Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 35 40 45 Thr Thr Ile Asp
Leu Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn 50 55 60 Leu Lys
Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro 65 70 75 80
Tyr Gly Met Arg Ser Lys Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly 85
90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile
Glu 100 105 110 Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
Ala Lys Pro 115 120 125 Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala Tyr Gly Ile 130 135 140 Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Gln Gln Lys His 145 150 155 160 Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 165 170 175 Ser Leu Ile Cys
Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro Ala Lys 180 185 190 Glu Thr
Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 195 200 205
Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala 210
215 220 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly 225 230 235 240 Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly
Asp Arg Thr Thr 245 250 255 Ile Asp Leu Gln Gln Lys His Thr Ala Tyr
Ser Ile Gly Asn Leu Lys 260 265 270 Pro Asp Thr Glu Tyr Glu Val Ser
Leu Ile Cys Phe Asp Pro Tyr Gly 275 280 285 Met Arg Ser Lys Pro Ala
Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 290 295 300 Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys 305 310 315 320 Asp
Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val 325 330
335 Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp
340 345 350 Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His
Thr Ala 355 360 365 Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
Glu Val Ser Leu 370 375 380 Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser
Lys Pro Ala Lys Glu Thr 385 390 395 400 Phe Thr Thr Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Thr Gly 405 410 415 Ser Ala Met Ala Ser
Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp 420 425 430 Val Thr Asp
Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp 435 440 445 Pro
Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val 450 455
460 Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr
465 470 475 480 Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val
Ser Leu Ile 485 490 495 Cys Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro
Ala Lys Glu Thr Phe 500 505 510 Thr Thr Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 515 520 525 Ser Ala Ile Glu Val Lys Asp
Val Thr Asp Thr Thr Ala Leu Ile Thr 530 535 540 Trp Ala Lys Pro Trp
Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 545 550 555 560 Ala Tyr
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 565 570 575
Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 580
585 590 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Met Arg
Ser 595 600 605 Lys Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly
Ser Gly Gly 610 615 620 Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu
Val Lys Asp Val Thr 625 630 635 640 Asp Thr Thr Ala Leu Ile Thr Trp
Ala Lys Pro Trp Val Asp Pro Pro 645 650 655 Pro Leu Trp Gly Cys Glu
Leu Ala Tyr Gly Ile Lys Asp Val Pro Gly 660 665 670 Asp Arg Thr Thr
Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser Ile 675 680 685 Gly Asn
Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe 690 695 700
Asp Pro Tyr Gly Met Arg Ser Lys Pro Ala Lys Glu Thr Phe Thr Thr 705
710 715 720 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala 725 730 735 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr Trp Ala 740 745 750 Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly Cys Glu Leu Ala Tyr 755 760 765 Gly Ile Lys Asp Val Pro Gly Asp
Arg Thr Thr Ile Asp Leu Gln Gln 770 775 780 Lys His Thr Ala Tyr Ser
Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 785 790 795 800 Glu Val Ser
Leu Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro 805 810 815 Ala
Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly 820 825
830 Ser Gly Thr Leu Gly His His His His His His His His 835 840 845
146417PRTHomo sapiens 146Ser Gly Gly Gly Gly Ser Ala Ile Glu Val
Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu Ile Thr Trp Ser Pro
Gly Glu Arg Ile Trp Met Phe Thr 20 25 30 Gly Cys Glu Leu Thr Tyr
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr 35 40 45 Thr Ile Asp Leu
Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu 50 55 60 Lys Pro
Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu 65 70 75 80
Arg Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 85
90 95 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val
Lys 100 105 110 Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser Pro
Gly Glu Arg 115 120 125 Ile Trp Met Phe Thr Gly Cys Glu Leu Thr Tyr
Gly Ile Lys Asp Val 130 135 140 Pro Gly Asp Arg Thr Thr Ile Asp Leu
Thr Glu Asp Glu Asn Gln Tyr 145 150 155 160 Ser Ile Gly Asn Leu Lys
Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile 165 170 175 Cys Pro Asn Tyr
Glu Arg Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr 180 185 190 Thr Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 195 200 205
Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 210
215 220 Ser Pro Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu Leu Thr
Tyr 225 230 235 240 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile
Asp Leu Thr Glu 245 250 255 Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu
Lys Pro Asp Thr Glu Tyr 260 265 270 Glu Val Ser Leu Ile Cys Pro Asn
Tyr Glu Arg Ile Ser Asn Pro Ala 275 280 285 Lys Glu Thr Phe Thr Thr
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 290 295 300 Gly Gly Gly Gly
Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr 305 310 315 320 Ala
Leu Ile Thr Trp Ser Pro Gly Glu Arg Ile Trp Met Phe Thr Gly 325 330
335 Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
340 345 350 Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn
Leu Lys 355 360 365 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Pro
Asn Tyr Glu Arg 370 375 380 Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr
Thr Gly Gly Gly Gly Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Thr
Leu Gly His His His His His His His 405 410 415 His 147626PRTHomo
sapiens 147Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr
Asp Thr 1 5 10 15 Thr Ala Leu Ile Thr Trp Ser Pro Gly Glu Arg Ile
Trp Met Phe Thr 20 25 30 Gly Cys Glu Leu Thr Tyr Gly Ile Lys Asp
Val Pro Gly Asp Arg Thr 35 40 45 Thr Ile Asp Leu Thr Glu Asp Glu
Asn Gln Tyr Ser Ile Gly Asn Leu 50 55 60 Lys Pro Asp Thr Glu Tyr
Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu 65 70 75 80 Arg Ile Ser Asn
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 85 90 95 Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys 100 105 110
Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser Pro Gly Glu Arg 115
120 125 Ile Trp Met Phe Thr Gly Cys Glu Leu Thr Tyr Gly Ile Lys Asp
Val 130 135 140 Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu
Asn Gln Tyr 145 150 155 160 Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu
Tyr Glu Val Ser Leu Ile 165 170 175 Cys Pro Asn Tyr Glu Arg Ile Ser
Asn Pro Ala Lys Glu Thr Phe Thr 180 185 190 Thr Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 195 200 205 Ala Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 210 215 220 Ser Pro
Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu Leu Thr Tyr 225 230 235
240 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu
245 250 255 Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr 260 265 270 Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu Arg Ile
Ser Asn Pro Ala 275 280 285 Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 290 295 300 Gly Gly Gly Gly Ser Ala Ile Glu
Val Lys Asp Val Thr Asp Thr Thr 305 310 315 320 Ala Leu Ile Thr Trp
Ser Pro Gly Glu Arg Ile Trp Met Phe Thr Gly 325 330 335 Cys Glu Leu
Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr 340 345 350 Ile
Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys 355 360
365 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu Arg
370 375 380 Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly
Gly Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Thr Gly Ser Ala Met
Ala Ser Gly Gly Gly 405 410 415 Gly Ser Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr 420 425 430 Trp Ser Pro Gly Glu Arg Ile
