U.S. patent application number 13/266430 was filed with the patent office on 2013-01-31 for ptp1b inhibitors.
This patent application is currently assigned to Cold Spring Harbor Laboratory. The applicant listed for this patent is Aftabul Haque, Nicholas Tonks. Invention is credited to Aftabul Haque, Nicholas Tonks.
Application Number | 20130029366 13/266430 |
Document ID | / |
Family ID | 42536360 |
Filed Date | 2013-01-31 |
United States Patent
Application |
20130029366 |
Kind Code |
A9 |
Tonks; Nicholas ; et
al. |
January 31, 2013 |
PTP1B INHIBITORS
Abstract
Described herein are agents, including antibodies, antibody
fragments, polypeptides and organic small molecules, that bind
reversibly inactive PTP1B (designated herein in the alternative as
PTP1B-SN or PTP1B-OX) and stabilize it (stabilize PTP1B-OX) in such
a manner that they inhibit reactivation of reversibly oxidized,
inactive PTP1B by reduction (by reducing agent) and have no
substantial direct inhibitory effect on phosphatase activity/PTP1B
activity (for example, as detectable in assays in vitro).
Inventors: |
Tonks; Nicholas; (Stony
Brook, NY) ; Haque; Aftabul; (Stony Brook,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tonks; Nicholas
Haque; Aftabul |
Stony Brook
Stony Brook |
NY
NY |
US
US |
|
|
Assignee: |
Cold Spring Harbor
Laboratory
Cold Spring Harbor
NY
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120164673 A1 |
June 28, 2012 |
|
|
Family ID: |
42536360 |
Appl. No.: |
13/266430 |
Filed: |
April 27, 2010 |
PCT Filed: |
April 27, 2010 |
PCT NO: |
PCT/US10/01251 PCKC 00 |
371 Date: |
March 9, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61173178 |
Apr 27, 2009 |
|
|
|
Current U.S.
Class: |
435/21 ; 435/188;
506/9; 530/387.3; 530/389.1 |
Current CPC
Class: |
C07K 16/40 20130101;
A61K 2039/505 20130101; C07K 2317/80 20130101; C07K 2317/622
20130101; G01N 33/573 20130101; G01N 2500/04 20130101; G01N
2333/9121 20130101 |
Class at
Publication: |
435/21 ;
530/389.1; 530/387.3; 506/9; 435/188 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C12Q 1/42 20060101 C12Q001/42; C40B 30/04 20060101
C40B030/04; C07K 16/40 20060101 C07K016/40; C07K 16/46 20060101
C07K016/46 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
CA53840 and grant GM55989, both awarded by the National Institutes
of Health. The government has certain rights in the invention.
Claims
1. An isolated antibody that specifically binds and stabilizes
PTP1B-OX or an antibody fragment that specifically binds and
stabilizes PTP1B-OX, but does not bind to or directly inhibit the
activity of reduced, active PTP1B.
2. The isolated antibody, or the antibody fragment of claim 1,
wherein the antibody comprises an amino acid sequence selected from
the scFv sequence of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or
SEQ ID NO: 34 to SEQ ID NO: 149 or is a variant antibody and
wherein the antibody fragment comprises a sufficient portion of the
scFv sequence of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ
ID NO: 34 to SEQ ID NO: 149 or is a variant antibody fragment.
3. The isolated antibody, or the antibody fragment of claim 1,
wherein the antibody comprises a V.sub.H fragment and/or a V.sub.L
fragment selected from the scFv sequence of any one of SEQ ID NO:
26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149 or a variant
of the scFv sequence and wherein the antibody fragment comprises a
V.sub.H fragment and/or a V.sub.L fragment selected from the scFv
sequence of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO:
34 to SEQ ID NO: 149 or a variant of the scFv sequence.
4. The isolated antibody, or the antibody fragment, of claim 1,
wherein the antibody or antigen binding fragment thereof is a
scFv.
5. (canceled)
6. An isolated PTP1B-OX binding polypeptide, comprising a CDR amino
acid sequence of the scFv sequence of any one of SEQ ID NO: 26 to
SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149.
7. An agent that specifically binds and stabilizes PTP1B-OX, but
does not bind to or directly inhibit the activity of reduced,
active PTP1B.
8. A method of identifying an agent that (a) specifically binds
PTP1B-OX, a mutant PTP1B that mimics the PTP1B-OX conformation or
both and (b) stabilizes PTP1B-OX, comprising: (a) combining (i)
PTP1B-OX and/or a mutant PTP1B that mimics the PTP1B-OX
conformation; (ii) a molecule that specifically binds and
stabilizes PTP1B-OX; and (iii) a candidate agent, under conditions
appropriate for binding of PTP1B-OX with the agent molecule that
specifically binds PTP1B-OX and stabilizes the conformation; (b)
assessing binding of the PTP1B-OX or mutant PTP1B that mimics the
PTP1B-OX conformation with the molecule a(ii); (c) comparing
binding assessed in (b) to (i) binding of PTP1B-OX or mutant PTP1B
that mimics the PTP1B-OX conformation with the molecule in the
absence of the candidate agent or (ii) a reference or control,
wherein if binding occurs to a lesser extent in the presence of the
candidate agent than in the absence of the candidate agent or to a
lesser extent as compared to the reference or control, the
candidate agent is an agent that specifically binds and stabilizes
PTP1B-OX or a mutant PTP1B that mimics PTP1B-OX conformation.