Trp Met Phe Thr Gly Cys Glu Leu Thr 435 440 445 Tyr Gly Ile Lys Asp
Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr 450 455 460 Glu Asp Glu
Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 465 470 475 480
Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu Arg Ile Ser Asn Pro 485
490 495 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly
Gly 500 505 510 Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val
Thr Asp Thr 515 520 525 Thr Ala Leu Ile Thr Trp Ser Pro Gly Glu Arg
Ile Trp Met Phe Thr 530 535 540 Gly Cys Glu Leu Thr Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr 545 550 555 560 Thr Ile Asp Leu Thr Glu
Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu 565 570 575 Lys Pro Asp Thr
Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu 580 585 590 Arg Ile
Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 595 600 605
Ser Gly Gly Gly Gly Ser Gly Thr Leu Gly His His His His His His 610
615 620 His His 625 148829PRTHomo sapiens 148Ser Gly Gly Gly Gly
Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu
Ile Thr Trp Ser Pro Gly Glu Arg Ile Trp Met Phe Thr 20 25 30 Gly
Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr 35 40
45 Thr Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu
50 55 60 Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn
Tyr Glu 65 70 75 80 Arg Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Gly 85 90 95 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Ile Glu Val Lys 100 105 110 Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr Trp Ser Pro Gly Glu Arg 115 120 125 Ile Trp Met Phe Thr Gly
Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val 130 135 140 Pro Gly Asp Arg
Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr 145 150 155 160 Ser
Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile 165 170
175 Cys Pro Asn Tyr Glu Arg Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr
180 185 190 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 195 200 205 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala
Leu Ile Thr Trp 210 215 220 Ser Pro Gly Glu Arg Ile Trp Met Phe Thr
Gly Cys Glu Leu Thr Tyr 225 230 235 240 Gly Ile Lys Asp Val Pro Gly
Asp Arg Thr Thr Ile Asp Leu Thr Glu 245 250 255 Asp Glu Asn Gln Tyr
Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 260 265 270 Glu Val Ser
Leu Ile Cys Pro Asn Tyr Glu Arg Ile Ser Asn Pro Ala 275 280 285 Lys
Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 290 295
300 Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr
305 310 315 320 Ala Leu Ile Thr Trp Ser Pro Gly Glu Arg Ile Trp Met
Phe Thr Gly 325 330 335 Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr 340
345 350 Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu
Lys 355 360 365 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn
Tyr Glu Arg 370 375 380 Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Gly Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Thr Gly
Ser Ala Met Ala Ser Gly Gly Gly 405 410 415 Gly Ser Ala Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile 420 425 430 Thr Trp Ser Pro
Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu Leu 435 440 445 Thr Tyr
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 450 455 460
Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 465
470 475 480 Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu Arg Ile
Ser Asn 485 490 495 Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly
Ser Gly Gly Gly 500 505 510 Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu
Val Lys Asp Val Thr Asp 515 520 525 Thr Thr Ala Leu Ile Thr Trp Ser
Pro Gly Glu Arg Ile Trp Met Phe 530 535 540 Thr Gly Cys Glu Leu Thr
Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 545 550 555 560 Thr Thr Ile
Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn 565 570 575 Leu
Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr 580 585
590 Glu Arg Ile Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly
595 600 605 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile
Glu Val 610 615 620 Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
Ser Pro Gly Glu 625 630 635 640 Arg Ile Trp Met Phe Thr Gly Cys Glu
Leu Thr Tyr Gly Ile Lys Asp 645 650 655 Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Thr Glu Asp Glu Asn Gln 660 665 670 Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu 675 680 685 Ile Cys Pro
Asn Tyr Glu Arg Ile Ser Asn Pro Ala Lys Glu Thr Phe 690 695 700 Thr
Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 705 710
715 720 Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile
Thr 725 730 735 Trp Ser Pro Gly Glu Arg Ile Trp Met Phe Thr Gly Cys
Glu Leu Thr 740 745 750 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Thr 755 760 765 Glu Asp Glu Asn Gln Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu 770 775 780 Tyr Glu Val Ser Leu Ile Cys
Pro Asn Tyr Glu Arg Ile Ser Asn Pro 785 790 795 800 Ala Lys Glu Thr
Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly 805 810 815 Ser Gly
Thr Leu Gly His His His His His His His His 820 825 149321PRTHomo
sapiens 149Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala 1 5 10 15 Lys Pro Glu Lys Trp Asp Gly Ser Ile Tyr Gly Cys
Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Asn Ser 35 40 45 Arg His Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys
Phe Thr Pro Tyr Gly Ala Lys Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr
Phe Thr Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys 85 90 95 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 100 105 110
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115
120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 130 135 140 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 165 170 175 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 225 230 235
240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 Lys 150321PRTHomo
sapiens 150Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala 1 5 10 15 Lys Pro Glu Lys Trp Asp Pro Pro Leu Trp Gly Cys
Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Asn Ser 35 40 45 Arg His Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys
Phe Thr Pro Tyr Gly Ala Lys Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr
Phe Thr Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys 85 90 95 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 100 105 110
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115
120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 130 135 140 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 165 170 175 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 225 230 235
240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 Lys 151321PRTHomo
sapiens 151Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala 1 5 10 15 Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys
Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Gln Gln 35 40 45 Lys His Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys
Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr
Phe Thr Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys 85 90 95 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 100 105 110
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115
120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 130 135 140 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 165 170 175 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 225 230 235
240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 Lys 152321PRTHomo
sapiens 152Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala 1 5 10 15 Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys
Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Gln Gln 35 40 45 Lys His Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys
Phe Asp Pro Tyr Asn Lys Arg Asn Val Pro 65 70 75 80 Ala Lys Glu Thr
Phe Thr Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys 85 90 95 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 100 105 110
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115
120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 130 135 140 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 165 170 175 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 225 230 235
240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 Lys 153321PRTHomo
sapiens 153Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala 1 5 10 15 Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys
Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu Gln Gln 35 40 45 Lys His Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys
Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro 65 70 75 80 Ala Lys Glu Thr
Phe Thr Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys 85 90 95 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 100 105 110
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115
120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 130 135 140 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 165 170 175 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 225 230 235
240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 Lys 154427PRTHomo
sapiens 154Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr 1 5 10 15 Trp Ala Lys Pro Glu Lys Trp Asp Gly Ser Ile Tyr
Gly Cys Glu Leu 20 25 30 Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp
Arg Thr Thr Ile Asp Leu 35 40 45 Asn Ser Arg His Thr Ala Tyr Ser
Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu
Ile Cys Phe Thr Pro Tyr Gly Ala Lys Ser 65 70 75 80 Asn Pro Ala Lys
Glu Thr Phe Thr Thr Gly Gly Gly Thr Pro Thr Ser 85 90 95 Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 100 105 110
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 115
120 125 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr 130 135 140 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr 145 150 155 160 Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln 165 170 175 Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp 180 185 190 Lys Arg Val Glu Pro Lys
Ser Cys Asp
Lys Thr His Thr Cys Pro Pro 195 200 205 Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro 210 215 220 Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 225 230 235 240 Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 245 250 255
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 260
265 270 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val 275 280 285 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser 290 295 300 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 305 310 315 320 Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu 325 330 335 Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe 340 345 350 Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 355 360 365 Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 370 375 380
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 385
390 395 400 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr 405 410 415 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 420
425 155202PRTHomo sapiens 155Ser Gln Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Glu Lys Trp
Asp Gly Ser Ile Tyr Gly Cys Glu Leu 20 25 30 Ala Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 35 40 45 Asn Ser Arg
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu
Tyr Glu Val Ser Leu Ile Cys Phe Thr Pro Tyr Gly Ala Lys Ser 65 70
75 80 Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Pro Thr
Arg 85 90 95 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 100 105 110 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 115 120 125 Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser 130 135 140 Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 145 150 155 160 Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 165 170 175 His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 180 185 190
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195 200 156427PRTHomo
sapiens 156Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr 1 5 10 15 Trp Ala Lys Pro Glu Lys Trp Asp Pro Pro Leu Trp
Gly Cys Glu Leu 20 25 30 Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp
Arg Thr Thr Ile Asp Leu 35 40 45 Asn Ser Arg His Thr Ala Tyr Ser
Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu
Ile Cys Phe Thr Pro Tyr Gly Ala Lys Ser 65 70 75 80 Asn Pro Ala Lys
Glu Thr Phe Thr Thr Gly Gly Gly Thr Pro Thr Ser 85 90 95 Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 100 105 110
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 115
120 125 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr 130 135 140 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr 145 150 155 160 Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln 165 170 175 Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp 180 185 190 Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro 195 200 205 Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 210 215 220 Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 225 230 235
240 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
245 250 255 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 260 265 270 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 275 280 285 Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser 290 295 300 Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys 305 310 315 320 Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 325 330 335 Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 340 345 350 Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 355 360
365 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
370 375 380 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly 385 390 395 400 Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 405 410 415 Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 420 425 157202PRTHomo sapiens 157Ser Gln Ile Glu Val Lys
Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro
Glu Lys Trp Asp Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Ala Tyr
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 35 40 45
Asn Ser Arg His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50
55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Thr Pro Tyr Gly Ala Lys
Ser 65 70 75 80 Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr
Pro Thr Arg 85 90 95 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 100 105 110 Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 115 120 125 Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 130 135 140 Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 145 150 155 160 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 165 170 175
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 180
185 190 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195 200
158427PRTHomo sapiens 158Ser Gln Ile Glu Val Lys Asp Val Thr Asp
Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp Val Asp Pro
Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Ala Tyr Gly Ile Lys Asp
Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 35 40 45 Gln Gln Lys His
Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr
Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser 65 70 75 80
Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Pro Thr Ser 85
90 95 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser 100 105 110 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp 115 120 125 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr 130 135 140 Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr 145 150 155 160 Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 165 170 175 Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 180 185 190 Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 195 200 205
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 210
215 220 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr 225 230 235 240 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 245 250 255 Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg 260 265 270 Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val 275 280 285 Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 290 295 300 Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 305 310 315 320 Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 325 330
335 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