9. A method of identifying an agent that (a) specifically binds
PTP1B-OX, a mutant PTP1B that mimics the PTP1B-OX conformation or
both and (b) stabilizes PTP1B-OX, comprising: (a) combining (i)
PTP1B-OX and/or a mutant PTP1B that mimics the PTP1B-OX
conformation; (ii) an antibody that specifically binds and
stabilizes PTP1B-OX or an antigen-binding antibody fragment that
specifically binds and stabilizes PTP1B-OX; and (iii) a candidate
agent, under conditions appropriate for binding of PTP1B-OX with
the antibody or antigen-binding antibody fragment; (b) assessing
binding of PTP1B-OX or mutant PTP1B that mimics the PTP1B-OX
conformation with the antibody or antigen-binding antibody
fragment; (c) comparing binding assessed in (b) to (i) binding of
PTP1B-OX or mutant PTP1B that mimics the PTP1B-OX conformation with
the antibody or the antigen-binding fragment in the absence of the
candidate agent or (ii) a reference or control, wherein if binding
occurs to a lesser extent in the presence of the candidate agent
than in the absence of the candidate agent or to a lesser extent as
compared to the reference or control, the candidate agent is an
agent that specifically binds and stabilizes PTP1B-OX or a mutant
PTP1B that mimics the PTP1B-OX conformation.
10. (canceled)
11. The method of claim 9, wherein the antibody comprises an amino
acid sequence selected from the scFv sequence of any one of SEQ ID
NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149 or a
variant thereof or the antigen-binding fragment comprises a
sufficient portion of the scFv sequence of any one of SEQ ID NO: 26
to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149 or a variant
thereof.
12-13. (canceled)
14. A method of identifying an agent that displaces a molecule that
binds to and stabilizes PTP1B-OX and binds to and stabilizes
PTP1B-OX, comprising: (a) combining (i) PTP1B-OX or a mutant PTP1B
that mimics PTP1B-OX conformation with (ii) a molecule that
specifically binds and stabilizes PTP1B-OX thereby producing a
(PTP1B-OX or mutant PTP1B):(molecule) complex; (b) combining the
complex produced in (a) with a candidate agent, under conditions
appropriate for detection of displacement of the molecule from the
complex; (c) assessing displacement of the molecule from PTP1B-OX
or mutant PTP1B in the complex by the candidate agent, wherein
displacement has occurred, the candidate agent is an agent that
displaces a molecule that binds to and stabilizes PTP1B-OX and
binds to and stabilizes PTP1B-OX.
15. A method of identifying an agent that displaces an antibody or
antibody fragment that binds to and stabilizes PTP1B-OX and binds
to and stabilizes PTP1B-OX, comprising: (a) combining (i) PTP1B-OX
or a mutant PTP1B that mimics PTP1B-OX conformation with (ii) an
antibody or antibody fragment that specifically binds and
stabilizes PTP1B-OX thereby producing a (PTP1B-OX or mutant
PTP1B):(antibody or antibody fragment) complex; (b) combining the
complex produced in (a) with a candidate agent, under conditions
appropriate for detection of displacement of the antibody or
antibody fragment from the complex; (c) assessing displacement of
the antibody or antibody fragment from PTP1B-OX or mutant PTP1B in
the complex by the candidate agent, wherein displacement has
occurred, the candidate agent is an agent that displaces an
antibody or antibody fragment that binds to and stabilizes PTP1B-OX
and binds to and stabilizes PTP1B-OX.
16. (canceled)
17. The method of claim 14, wherein the antibody comprises an amino
acid sequence selected from the scFv sequence of any one of SEQ ID
NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149 or a
variant thereof or the antigen-binding fragment thereof comprises a
sufficient portion of the scFv sequence of any one of SEQ ID NO: 26
to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149 or a variant
thereof.
18-19. (canceled)
20. A method of stabilizing reversibly oxidized PTP1B (PTP1B-OX),
comprising combining reversibly oxidized PTP1B with an agent that
binds PTP1B-OX and inhibits reactivation of PTP1B-OX by a reducing
agent, under conditions under which the agent binds PTP1B-OX.
21. The method of claim 20, wherein the agent is an antibody that
specifically binds and stabilizes PTP1B-OX or an antigen-binding
antibody fragment that specifically binds and stabilizes
PTP1B-OX.
22. The method of claim 21, wherein the antibody is an antibody
that comprises an amino acid sequence selected from the scFv
sequence of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO:
34 to SEQ ID NO: 149 or a variant antibody or the antibody-binding
fragment is a fragment that comprises a sufficient portion of the
scFv sequence of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ
ID NO: 34 to SEQ ID NO: 149 or a variant antibody fragment.
23. The method of claim 20, wherein the agent is a small molecule
that binds and stabilizes PTP1B-OX.
24. The method of claim 20, wherein the reversibly oxidized PTP1B
is in a cell.
25-40. (canceled)
41. A method of identifying an agent that binds to and stabilizes
PTP1B-OX, but does not inhibit enzymatic activity of PTP1B in its
reduced, active form, comprising contacting a PTP1B molecule with
an agent that binds and stabilizes PTP1B-OX and measuring the
activity of said PTP1B in the presence of the agent, wherein, if
PTP1B activity is not decreased in the presence of the agent, the
agent is identified as an agent that binds to and stabilizes
PTP1B-OX, but does not directly inhibit enzymatic activity of
PTP1B.
42-43. (canceled)
44. An isolated PTP1B-OX:antibody complex or isolated
PTP1B-OX:antibody fragment complex.
45. (canceled)
46. A method of identifying a binding agent that (a) specifically
binds PTP1B-OX and (b) inhibits the reactivation of PTP1B-OX to
active, reduced PTP1B, comprising: (a) combining (i) PTP1B-OX and
(ii) a candidate binding agent under conditions appropriate for
reactivation of PTP1B-OX to active, reduced PTP1B; (b) assessing
PTP1B activity; and (c) comparing the PTP1B activity assessed in
(b) to (i) activity of PTP1B-OX under the same conditions but in
absence of the candidate binding agent or (ii) a reference or
control activity, wherein if PTP1B activity in the presence of the
candidate binding agent is lower than in the absence of the
candidate binding agent or than the reference or control level,
then the candidate binding agent is an agent that specifically
binds PTP1B-OX and inhibits the reactivation of PTP1B-OX to active,
reduced PTP1B.