340 345 350 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu 355 360 365 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe 370 375 380 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly 385 390 395 400 Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 405 410 415 Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 420 425 159202PRTHomo sapiens 159Ser Gln
Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15
Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20
25 30 Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp
Leu 35 40 45 Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys
Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly Ala Lys Ser 65 70 75 80 Asn Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Thr Pro Thr Arg 85 90 95 Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 100 105 110 Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125 Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 130 135 140 Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 145 150
155 160 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys 165 170 175 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro 180 185 190 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195
200 160427PRTHomo sapiens 160Ser Gln Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Ala Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 35 40 45 Gln Gln Lys
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Asn Lys Arg Asn 65 70
75 80 Val Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Pro Thr
Ser 85 90 95 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser 100 105 110 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp 115 120 125 Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr 130 135 140 Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr 145 150 155 160 Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 165 170 175 Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 180 185 190
Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 195
200 205 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro 210 215 220 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr 225 230 235 240 Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn 245 250 255 Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg 260 265 270 Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val 275 280 285 Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 290 295 300 Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 305 310 315
320 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
325 330 335 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe 340 345 350 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu 355 360 365 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe 370 375 380 Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly 385 390 395 400 Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr 405 410 415 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 420 425 161202PRTHomo sapiens
161Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
1 5 10 15 Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys
Glu Leu 20 25 30 Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu 35 40 45 Gln Gln Lys His Thr Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys
Phe Asp Pro Tyr Asn Lys Arg Asn 65 70 75 80 Val Pro Ala Lys Glu Thr
Phe Thr Thr Gly Gly Gly Thr Pro Thr Arg 85 90 95 Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 100 105 110 Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 130
135 140 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 145 150 155 160 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys 165
170 175 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 180 185 190 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195 200
162427PRTHomo sapiens 162Ser Gln Ile Glu Val Lys Asp Val Thr Asp
Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp Val Asp Pro
Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Ala Tyr Gly Ile Lys Asp
Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 35 40 45 Gln Gln Lys His
Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr
Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser 65 70 75 80
Lys Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Pro Thr Ser 85
90 95 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser 100 105 110 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp 115 120 125 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr 130 135 140 Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr 145 150 155 160 Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 165 170 175 Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 180 185 190 Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 195 200 205
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 210
215 220 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr 225 230 235 240 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 245 250 255 Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg 260 265 270 Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val 275 280 285 Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 290 295 300 Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 305 310 315 320 Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 325 330
335 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
340 345 350 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu 355 360 365 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe 370 375 380 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly 385 390 395 400 Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 405 410 415 Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 420 425 163202PRTHomo sapiens 163Ser Gln
Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15
Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20
25 30 Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp
Leu 35 40 45 Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys
Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly Met Arg Ser 65 70 75 80 Lys Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Thr Pro Thr Arg 85 90 95 Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 100 105 110 Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125 Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 130 135 140 Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 145 150
155 160 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys 165 170 175 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro 180 185 190 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195
200 164446PRTHomo sapiens 164Ala Met Ala Ser Gly Gly Gly Gly Ser
Ala Ile Glu Val Lys Asp Val 1 5 10 15 Thr Asp Thr Thr Ala Leu Ile
Thr Trp Ala Lys Pro Trp Val Asp Pro 20 25 30 Pro Pro Leu Trp Gly
Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro 35 40 45 Gly Asp Arg
Thr Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser 50 55 60 Ile
Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys 65 70
75 80 Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro Ala Lys Glu Thr Phe
Thr 85 90 95 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 100 105 110 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr
Ala Leu Ile Thr Trp 115 120 125 Ala Lys Pro Trp Val Asp Pro Pro Pro
Leu Trp Gly Cys Glu Leu Ala 130 135 140 Tyr Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Asp Leu Gln 145 150 155 160 Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 165 170 175 Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn 180 185 190
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly 195
200 205 Gly Ser Gly Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys Thr His
Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315
320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
165652PRTHomo sapiens 165Ala Met Ala Ser Gly Gly Gly Gly Ser Ala
Ile Glu Val Lys Asp Val 1 5 10 15 Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ala Lys Pro Trp Val Asp Pro 20 25 30 Pro Pro Leu Trp Gly Cys
Glu Leu Ala Tyr Gly Ile Lys Asp Val Pro 35 40 45 Gly Asp Arg Thr
Thr Ile Asp Leu Gln Gln Lys His Thr Ala Tyr Ser 50 55 60 Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys 65 70 75 80
Phe Asp Pro Tyr Gly Ala Lys Ser Asn Pro Ala Lys Glu Thr Phe Thr 85