47-53. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/173,178, filed Apr. 27, 2009. The entire
teachings of the referenced application are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
PTP1B Signaling
[0003] PTP1B is an intracellular protein tyrosine phosphatase. PTP
activity is regulated by modifications of several amino acid
residues in the polypeptide, such as phosphorylation of Ser
residues (Brautigan and Pinault, 1993; Dadke et al., 2001; Flint et
al., 1993) and oxidation of the active Cys residue in its catalytic
motif (Lee et al., 1998; Meng et al., 2002), which is
evolutionarily conserved among protein tyrosine phosphatases and
dual specificity phosphatase family members (Andersen et al.,
2001). In its reduced, active state, which is the basal state of
the enzyme, PTP1B regulates a number of signaling pathways. For
example, PTP1B activity antagonizes insulin signaling. Upon
reception of an insulin signal by a target cell, PTP1B is
reversibly oxidized, which removes the inhibitory regulation of
insulin signaling by PTP1B until the enzyme returns to its reduced,
active state. Other signaling pathways, for example EGF and leptin
signaling, are also modulated by PTP1B activity. PTP1B is indicated
in a number of human diseases, such as obesity and diabetes.
[0004] Many small molecule PTP1B inhibitors have been identified.
The catalytic pocket of PTP1B tends to be highly charged and, as a
result, small molecule inhibitors of PTP1B tend to be highly polar
molecules. Such compounds are not passively absorbed by the human
body and, thus, cannot be used as drugs via standard enteral or
parenteral delivery routes. Because of these characteristics, PTP1B
is seen to be an "undruggable" target (See, e.g., Cheng et al., Nat
Biotech 2007).
[0005] The inability to find drugs that target this well-validated
target is illustrated by the lack of clinical trials successfully
completed by any of the companies pursuing PTP inhibitors. The
identification and development of PTP inhibitors is highly
desirable in order to address the major unmet medical need to treat
PTP1B-related disorders, such as obesity and diabetes.
SUMMARY OF THE INVENTION
[0006] Described herein are agents, including antibodies, antibody
fragments, polypeptides and other molecules, such as small organic
compounds, that bind and stabilize reversibly oxidized, inactive
PTP in such a manner that they inhibit (partially or completely)
reactivation of the reversibly oxidized, inactive PTP1B by
reduction (by reducing agent) and do not directly inhibit PTP1B
activity (have no substantial direct inhibitory effect on
phosphatase activity, as assessed, for example, in assays in
vitro). Reversibly oxidized, inactive PTP is also referred to
herein as PTP in reversibly oxidized, inactive form; PTP1B in
cyclic sulphenamide conformation; and reversibly oxidized, inactive
conformation of PTP1B. All are designated in the alternative and
interchangeably herein as PTP1B-OX or PTP1B-SN. Without wishing to
be bound by theory, Applicant describes herein binding to and
stabilization of reversibly oxidized, inactive PTP1B. Methods of
binding to and stabilizing PTP1B-OX, as well as agents, such as
antibodies, antibody fragments, polypeptides and small molecules
(such as small organic molecules), that bind to and stabilize
PTP1B-OX and inhibit (partially or completely) its reactivation by
reduction are encompassed within the scope of the invention
described and claimed herein, regardless of the conformation of the
inactive form they bind and stabilize.
[0007] Also described herein are methods of identifying agents that
bind PTP1B-OX and stabilize it in its reversibly oxidized, inactive
conformation and methods of screening for molecules that bind,
modulate the stability of (e.g., stabilize) PTP1B-OX and do not
directly inhibit native PTP enzyme activity (enzyme activity of PTP
in its reduced, active form). Such agents may be any type of
molecule or belong to any class of molecules. For example, such
PTP1B-OX binding molecules may be amino acids, proteins,
polypeptides, peptides, antibodies, antibody fragments,
carbohydrates, glycans, nucleotides, nucleosides, nucleic acids,
saccharides, polysaccharides, glycoproteins, lipids, organic
compounds, such as small organic compounds, or any other molecule
that binds PTP1B-OX. Further, PTP1B-OX-binding molecules may be in
monomeric, dimeric, oligomeric or polymeric form; they may be amino
acids, dipeptides, oligopeptides, polypeptides or proteins and
analogues and derivatives thereof. Specific embodiments described
herein are methods of identifying agents, including small
molecules, that specifically bind PTP1B-OX and stabilize it in its
reversibly oxidized, inactive conformation and methods of screening
for small molecules that specifically bind PTP1B-OX, modulate the
stability of (e.g., stabilize) PTP1B-OX and do not directly inhibit
native PTP1B enzyme activity (enzyme activity of PTP1B in its
reduced, active form). Also described herein are methods of
stabilizing reversibly oxidized, inactive PTP1B (PTP1B-OX) and
inhibiting its reactivation; methods of modulating PTP1B signaling
by modulating (e.g., maintaining or enhancing) the stability of
PTP1B-OX (rather than by direct modulation of PTP1B enzyme
activity); methods of controlling the size of the pool of active
PTP1B in a cell, in which reduction of reversibly oxidized,
inactive PTP1B in the cell is hindered or decreased; methods of
prolonging inactivation of PTP1B-OX; methods of prolonging the
existence of PTP1B in its reversibly oxidized, inactive
conformation; and methods of inhibiting reactivation of the
reversibly oxidized, inactive form of PTP1B. Also described are
methods of controlling (modulating) PTP1B-mediated regulation of
signaling in a cell. Methods described herein apply to any pathway
in which a stimulus induces transient oxidation and inactivation of
a PTP, such as PTP1B (PTP1B-mediated regulation of any signal in a
cell that is in response to a stimulus that induces such transient
oxidation and inactivation of PTP1B), which, in turn, contributes
to enhanced phosphorylation of a member or a target of the
signaling pathway. Described herein are methods of controlling
(modulating) PTP1B-mediated regulation of insulin, EGF and leptin
signaling in cells; methods of controlling or modulating insulin,
EGF or leptin signaling-mediated redox regulation of PTP1B
activity; and methods of treating a condition (for example insulin
resistance, diabetes or obesity) in which PTP1-B-mediated
regulation of signaling is involved. Described herein are methods
of augmenting a hormone response that is inhibited by PTP1B
activity, for example methods of augmenting insulin response by
stabilizing reversibly oxidized, inactive PTP1B (PTP1B-OX).
[0008] PTP1B-OX-binding polypeptides include all polypeptides that
bind and stabilize PTP1B-OX. In certain embodiments,
PTP1B-OX-binding polypeptides bind PTP1B-OX specifically (bind
PTP1B-OX but do not bind PTP in its reduced state or
conformation/active form). In some embodiments, such
PTP1B-OX-binding polypeptides mimic the activity of an antibody or
antibody fragment provided herein. In some embodiments,
PTP1B-OX-binding polypeptides comprise a PTP1B-OX-binding domain or
PTP1B-OX targeting domain. In some embodiments, PTP1B-binding
polypeptides are fibronectin-like polypeptides comprising a
PTP1B-OX-binding domain or PTP1B-OX targeting domain. In specific
embodiments, PTP1B-OX binding polypeptides are adnectins or
antibodies. These antibodies, antibody fragments and
PTP1B-OX-binding polypeptides bind PTP1B-OX and stabilize the
reversibly oxidized, inactive form such that reduction and
reactivation of PTP1B-OX (and formation of reduced, active PTP1B)
is inhibited. Such antibodies, antibody fragments and polypeptides
can comprise any amino acid sequence, provided that the antibody,
antibody fragment or polypeptide binds reversibly oxidized,
inactive PTP1B (PTP1B-OX) and inhibits (partially or completely)
its reduction and reactivation (conversion of PTP1B-OX) to reduced,
active PTP1B. In certain embodiments, the antibodies, antibody
fragments or polypeptides specifically bind PTP1B-OX (bind PTP1B-OX
but do not bind PTP1B in its reduced state or conformation/active
form). Some embodiments include single chain Fvs (scFvs), for
example those provided herein (see scFvs included, as described
below, within SEQ ID NO: 26 to SEQ ID NO: 29 and SEQ ID NO: 34 to
SEQ ID NO: 149), which bind PTP1B-OX conformation and stabilize the
reversibly oxidized, inactive conformation. The sequences provided
in SEQ ID NO: 26 to SEQ ID NO: 29 and SEQ ID NO: 34 to SEQ ID
NO:149 represent scFv sequences flanked by an N-terminal leader
peptide and a C-terminal tag peptide. The N-terminal 22 amino acids
MKKTAIAIAVALAGFATVAQAA (SEQ ID NO: 150) and the C-terminal 23 amino
acids GQAGQHHHHHHGAYPYDVPDYAS (SEQ ID NO: 151) are coded for by
components of the phagemid pComb3XSS (SEQ ID NO: 152) or the
mammalian expression vector pcDNA3.2N5/GW/D-TOPO (SEQ ID NO: 23),
into which the scFv sequences were cloned. The sequences present
between these N- and C-terminal amino acids are the scFv sequences.
The sequences are displayed herein with the N-terminal leader
peptide, the C-terminal tag and the linker sequences underlined.
The linker sequence is used to join a variable light (V.sub.L)
chain and a variable heavy (V.sub.H) chain fragment to produce a
scFv.
[0009] The scFv sequences provided herein are of the general
pattern: NH.sub.2--V.sub.L-linker-V.sub.H--COOH. The N-terminal
leader peptide and the C-terminal tag, as well as the linkers, are
identified in the provided scFv sequences (provided within SEQ ID
NO: 26 to SEQ ID NO: 29 and SEQ ID NO: 34 to SEQ ID NO: 149, as
described herein). The sequence between the N-terminal leader
peptide and the linker is the V.sub.L fragment of the respective
scFv, and the sequence between the linker and the C-terminal tag is
the V.sub.H fragment of the respective scFv.
[0010] For example:
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSSAYGYGWYQ
QKSPGSAPVTVIYNNNKRPSNIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSED
SSTDAIFGAGTTLTVLGQSSRSSTVTLDESGGGLQAPGGALSLVCKASGFTFSSY
DMGWIRQAPGKGLEYVAGITDNGRYASYGSAVDGRATISRDNGQSSVRLQLNN
LRAEDTGTYYCARDDGSGWTGNSIDAWGHGTEIIVSSTSGQAGQHHHHHHGAY PYDVPDYAS
(SEQ ID NO: 78). In this sequence, the N-terminal tag, the
C-terminal tag, and the linker sequence are underlined. The scFv
sequence is the sequence starting with "LTQ . . . " and ending with
" . . . STS" between the N-terminal leader peptide and the
C-terminal tag. Accordingly, the V.sub.L fragment of this scFv is
the sequence starting with "LTQ . . . " and ending with " . . .
TVL" between the N-terminal leader peptide and the linker, and the
V.sub.H fragment of this scFv is the sequence starting with "TVT .
. . " and ending with " . . . STS" between the linker and the
C-terminal tag. Where possible, the C-terminal tag, the N-terminal
leader peptide, the linker and the V.sub.L and V.sub.H fragments
are identified in a similar manner in the scFv sequences provided
herein.
[0011] Fifteen of the listed scFvs (scFv4, scFv28, scFv9, scFv10,
scFv29, scFv31, scFv11, scFv18, scFv63, scFv75, scFv79, scFv81,
scFv88, scFv89 and scFv91) display truncated amino acid sequences
because of incomplete DNA sequences from the sequencing
results.
[0012] Antigen-binding antibodies, antibody fragments and
PTP1B-OX-binding polypeptides can comprise a portion or segment
(continuous or not continuous) of a scFv of any one of SEQ ID NO:
26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149, bind
PTP1B-OX and stabilize reversibly oxidized, inactive conformation.