90 95 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 100 105 110 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr Trp 115 120 125 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly Cys Glu Leu Ala 130 135 140 Tyr Gly Ile Lys Asp Val Pro Gly Asp
Arg Thr Thr Ile Asp Leu Gln 145 150 155 160 Gln Lys His Thr Ala Tyr
Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 165 170 175 Tyr Glu Val Ser
Leu Ile Cys Phe Asp Pro Tyr Gly Ala Lys Ser Asn 180 185 190 Pro Ala
Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly 195 200 205
Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp 210
215 220 Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro 225 230 235 240 Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp
Val Pro Gly Asp 245 250 255 Arg Thr Thr Ile Asp Leu Gln Gln Lys His
Thr Ala Tyr Ser Ile Gly 260 265 270 Asn Leu Lys Pro Asp Thr Glu Tyr
Glu Val Ser Leu Ile Cys Phe Asp 275 280 285 Pro Tyr Gly Ala Lys Ser
Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly 290 295 300 Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile 305 310 315 320 Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys 325 330
335 Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly
340 345 350 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln
Gln Lys 355 360 365 His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp
Thr Glu Tyr Glu 370 375 380 Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly
Ala Lys Ser Asn Pro Ala 385 390 395 400 Lys Glu Thr Phe Thr Thr Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 405 410 415 Gly Thr Gly Ala Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 420 425 430 Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 435 440 445 Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 450 455
460 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
465 470 475 480 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro 485 490 495 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr 500 505 510 Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val 515 520 525 Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 530 535 540 Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 545 550 555 560 Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 565 570 575
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 580
585 590 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser 595 600 605 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 610 615 620 Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 625 630 635 640 Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 645 650 166770PRTHomo sapiens 166Ser Gln Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala
Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30
Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu 35
40 45 Gln Gln Lys His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp
Thr 50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly
Leu Lys Ser 65 70 75 80 Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly
Gly Gly Ser Gly Gly 85 90 95 Gly Gly Ser Ile Glu Val Lys Asp Val
Thr Asp Thr Thr Ala Leu Ile 100 105 110 Thr Trp Ala Lys Pro Trp Val
Asp Pro Pro Pro Leu Trp Gly Cys Glu 115 120 125 Leu Thr Tyr Gly Ile
Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly 130 135 140 Leu Gln Gln
Lys His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp 145 150 155 160
Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu Lys 165
170 175 Ser Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser
Gly 180 185 190 Gly Gly Gly Ser Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu 195 200 205 Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly Cys 210 215 220 Glu Leu Thr Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile 225 230 235 240 Gly Leu Gln Gln Lys His
Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro 245 250 255 Asp Thr Glu Tyr
Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu 260 265 270 Lys Ser
Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser 275 280 285
Gly Gly Gly Gly Ser Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala 290
295 300 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly 305 310 315 320 Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly
Asp Arg Thr Thr 325 330 335 Ile Gly Leu Gln Gln Lys His Asn Gln Tyr
Ser Ile Gly Asn Leu Lys 340 345 350 Pro Asp Thr Glu Tyr Glu Val Ser
Leu Ile Cys Phe Asp Pro Tyr Gly 355 360 365 Leu Lys Ser Arg Pro Ala
Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly
Gly Ser Ile Glu Val Lys Asp Val Thr Asp Thr Thr 385 390 395 400 Ala
Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp 405 410
415 Gly Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
420 425 430 Thr Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser Ile Gly
Asn Leu 435 440 445 Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys
Phe Asp Pro Tyr 450 455 460 Gly Leu Lys Ser Arg Pro Ala Lys Glu Thr
Phe Thr Thr Gly Gly Gly 465 470 475 480 Gly Ser Gly Gly Gly Gly Ser
Ile Glu Val Lys Asp Val Thr Asp Thr 485 490 495 Thr Ala Leu Ile Thr
Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu 500 505 510 Trp Gly Cys
Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 515 520 525 Thr
Thr Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser Ile
Gly Asn 530 535 540 Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile
Cys Phe Asp Pro 545 550 555 560 Tyr Gly Leu Lys Ser Arg Pro Ala Lys
Glu Thr Phe Thr Thr Gly Gly 565 570 575 Gly Gly Ser Gly Gly Gly Gly
Ser Ile Glu Val Lys Asp Val Thr Asp 580 585 590 Thr Thr Ala Leu Ile
Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro 595 600 605 Leu Trp Gly
Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp 610 615 620 Arg
Thr Thr Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser Ile Gly 625 630
635 640 Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe
Asp 645 650 655 Pro Tyr Gly Leu Lys Ser Arg Pro Ala Lys Glu Thr Phe
Thr Thr Gly 660 665 670 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Glu
Val Lys Asp Val Thr 675 680 685 Asp Thr Thr Ala Leu Ile Thr Trp Ala
Lys Pro Trp Val Asp Pro Pro 690 695 700 Pro Leu Trp Gly Cys Glu Leu
Thr Tyr Gly Ile Lys Asp Val Pro Gly 705 710 715 720 Asp Arg Thr Thr
Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser Ile 725 730 735 Gly Asn
Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe 740 745 750
Asp Pro Tyr Gly Leu Lys Ser Arg Pro Ala Lys Glu Thr Phe Thr Thr 755
760 765 Gly Leu 770 167576PRTHomo sapiens 167Ser Gln Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys
Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Thr
Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu 35 40
45 Gln Gln Lys His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu
Lys Ser 65 70 75 80 Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly
Gly Ser Gly Gly 85 90 95 Gly Gly Ser Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile 100 105 110 Thr Trp Ala Lys Pro Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu 115 120 125 Leu Thr Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Gly 130 135 140 Leu Gln Gln Lys
His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp 145 150 155 160 Thr
Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu Lys 165 170
175 Ser Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly
180 185 190 Gly Gly Gly Ser Ile Glu Val Lys Asp Val Thr Asp Thr Thr
Ala Leu 195 200 205 Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro
Leu Trp Gly Cys 210 215 220 Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile 225 230 235 240 Gly Leu Gln Gln Lys His Asn
Gln Tyr Ser Ile Gly Asn Leu Lys Pro 245 250 255 Asp Thr Glu Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu 260 265 270 Lys Ser Arg
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser 275 280 285 Gly
Gly Gly Gly Ser Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala 290 295
300 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly
305 310 315 320 Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp
Arg Thr Thr 325 330 335 Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser
Ile Gly Asn Leu Lys 340 345 350 Pro Asp Thr Glu Tyr Glu Val Ser Leu
Ile Cys Phe Asp Pro Tyr Gly 355 360 365 Leu Lys Ser Arg Pro Ala Lys
Glu Thr Phe Thr Thr Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly
Ser Ile Glu Val Lys Asp Val Thr Asp Thr Thr 385 390 395 400 Ala Leu
Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp 405 410 415
Gly Cys Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr 420
425 430 Thr Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser Ile Gly Asn
Leu 435 440 445 Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr 450 455 460 Gly Leu Lys Ser Arg Pro Ala Lys Glu Thr Phe
Thr Thr Gly Gly Gly 465 470 475 480 Gly Ser Gly Gly Gly Gly Ser Ile
Glu Val Lys Asp Val Thr Asp Thr 485 490 495 Thr Ala Leu Ile Thr Trp
Ala Lys Pro Trp Val Asp Pro Pro Pro Leu 500 505 510 Trp Gly Cys Glu
Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 515 520 525 Thr Thr
Ile Gly Leu Gln Gln Lys His Asn Gln Tyr Ser Ile Gly Asn 530 535 540
Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro 545
550 555 560 Tyr Gly Leu Lys Ser Arg Pro Ala Lys Glu Thr Phe Thr Thr
Gly Leu 565 570 575 16812PRTHomo sapiens 168Ala Lys Pro Glu Lys Trp
Asp Gly Pro Pro Leu Trp 1 5 10 16911PRTHomo sapiens 169Phe Asp Pro
Tyr Asn Lys Arg Asn Val Pro Ala 1 5 10 17011PRTHomo sapiens 170Phe
Asp Pro Tyr Gly Leu Lys Ser Arg Pro Ala 1 5 10 17188PRTHomo sapiens
171Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Glu Lys Trp Asp Gly Ser Ile Tyr Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Asn 35 40 45 Ser Arg His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Thr Pro Tyr Gly Ala Lys Ser Asn 65 70 75 80 Pro Ala Lys Glu Thr Phe
Thr Thr 85 17288PRTHomo sapiens 172Ala Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Glu Lys Trp
Asp Pro Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn 35 40 45 Ser Arg
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60
Tyr Glu Val Ser Leu Ile Cys Phe Thr Pro Tyr Gly Ala Lys Ser Asn 65
70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr 85 17388PRTHomo sapiens
173Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr Gly Ala Lys Ser Asn 65 70 75 80 Pro Ala Lys Glu Thr Phe
Thr Thr 85 17488PRTHomo sapiens 174Ala Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln 35 40 45 Gln Lys
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Asn Lys Arg Asn Val 65
70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr 85 17588PRTHomo sapiens
175Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr Gly Met Arg Ser Lys 65 70 75 80 Pro Ala Lys Glu Thr Phe
Thr Thr 85 17688PRTHomo sapiens 176Ala Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln 35 40 45 Gln Lys
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu Lys Ser Arg 65
70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr 85 17788PRTHomo sapiens
177Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Gly Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr Gly Leu Lys Ser Arg 65 70 75 80 Pro Ala Lys Glu Thr Phe
Thr Thr 85 17888PRTHomo sapiens 178Ala Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys
Asp Val Ser Gly Asp Arg Thr Thr Ile Gly Leu Gln 35 40 45 Gln Lys
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Arg Lys Ser Gln 65
70 75 80 Pro Thr Lys Glu Thr Phe Thr Thr 85 1796PRTHomo sapiens
179Gln Gln Lys His Asn Gln 1 5 180100PRTHomo sapiens 180Ala Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser
Pro Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu Leu Thr Tyr 20 25
30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu
35 40 45 Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu Arg Ile
Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr
Leu Gly His His His His 85 90 95 His His His His 100 181319PRTHomo
sapiens 181Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Ser 1 5 10 15 Pro Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu
Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Thr Glu Asp 35 40 45 Glu Asn Gln Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Pro
Asn Tyr Glu Arg Ile Ser Asn Pro Ala Lys 65 70 75 80 Glu Thr Phe Thr
Thr Gly Ala Glu Pro Lys Ser Cys Asp Lys Thr His 85 90 95 Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 100 105 110
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 115
120 125 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu 130 135 140 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys 145 150 155 160 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 165 170 175 Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 180 185 190 Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 195 200 205 Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 210 215 220 Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 225 230 235
240 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
245 250 255 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser 260 265 270 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 275 280 285 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 290 295 300 His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 305 310 315 182425PRTHomo sapiens
182Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
1 5 10 15 Trp Ser Pro Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu
Leu Thr 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Asp Leu Thr 35 40 45 Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Pro
Asn Tyr Glu Arg Ile Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr
Thr Gly Gly Gly Thr Pro Thr Ser Ser Ala 85 90 95 Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 100 105 110 Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 115 120 125
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 130
135 140 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu 145 150 155 160 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr 165 170 175 Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg 180 185 190 Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro 195 200 205 Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 245 250
255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
260 265 270 Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 325 330
335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 370 375 380 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu
Ser Pro Gly Lys 420 425 183200PRTHomo sapiens 183Ser Gln Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ser
Pro Gly Glu Arg Ile Trp Met Phe Thr Gly Cys Glu Leu Thr 20 25 30
Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr 35
40 45 Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Pro Asn Tyr Glu Arg Ile
Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr
Pro Thr Arg Thr Val 85 90 95 Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys 100 105 110 Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg 115 120 125 Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 130 135 140 Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 145 150 155 160
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 165
170 175 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr 180 185 190 Lys Ser Phe Asn Arg Gly Glu Cys 195 200
184101PRTHomo sapiens 184Ile Glu Val Lys Asp Val Thr Asp Thr Thr
Ala Leu Ile Thr Trp Ile 1 5 10 15 Pro Pro His Asn Ala Asp Ser Ser
Ile Ile Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Asp Leu Tyr Asp 35 40 45 Val Ala Phe Asp
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val
Ser Leu Ile Cys Asp Thr Phe Tyr Gly Phe Asp Ser Asn Pro 65 70 75 80
Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85
90 95 His His His His His 100 185101PRTHomo sapiens 185Ala Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Asp
Leu Ala Ala Tyr Ser Val Ala Tyr Asn Gly Cys Glu Leu Thr Tyr 20 25
30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Phe Asn
35 40 45 Asp Asn Asn Pro Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Asn Asn Phe Tyr Gly Ile
Leu Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly
Thr Leu Gly His His His 85 90 95 His His His His His 100
186100PRTHomo sapiens 186Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Val Pro Pro Ile Asn Ile Asn Gly
Val Ile Ala Gly Cys Glu Leu Thr 20 25 30 Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Phe 35 40 45 Asp Phe Ser Tyr
Val Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu
Val Ser Leu Ile Cys Phe Thr Thr Asn Gly Asp Ala Val Ser 65 70 75 80
Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His 85
90 95 His His His His 100 187101PRTHomo sapiens 187Ala Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Asn Pro