For example, some embodiments include antibodies, antibody
fragments or PTP1B-OX-binding polypeptides whose amino acid
composition is sufficiently similar to all or a portion of a scFv
of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34 to
SEQ ID NO: 149 that they bind reversibly oxidized, inactive
PTP1B-OX in such a manner that they inhibit its reduction
(reactivation by reducing agent to reduced, active PTP1B). Any of
these sequences can be changed by deletion, addition, substitution
or replacement of one or more amino acid residues. For example,
individual amino acid residues (one or more amino acid residues) of
a scFv of any one of SEQ ID NO: 26 to SEQ ID NO: 29 and SEQ ID NO:
34 to SEQ ID NO: 149 may be substituted with or replaced by another
amino acid residue, such as by an amino acid residue whose presence
results in a silent substitution or a change in sequence that does
not substantially alter the ability of the resulting antibody,
antibody fragment or polypeptide to bind and stabilize PTP1B-OX.
Such silent substitutions are well known to those of skill in the
art Amino acid substitutions, additions and replacements can be
naturally-occurring and/or non-naturally-occurring amino acid
residues, including modified amino acid residues. Antibodies and
antibody fragments can be of any length (e.g., shorter or longer
than a sequence provided herein) sufficient to bind and stabilize
PTP1B-OX. As is well known to the skilled artisan, variable regions
can be of varying length (e.g., shorter or longer than the variable
region of an antibody described herein). Deletion, substitution, or
addition of one or more amino acid residues can result in
functional antibodies or antibody fragments. For example, scFvs can
comprise linker sequences of varying length and amino acid
composition; framework and/or complementarity determining regions
(CDR) of varying length and amino acid composition and, optionally,
additional N-terminal or C-terminal sequences, for example leader
peptides or tags. For example, a scFv can comprise any of the ScFvs
whose amino acid sequences are provided herein (e.g., SEQ ID NO:
26-SEQ ID NO: 29, SEQ ID NO: 34-SEQ ID NO: 149) or a variant of any
of the scFv sequences represented. Variants can differ from the
ScFvs represented in any of SEQ ID NO: 26-29 and SEQ ID NO: 34-149
by one or more (at least one) alteration, which can be a
substitution, deletion, addition and/or replacement, as described
herein. Variants can be of any length (e.g., shorter or longer than
the scFvs whose sequences are provided herein). The one or more
alteration can be a naturally-occurring or a non-naturally
occurring amino acid residue, including modified amino acid
residues. In some embodiments described herein, an antibody,
antibody fragment or PTP1B-OX-binding polypeptide can comprise any
of the sequences presented herein or a variant thereof. The
antibody, antibody fragment or PTP1B-OX-binding polypeptide that
binds (specifically binds) PTP1B-OX and stabilizes it in the
reversibly oxidized inactive form can comprise any of the sequences
presented or a variant thereof. Variants of the amino acid
sequences provided herein are referred to, respectively, as variant
antibodies, variant antibody fragments or variant PTP1B-OX-binding
polypeptides. The phrases antibody that specifically binds PTP1B-OX
and stabilizes PTP1B-OX (also referred to as PTP1B-OX conformation
or form), antibody fragment that specifically binds PTP1B-OX and
stabilizes PTP1B-OX (also referred to as PTP1B-OX conformation or
form) or polypeptide that specifically binds PTP1B-OX and
stabilizes PTP1B-OX (also referred to as PTP1B-OX conformation or
form) or substantially equivalent terms or phrases are intended to
encompass antibodies, antibody fragments and PTP1B-OX-binding
polypeptides that bind PTP1B-OX and whose sequences are all or a
portion of a sequence presented herein or a variant thereof. A
variant antibody, a variant antibody fragment and a variant
polypeptide are, respectively, an antibody, antibody fragment and
polypeptide whose amino acid composition differs from a sequence
presented herein and that binds PTP1B-OX and stabilizes it in its
reversibly oxidized, inactive conformation, without directly
inhibiting PTP1B. Amino acid deletions, additions, replacements or
substitutions can occur within the framework and/or complementarity
determining regions of the scFvs described herein. Many leader
peptides and protein tags are known to the skilled artisan. Leader
peptides and/or protein tags may be used for protein purification,
detection, solubilization, targeting of a protein or peptide to a
subcellular localization or for secretion, and other purposes well
known to those of skill in the art. It is well known in the art
that scFv linker sequences may vary in length and amino acid
sequence.
[0013] Typically, the antibody, antibody fragment or
PTP1B-OX-binding polypeptide does not bind or directly affect the
activity of reduced, active PTP1B. Binding of such antibodies,
antibody fragments or polypeptides is referred to herein as
specific binding and the antibodies, antibody fragments and
polypeptides as antibodies, antibody fragments and polypeptides
that specifically bind PTP1B-OX. Antibodies, antibody fragments and
polypeptides that specifically bind PTP1B-OX produce their effects
on PTP1B-mediated regulation of signaling by stabilizing PTP1B-OX
and on their own, do not inhibit (do not directly inhibit) reduced,
active PTP1B. Antibodies, antibody fragments and polypeptides that
specifically bind PTP1B-OX do not bind the active site of reduced,
active PTP1B. They do not bind the active site of the enzyme in its
reduced, active state. They may, however, bind residues that are
part of the active site, but binding to these residues is
restricted to the reversibly oxidized, PTP1B-OX state. In some
embodiments, the antibody is an intrabody, such as a scFv expressed
inside a cell. In some embodiments, the antibody, antibody fragment
or polypeptide is further characterized in that it binds PTP1B-CASA
mutant protein.
[0014] Also described herein are methods of identifying agents that
bind PTP1B-OX and stabilize this inactive form by assessing
interference with PTP-antibody binding, PTP-antibody fragment
binding, PTP1B-polypeptide binding or PTP1B-small molecule binding.
Such agents bind reversibly oxidized, inactive PTP1B (PTP1B-OX) and
stabilize it in such a manner that they inhibit reactivation of
PTP1B-OX by reduction (by reducing agent) and have no substantial
direct inhibitory effect on phosphatase activity/PTP1B activity. In
some embodiments, the method is one of identifying or screening for
an agent (e.g., an antibody, an antibody fragment, a polypeptide or
a small (organic) molecule) that specifically binds PTP1B-OX and
stabilizes its conformation. In some embodiments, the ability of a
candidate agent to bind to and stabilize PTP1B-OX is assessed by
determining whether it is able to interfere with binding to
PTP1B-OX by an antibody or antibody-fragment that specifically
binds PTP1B-OX and stabilizes its conformation. In some
embodiments, the method comprises [0015] (a) combining (i)
PTP1B-OX; (ii) a molecule that specifically binds PTP1B-OX and
stabilizes the conformation (e.g., an antibody that specifically
binds PTP1B-OX and stabilizes the conformation; an antibody
fragment that specifically binds PTP1B-OX and stabilizes the
conformation; a polypeptide that specifically binds PTP1B-OX and
stabilizes the conformation; or a small molecule that specifically
binds PTP1B-OX and stabilizes the conformation); and (iii) a
candidate agent, under conditions appropriate for binding of
PTP1B-OX with a molecule that specifically binds PTP1B-OX and
stabilizes the conformation; [0016] (b) assessing binding of
PTP1B-OX with the molecule a(ii); and [0017] (c) comparing binding
assessed in (b) with binding of PTP1B-OX with the molecule a(ii)
under substantially the same conditions, but in the absence of the
candidate agent (also referred to as to an appropriate reference or
control), wherein if binding of PTP1B-OX to the molecule a(ii)
occurs to a lesser extent in the presence of the candidate agent
than in the absence of the candidate agent (or as compared to the
reference or control), the candidate agent is an agent that
specifically binds PTP1B-OX.
[0018] In one embodiment, the method comprises (a) combining (i)
PTP1B-OX; (ii) a molecule that is an antibody that specifically
binds and stabilizes PTP1B-OX; and (iii) a candidate agent, (e.g.,
in a cell, in assay media or in a solution). Conditions used are
those appropriate for binding of PTP1B-OX with the antibody. In
addition, if the candidate agent is an agent that binds PTP1B-OX,
binding occurs, forming PTP1B-OX-bound candidate agent. In other
embodiments of the method, the molecule of (ii) is an antibody
fragment that specifically binds and stabilizes PTP1B-OX, a
polypeptide that specifically binds and stabilizes PTP1B-OX, or any
molecule that specifically binds and stabilizes PTP1B-OX. In some
embodiments, the molecule is a small organic compound.
[0019] In certain embodiments, the candidate agent is a small
molecule (e.g., a small organic molecule) and the agent identified
is a small molecule (e.g., small organic molecule) that
specifically binds PTP1B-OX and stabilizes it. The candidate agent
can be any of a wide variety of agents, such as but not limited to,
an antibody, an antibody fragment (e.g., a scFv intrabody or
antigen-binding antibody fragment) or a polypeptide.
[0020] In step (c), comparison can be to an appropriate reference
or control. An appropriate reference or control, to which binding
is compared in the method, is the results of assessing binding
under the same conditions as those used for assessing binding of
the candidate agent (as described above and elsewhere herein), but
in the absence of the candidate agent. Assessment in the absence of
the candidate agent can be carried out at the same time the
candidate agent is assessed for its ability to bind PTP1B-OX,
previously or subsequently, provided that substantially the same
conditions (other than presence of the candidate agent) are used.
The reference or control can be a pre-established value or set of
values; a value or set of values established at the time the
assessment is being carried out; or a value or set of values
established after assessment of the candidate agent has been
completed.
[0021] In some embodiments, agents that specifically bind PTP1B-OX
are identified by assessing the ability of a candidate agent to
disrupt binding of a molecule (for example, an antibody or antibody
fragment) to PTP1B-OX. Such an agent can be any molecule that
specifically binds to PTP1B-OX. Agents, for example small molecules
(e.g., small organic molecules), identified by this method exhibit
high affinity for binding the reversibly oxidized, inactive form,
as evidenced by their ability to displace a molecule bound to
PTP1B-OX. In some embodiments, the method is a method of
identifying or screening for an agent that (a) displaces a molecule
(e.g., an antibody, antibody fragment, polypeptide or small
molecule) that binds to and stabilizes (is bound to and has
stabilized) PTP1B-OX and (b) binds to and stabilizes PTP1B-OX. The
method comprises: [0022] (a) combining (i) a PTP1B-OX-molecule
complex that comprises PTP1B-OX having bound thereto a molecule
(e.g., an antibody that specifically binds PTP1B-OX and stabilizes
the reversibly oxidized, inactive conformation of PTP1B-OX; an
antibody fragment that specifically binds PTP1B-OX and stabilizes
the reversibly oxidized, inactive conformation; a polypeptide that
specifically binds PTP1B-OX and stabilizes the reversibly oxidized,
inactive conformation; a small molecule that specifically binds
PTP1B-OX and stabilizes the reversibly oxidized, inactive
conformation) and (ii) a candidate agent, under conditions
appropriate for detecting displacement of the molecule from
PTP1B-OX in the PTP1B-OX molecule complex; and [0023] (b) assessing
displacement of the molecule from PTP1B-OX in the complex by the
candidate agent, wherein if displacement has occurred, the
candidate agent is an agent that displaces a molecule (e.g., an
antibody, antibody fragment, polypeptide or small molecule) that
binds to and stabilizes (is bound to and has stabilized)
PTP1B-OX.
[0024] Conditions used are those appropriate for detecting
displacement of the molecule from the complex.