Ser Val Leu Ser Val Tyr Tyr His Gly Cys Glu Leu Thr Tyr 20 25 30
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Phe Leu 35
40 45 Asp Val Ser Pro Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu
Tyr 50 55 60 Glu Val Ser Leu Ile Cys Ser Thr Phe Thr Gly Asp Tyr
Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr
Leu Gly His His His 85 90 95 His His His His His 100 18898PRTHomo
sapiens 188Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile
Thr Trp 1 5 10 15 Phe Phe Pro Tyr Ala Tyr His Asp Gly Cys Glu Leu
Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile
Asp Leu Asp Leu Leu Leu 35 40 45 Pro Phe Tyr Ser Ile Gly Asn Leu
Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Ile Thr
Asn Ser Phe Asp Gly Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr
Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His
189101PRTHomo sapiens 189Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Phe Pro His Phe Leu Asp Val
Leu Thr Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Asp Leu Asp Asn 35 40 45 Asp Tyr Ile Ala
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val
Ser Leu Ile Cys Phe Lys Asp Leu Gly Thr Tyr Ser Asn Pro 65 70 75 80
Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85
90 95 His His His His His 100 190100PRTHomo sapiens 190Ala Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ile
Pro Pro Asn Asn Ile Gly Gly Phe Ile Leu Gly Cys Glu Leu Thr 20 25
30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Tyr
35 40 45 Asp Phe Ala Asn Ile Tyr Ser Ile Gly Asn Leu Lys Pro Asp
Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Ile Thr Asn Asn Gly
Tyr Leu Asn Ser 65 70 75 80 Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr
Leu Gly His His His His 85 90 95 His His His His 100 19199PRTHomo
sapiens 191Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile
Thr Trp 1 5 10 15 Asp Ile Ser Leu Asp Phe Asp Tyr Ser Ile Gly Cys
Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr
Thr Ile Asp Leu His Tyr 35 40 45 Phe Asp Phe Ala Tyr Ser Ile Gly
Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys
Phe Thr Phe Asp Gly Phe Asn Val Ala Lys 65 70 75 80 Glu Thr Phe Thr
Thr Gly Gly Gly Thr Leu Gly His His His His His 85 90 95 His His
His 192639PRTHomo sapiens 192Ser Gly Gly Gly Gly Ser Ala Ile Glu
Val Lys Asp Val Thr Asp Thr 1 5 10 15 Thr Ala Leu Ile Thr Trp Ala
Lys Pro Trp Val Asp Pro Pro Pro Leu 20 25 30 Trp Gly Cys Glu Leu
Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg 35 40 45 Thr Thr Ile
Asp Leu Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn 50 55 60 Leu
Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro 65 70
75 80 Tyr Gly Met Arg Ser Lys Pro Ala Lys Glu Thr Phe Thr Thr Gly
Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ala Ile Glu 100 105 110 Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile
Thr Trp Ala Lys Pro 115 120 125 Trp Val Asp Pro Pro Pro Leu Trp Gly
Cys Glu Leu Ala Tyr Gly Ile 130 135 140 Lys Asp Val Pro Gly Asp Arg
Thr Thr Ile Asp Leu Gln Gln Lys His 145 150 155 160 Thr Ala Tyr Ser
Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 165 170 175 Ser Leu
Ile Cys Phe Asp Pro Tyr Gly Met Arg Ser Lys Pro Ala Lys 180 185 190
Glu Thr Phe Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 195
200 205 Gly Gly Gly Ser Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr
Ala 210 215 220 Leu Ile Thr Trp Ala Lys Pro Trp Val Asp Pro Pro Pro
Leu Trp Gly 225 230 235 240 Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr 245 250 255 Ile Asp Leu Gln Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys 260 265 270 Pro Asp Thr Glu Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly 275 280 285 Met Arg Ser Lys
Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Gly 290 295 300 Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys 305 310 315
320 Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val
325 330 335 Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile
Lys Asp 340 345 350 Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln
Lys His Thr Ala 355 360 365 Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr Glu Val Ser Leu 370 375 380 Ile Cys Phe Asp Pro Tyr Gly Met
Arg Ser Lys Pro Ala Lys Glu Thr 385 390 395 400 Phe Thr Thr Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Thr Gly 405 410 415 Ser Ala Met
Ala Ser Gly Gly Gly Gly Ser Ala Ile Glu Val Lys Asp 420 425 430 Val
Thr Asp Thr Thr Ala Leu Ile Thr Trp Ala Lys Pro Trp Val Asp 435 440
445 Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala Tyr Gly Ile Lys Asp Val
450 455 460 Pro Gly Asp Arg Thr Thr Ile Asp Leu Gln Gln Lys His Thr
Ala Tyr 465 470 475 480 Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
Glu Val Ser Leu Ile 485 490 495 Cys Phe Asp Pro Tyr Gly Met Arg Ser
Lys Pro Ala Lys Glu Thr Phe 500 505 510 Thr Thr Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 515 520 525 Ser Ala Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 530 535 540 Trp Ala Lys
Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 545 550 555 560
Ala Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 565
570 575 Gln Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp
Thr 580 585 590 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly
Met Arg Ser 595 600 605 Lys Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly
Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gly Thr Leu Gly His His
His His His His His His 625 630 635 193102PRTHomo sapiens 193Ala
Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10
15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala
20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly
Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys
Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly Leu Lys Ser Arg 65 70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr
Gly Gly Gly Thr Leu Gly His His 85 90 95 His His His His His His
100 194103PRTHomo sapiens 194Ser Gln Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp Val Asp
Pro Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Thr Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu 35 40 45 Gln Gln Lys
His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu Lys Ser 65 70
75 80 Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Leu Gly Thr Leu Gly
His 85 90 95 His His His His His His His 100 19588PRTHomo sapiens
195Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Gly Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr Gly Arg Ser Ser Arg 65 70 75 80 Arg Ile Lys Glu Thr Phe
Thr Thr 85 19688PRTHomo sapiens 196Ala Ile Glu Val Lys Asp Val Thr
Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Gly Gln Pro Trp Val Ser
Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys
Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu Gln 35 40 45 Gln Lys
His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60
Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu His Ser Arg 65
70 75 80 Leu Thr Lys Glu Thr Phe Thr Thr 85 19788PRTHomo sapiens
197Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Gly Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr Gly Arg Lys Ser Gln 65 70 75 80 Pro Thr Lys Glu Thr Phe
Thr Thr
85 19888PRTHomo sapiens 198Ala Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Gly Leu Gln 35 40 45 Gln Lys His Thr
Ala Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Met Lys Ser Lys 65 70 75 80
Pro Ser Lys Glu Thr Phe Thr Thr 85 19988PRTHomo sapiens 199Ala Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15
Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu Ala 20
25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu
Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr
Gly Gly Lys Ser Arg 65 70 75 80 Pro Thr Lys Glu Thr Phe Thr Thr 85
20088PRTHomo sapiens 200Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr
Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro
Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Gly Leu Gln 35 40 45 Gln Lys His Thr Ala
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val
Ser Leu Ile Cys Phe Asp Pro Tyr Gly His Ser Thr His 65 70 75 80 Pro
Val Lys Gly Thr Phe Thr Thr 85 20187PRTHomo sapiens 201Ser Gln Ile
Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp
Phe Lys Pro Leu Ala Glu Ile Asp Gly Cys Glu Leu Thr Tyr Gly 20 25