[0025] In one embodiment, the method comprises (a) combining (i) a
complex (a PTP1B-OX-antibody complex) that comprises PTP1B-OX
having bound thereto a molecule that is an antibody that
specifically binds PTP1B-OX and stabilizes the conformation; and
(ii) a candidate agent. Conditions used are those appropriate for
assessing or detecting displacement of the antibody from the
complex. If the candidate agent is an agent that displaces the
antibody from the PTP1B-OX-antibody complex, PTP1B-OX-candidate
agent complex will form and the antibody will be displaced. In
other embodiments of the method, the molecule of (i) (in the
complex) is an antibody fragment that specifically binds PTP1B-OX
and stabilizes the conformation, a polypeptide that specifically
binds PTP1B-OX and stabilizes the conformation, or a small molecule
that specifically binds PTP1B-OX and stabilizes the
conformation.
[0026] In some embodiments, the method further comprises comparing
displacement assessed in (b) with displacement of the molecule from
PTP1B-OX in the complex under substantially the same conditions,
but in the absence of the candidate agent, wherein if displacement
occurs to the same or a lesser extent in the presence of the
candidate agent than in the absence of the candidate agent, the
candidate agent is not an agent that displaces a molecule that
binds to and stabilizes (is bound to and has stabilized) PTP1B-OX
and binds to and stabilizes PTP1B-OX.
[0027] In step (b), comparison can be made to an appropriate
reference or control. An appropriate control is described above.
Agents can also be identified by insilico screening, such as by
using docking methods to design or identify molecules or compounds
that bind the reversibly oxidized, inactive form of PTP1B (e.g.,
with reference to its crystal structure).
[0028] Agents identified by methods described herein can be further
assessed for their ability to bind and stabilize PTP1B-OX, for
example in in vitro assays and in appropriate animal models.
[0029] Also described herein are an isolated PTP1B-OX:antibody
complex, an isolated PTP1B-OX:antibody fragment complex, and an
isolated PTP1B-OX:PTP1B-OX-binding polypeptide complex. In specific
embodiments, the antibody component of the isolated
PTP1B-OX:antibody complex comprises an amino acid sequence
described herein, for example a sequence selected from any of the
scFv sequences of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34
to SEQ ID NO: 149. In some embodiments, the antibody fragment
component of the isolated PTP1B-OX:antibody fragment complex
comprises a sufficient portion or segment of the amino acid
sequences described herein, for example a sequence selected from
any of the scFv sequences of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ
ID NO: 34 to SEQ ID NO: 149. In some embodiments, the
PTP1B-OX-binding polypeptide component of the isolated PTP1B-OX:
PTP1B-OX-binding polypeptide complex comprises a sufficient portion
or segment of the amino acid sequences described herein, for
example a sequence selected from a CDR of any of the scFv sequences
of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO:
149, to specifically bind reversibly oxidized, inactive PTP1B
(PTP1B-OX) and stabilize its conformation such that reduction is
inhibited and reactivation of the inactive form is inhibited. Such
antibodies, antibody fragments and PTP1B-OX-binding polypeptide can
comprise any of the sequences described herein, including variants
thereof as described herein.
[0030] Further described herein is a method of stabilizing
reversibly oxidized, inactive PTP1B (PTP1B-OX) in which reversibly
oxidized, inactive PTP is combined with an agent that binds the
oxidized conformation and inhibits reactivation of PTP1B-OX by a
reducing agent. Here, the agent can be, for example, an antibody
that specifically binds reversibly oxidized, inactive PTP1B and
stabilizes the inactive conformation; an antibody fragment that
specifically binds reversibly oxidized, inactive PTP1B and
stabilizes the inactive conformation; a polypeptide that
specifically binds reversibly oxidized, inactive PTP1B and
stabilizes the inactive conformation; or a small molecule that
binds reversibly oxidized, inactive PTP and stabilizes the inactive
conformation. Such antibodies, antibody fragments and polypeptides
can comprise any amino acid sequence, provided that the antibody,
antibody fragment or polypeptide binds reversibly oxidized,
inactive PTP1B (PTP1B-OX) and inhibits (partially or completely)
its reduction (reactivation by reducing agent to the reduced,
active PTP1B). Antibodies, antibody fragments and PTP1B-OX-binding
polypeptides used in the method can comprise any of the ScFvs of
any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ
ID NO: 149, or any variant thereof, as described herein.
[0031] In specific embodiments, the method is a method of
stabilizing reversibly oxidized, inactive PTP1B in a cell (e.g.,
human or other mammalian cell), such as in an individual (e.g. a
human or other mammal or vertebrate). In some embodiments, the
method is one of stabilizing reversibly oxidized, inactive PTP1B in
cells. In some embodiments, the agent is administered by a route
and in such a quantity that it enters cells in sufficient amount or
concentration to have the desired effect (e.g., inhibiting or
preventing reduction of inactive PTP1B-OX and its reactivation).
The agent, for example an intrabody (e.g., a scFv), can be
administered as a result of/by means of gene expression in cells
(e.g., gene therapy).
[0032] A further method described herein is a method of decreasing
the pool of reduced, active PTP1B in a cell, comprising inhibiting
the reduction of reversibly oxidized, inactive PTP1B (PTP1B-OX) in
the cell to reduced, active PTP1B. This can be carried out, for
example, by contacting reversibly oxidized, inactive PTP1B in the
cell with an agent that binds reversibly oxidized, inactive PTP1B
and stabilizes the inactive conformation. The agent can be an
antibody that specifically binds reversibly oxidized, inactive
PTP1B and stabilizes the inactive conformation, an antibody
fragment that specifically binds reversibly oxidized, inactive PTP
and stabilizes the inactive conformation, a polypeptide that
specifically binds reversibly oxidized, inactive PTP1B and
stabilizes the inactive conformation or a small molecule that
specifically binds reversibly oxidized, inactive PTP1B and
stabilizes the inactive conformation. In certain embodiments, the
agent is an intrabody, for example a intracellular scFv. The
composition of the antibody, antibody fragment or polypeptide can
comprise any of the sequences presented herein or a variant
thereof, as described herein.