30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp
35 40 45 Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu
Tyr Glu 50 55 60 Val Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser
Asn Pro Ala Lys 65 70 75 80 Glu Thr Phe Thr Thr Gly Leu 85
20290PRTHomo sapiens 202Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr
Ala Leu Ile Thr Trp 1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro
Leu Trp Gly Cys Glu Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Asp Leu Gln 35 40 45 Gln Lys His Thr Ala
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val
Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu Lys Ser Arg 65 70 75 80 Pro
Ala Lys Glu Thr Phe Thr Thr Gly Gly 85 90 20390PRTHomo sapiens
203Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15 Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu
Leu Ala 20 25 30 Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr
Ile Gly Leu Gln 35 40 45 Gln Lys His Thr Ala Tyr Ser Ile Gly Asn
Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Phe
Asp Pro Tyr Gly Leu Lys Ser Arg 65 70 75 80 Pro Ala Lys Glu Thr Phe
Thr Thr Gly Gly 85 90 20491PRTHomo sapiens 204Ser Gln Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys
Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Thr
Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu 35 40
45 Gln Gln Lys His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu
Lys Ser 65 70 75 80 Arg Pro Ala Lys Glu Thr Phe Thr Thr Gly Leu 85
90 20591PRTHomo sapiens 205Ser Gln Ile Glu Val Lys Asp Val Thr Asp
Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Gly Gln Pro Trp Val Ser Pro
Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Thr Tyr Gly Ile Lys Asp
Val Pro Gly Asp Arg Thr Thr Ile Gly Leu 35 40 45 Gln Gln Lys His
Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr
Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu His Ser 65 70 75 80
Arg Leu Thr Lys Glu Thr Phe Thr Thr Gly Leu 85 90 20691PRTHomo
sapiens 206Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp
Gly Cys Glu Leu 20 25 30 Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp
Arg Thr Thr Ile Gly Leu 35 40 45 Gln Gln Lys His Asn Gln Tyr Ser
Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu
Ile Cys Phe Asp Pro Tyr Gly Arg Lys Ser 65 70 75 80 Gln Pro Thr Lys
Glu Thr Phe Thr Thr Gly Leu 85 90 20791PRTHomo sapiens 207Ser Gln
Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15
Trp Ala Lys Pro Trp Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20
25 30 Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly
Leu 35 40 45 Gln Gln Lys His Asn Gln Tyr Ser Ile Gly Asn Leu Lys
Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro
Tyr Gly Gly Lys Ser 65 70 75 80 Arg Pro Thr Lys Glu Thr Phe Thr Thr
Gly Leu 85 90 20891PRTHomo sapiens 208Ser Gln Ile Glu Val Lys Asp
Val Thr Asp Thr Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp
Val Asp Pro Pro Pro Leu Trp Gly Cys Glu Leu 20 25 30 Thr Tyr Gly
Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Gly Leu 35 40 45 Gln
Gln Lys His Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55
60 Glu Tyr Glu Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Met Lys Ser
65 70 75 80 Lys Pro Ser Lys Glu Thr Phe Thr Thr Gly Leu 85 90
20989PRTHomo sapiens 209Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr
Thr Ala Leu Ile Thr 1 5 10 15 Trp Ala Lys Pro Trp Val Asp Pro Pro
Pro Leu Trp Gly Cys Glu Leu 20 25 30 Thr Tyr Gly Ile Lys Asp Val
Pro Gly Asp Arg Thr Thr Ile Gly Leu 35 40 45 Gln Gln Lys His Asn
Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr Glu
Val Ser Leu Ile Cys Phe Asp Pro Tyr Gly Leu Lys Ser 65 70 75 80 Arg
Pro Ala Lys Glu Thr Phe Thr Thr 85 21014PRTHomo sapiens 210Val Thr
Leu Arg Gly Asp Trp Ser Glu Asp Ser Lys Pro Ile 1 5 10 21114PRTHomo
sapiens 211Val Thr Val Arg Gly Asp Trp Tyr Glu Tyr Ser Lys Pro Ile
1 5 10 21214PRTHomo sapiens 212Val Thr Gly Arg Gly Asp Trp Thr Glu
His Ser Lys Pro Ile 1 5 10 21314PRTHomo sapiens 213Val Thr Ala Arg
Gly Asp Trp Val Glu Gly Ser Lys Pro Ile 1 5 10 21414PRTHomo sapiens
214Val Thr Pro Arg Gly Asp Trp Thr Glu Gly Ser Lys Pro Ile 1 5 10
21514PRTHomo sapiens 215Val Thr Pro Arg Gly Asp Trp Ile Glu Phe Ser
Lys Pro Ile 1 5 10 21614PRTHomo sapiens 216Val Thr Gly Arg Gly Asp
Trp Asn Glu Gly Ser Lys Pro Ile 1 5 10 21714PRTHomo sapiens 217Val
Thr Phe Arg Gly Asp Trp Ile Glu Leu Ser Lys Pro Ile 1 5 10
21814PRTHomo sapiens 218Val Ala Ala Thr Pro Trp Thr Trp Val Leu Arg
Glu Thr Ser 1 5 10 21914PRTHomo sapiens 219Val Ala Ala Thr Pro Trp
Val Leu Ile Thr Arg Ser Thr Ser 1 5 10 2209PRTHomo sapiens 220Asp
Ala Pro Trp Tyr Gln Gly Arg Tyr 1 5 22114PRTHomo sapiens 221Val Thr
Gly Arg Leu Arg Ala Gln Leu Val Ser Lys Pro Ile 1 5 10 2229PRTHomo
sapiens 222Asp Ala Pro Pro Arg Thr Lys Gln Tyr 1 5 22314PRTHomo
sapiens 223Val Thr Gly Arg Leu Arg Asp Leu Leu Gln Ser Lys Pro Ile
1 5 10 22423PRTHomo sapiens 224Val Thr Gly Leu Val Arg Phe Arg Val
Val Asn Ser Ser Leu Cys Met 1 5 10 15 Trp Ala Arg Ser Lys Pro Ile
20 2259PRTHomo sapiens 225Arg His Pro His Phe Pro Thr Arg Tyr 1 5
2266PRTHomo sapiens 226Pro Leu Gln Pro Pro Leu 1 5 22714PRTHomo
sapiens 227Val Thr Lys Glu Arg Asn Gly Arg Glu Leu Phe Thr Pro Ile
1 5 10 2286PRTHomo sapiens 228Pro Leu Gln Pro Pro Thr 1 5
22914PRTHomo sapiens 229Val Thr Asp Gly Arg Asn Gly Arg Leu Leu Ser
Ile Pro Ile 1 5 10 23015PRTHomo sapiens 230Val Thr Met Gly Leu Tyr
Gly His Glu Leu Leu Thr Pro Pro Ile 1 5 10 15 23114PRTHomo sapiens
231Val Thr Asp Gly Glu Asn Gly Gln Phe Leu Leu Val Pro Ile 1 5 10
2329PRTHomo sapiens 232His Glu Arg Asp Gly Ser Arg Gln Tyr 1 5
2336PRTHomo sapiens 233Pro Gly Gly Val Arg Thr 1 5 23419PRTHomo
sapiens 234Val Thr Asp Tyr Phe Asn Pro Thr Thr His Glu Tyr Ile Tyr
Gln Thr 1 5 10 15 Thr Pro Ile 2359PRTHomo sapiens 235Trp Ala Pro
Val Asp Arg Tyr Gln Tyr 1 5 2366PRTHomo sapiens 236Pro Arg Asp Val
Tyr Thr 1 5 23719PRTHomo sapiens 237Val Thr Asp Tyr Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu 1 5 10 15 Ser Pro Ile 2389PRTHomo
sapiens 238Thr Gln Gly Ser Thr His Tyr Gln Tyr 1 5 2396PRTHomo
sapiens 239Pro Gly Met Val Tyr Thr 1 5 24019PRTHomo sapiens 240Val
Thr Asp Tyr Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr Arg Thr 1 5 10
15 Thr Pro Ile 2419PRTHomo sapiens 241Tyr Trp Glu Gly Leu Pro Tyr
Gln Tyr 1 5 2426PRTHomo sapiens 242Pro Arg Asp Val Asn Thr 1 5
24319PRTHomo sapiens 243Val Thr Asp Trp Tyr Asn Pro Asp Thr His Glu
Tyr Ile Tyr His Thr 1 5 10 15 Ile Pro Ile 2449PRTHomo sapiens
244Ser Pro Tyr Leu Arg Val Ala Arg Tyr 1 5 2456PRTHomo sapiens
245Pro Ser Ser Ala Arg Thr 1 5 24614PRTHomo sapiens 246Val Thr Pro
Ser Asn Ile Ile Gly Arg His Tyr Gly Pro Ile 1 5 10 2479PRTHomo
sapiens 247Val Asn Asp Pro Gln Arg Asn Arg Tyr 1 5 2486PRTHomo
sapiens 248Pro Ala Tyr Tyr Pro Thr 1 5 24914PRTHomo sapiens 249Val
Thr Tyr Ser His Ile Lys Tyr Leu Tyr His Lys Pro Ile 1 5 10
2509PRTHomo sapiens 250Ser Asp Ser Leu Lys Val Ser Arg Tyr 1 5
2516PRTHomo sapiens 251Pro Lys Gln Tyr His Thr 1 5 25214PRTHomo
sapiens 252Val Thr Pro Ser Asn Ile Ile Gly Arg His Tyr Gly Pro Ile
1 5 10 2539PRTHomo sapiens 253Ser Ala Pro Leu Lys Val Ala Arg Tyr 1
5 2546PRTHomo sapiens 254Pro Lys Asn Val Tyr Thr 1 5 25510PRTHomo
sapiens 255Val Thr Lys Met Arg Asp Tyr Arg Pro Ile 1 5 10
25689PRTHomo sapiens 256Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Thr Ala Pro Asp
Ala Ala Phe Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser 65 70 75 80 Asn
Pro Leu Ser Ala Glu Phe Thr Thr 85 2577PRTHomo sapiens 257Ser Glu
Val Thr Glu Asp Ser 1 5 2589PRTHomo sapiens 258Thr Ala Pro Asp Ala
Ala Phe Asp Ser 1 5 2597PRTHomo sapiens 259Ser Glu Lys Val Gly Glu
Ala 1 5 2606PRTHomo sapiens 260Pro Gly Ser Glu Arg Ser 1 5
2618PRTHomo sapiens 261Thr Gly Leu Lys Pro Gly Thr Glu 1 5
26210PRTHomo sapiens 262Val Lys Gly Gly His Arg Ser Asn Pro Leu 1 5
10 2639PRTHomo sapiens 263Leu Pro Ala Pro Lys Asn Leu Val Val 1 5
2643PRTHomo sapiens 264Leu Val Val 1 2655PRTHomo sapiens 265Leu Arg
Leu Ser Trp 1 5 2667PRTHomo sapiens 266Phe Leu Ile Gln Tyr Gln Glu
1 5 2675PRTHomo sapiens 267Ile Asn Leu Thr Val 1 5 2683PRTHomo
sapiens 268Tyr Asp Leu 1 2697PRTHomo sapiens 269Tyr Thr Val Ser Ile
Tyr Gly 1 5 2706PRTHomo sapiens 270Ser Ala Glu Phe Thr Thr 1 5
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