[0033] Such antibodies, antibody fragments and polypeptides can
comprise any amino acid sequence, provided that the antibody,
antibody fragment or polypeptide binds reversibly oxidized,
inactive PTP1B (PTP1B-OX) and inhibits (partially or completely)
its reduction to reduced, active PTP (reactivation of PTP1B-OX by
reduction). Specific embodiments include antibodies that comprise
all or a portion (continuous or not continuous) of a sequence
selected from the scFv sequences of any one of SEQ ID NO: 26 to SEQ
ID NO: 29 on SEQ ID NO. 34 to SEQ ID NO. 149, bind PTP1B-OX
conformation and stabilize the reversibly oxidized, inactive
conformation. Antibody fragments or PTP1B-OX-binding polypeptides
can comprise a portion or segment (continuous or discontinuous) of
any of the scFv sequences provided in SEQ ID NO: 26 to SEQ ID NO:
29 or SEQ ID NO. 34 to SEQ ID NO. 145, bind PTP1B-OX and stabilize
reversibly oxidized, inactive conformation. For example,
embodiments include antibodies, antibody fragments and
PTP1B-OX-binding polypeptides whose amino acid composition is
sufficiently similar to all or a portion of the scFv sequence of
any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO. 34 to SEQ
ID NO. 149 that they bind reversibly oxidized, inactive PTP1B-OX in
such a manner that they inhibit (partially or completely) its
reduction (reactivation by reducing agent to reduced, active
PTP1B). Such antibodies, antibody fragments and PTP1B-OX-binding
polypeptides, as well as variant antibodies, variant antibody
fragments and variant polypeptides, are described herein.
[0034] Additional embodiments of methods are a method of prolonging
existence of PTP1B in its oxidized/inactive conformation or
prolonging inactivation of PTP1B by stabilizing the inactive form
(PTP1B-OX), and a method of inhibiting reactivation of reduced,
active PTP1B. In each of these embodiments, an agent (e.g., an
antibody, an antibody fragment, a polypeptide, a small organic
molecule) that binds to and stabilizes PTP1B-OX and inhibits its
reactivation is administered, for example as described above in the
context of the method of stabilizing reversibly oxidized, inactive
PTP1B in cells.
[0035] A method of controlling PTP1B-mediated regulation of
signaling in a cell is also described herein. Such a method
comprises modulating the pool of reduced, active PTP1B and
reversibly oxidized, inactive PTP1B in the cell by controlling the
extent to which PTP1B-OX is reduced. In the method, an agent that
specifically binds PTP1B-OX and stabilizes the inactive
conformation is introduced into or expressed in the cell in
sufficient quantity to inhibit or prevent reduction (reactivation)
of inactive PTP1B-OX. The agent can be of any type, including but
not limited to, proteins, polypeptides, peptides, glycoproteins,
glycans, carbohydrates, lipids, antibodies, antibody fragments, or
small molecules (e.g., small organic molecules). In a specific
embodiment, the agent can be an Adnectin.TM.. In specific
embodiments, the agent can be, for example, a small molecule that
specifically binds PTP1B-OX and stabilizes the inactive
conformation, an antibody that specifically binds PTP1B-OX and
stabilizes the inactive conformation, an antibody fragment that
specifically binds PTP1B-OX and stabilizes the inactive
conformation, or a polypeptide that specifically binds PTP1B-OX and
stabilizes the inactive conformation. In specific embodiments, the
agent is an intrabody, such as a scFv. Such antibodies, antibody
fragments and polypeptides can comprise any amino acid sequence,
provided that the antibody, antibody fragment or polypeptide binds
reversibly oxidized, inactive PTP1B (PTP1B-OX) and inhibits
(partially or completely) its reduction by reducing agent to
reduced active PTP1B (reactivation of PTP1B-OX by reduction).
Specific embodiments include antibodies that comprise all or a
portion of a sequence selected from any of the scFv sequences of
any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO. 34 to SEQ
ID NO. 149, bind PTP1B-OX conformation and stabilize the reversibly
oxidized, inactive conformation, as well as antibody fragments or
PTP1B-OX-binding polypeptides that comprise a portion or segment
(contiguous or discontinuous) of any one of SEQ ID NO: 26 to SEQ ID
NO: 29 or SEQ ID NO. 34 to SEQ ID NO. 149, bind PTP1B-OX and
stabilize the reversibly oxidized, inactive conformation. Further
embodiments include antibodies, antibody fragments and polypeptides
whose amino acid composition is sufficiently similar to all or a
portion of any one of SEQ ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO.
34 to SEQ ID NO. 149 that they bind reversibly oxidized, inactive
PTP1B in such a manner that they inhibit its reduction to reduced,
active PTP1B (reactivation by reducing agent). Such antibodies,
antibody fragments and PTP1B-OX-binding polypeptides, as well as
variant antibodies, variant antibody fragments and variant
polypeptides, are described herein.
[0036] In some embodiments, the method is one of controlling or
modulating redox regulation, for example reversible oxidation of
PTP1B, such as signaling-mediated redox regulation, of PTP1B. Any
PTP1B-mediated regulation of a signaling pathway, for example a
signaling pathway involving and/or affected by PTP redox
regulation, such as reversible oxidation of PTP1B, may be modulated
by methods and agents provided herein. Non-limiting examples of
signaling pathways that involve PTP redox regulation are insulin,
EGF, and leptin signaling. In some embodiments, the method is a
method of controlling PTP1B-mediated regulation of insulin
signaling. In some embodiments, the method is a method of
controlling PTP1B-mediated regulation of EGF signaling or a method
of controlling PTP1B-mediated regulation of leptin signaling.
[0037] Also described herein are methods of identifying an agent
that binds to and stabilizes PTP1B-OX and does not bind PTP in its
reduced, active form (PTP1B). In some embodiments, the method
comprises contacting PTP1B with an agent that binds to and
stabilizes PTP1B-OX and assessing binding of the agent to PTP1B,
wherein if the agent does not bind PTP1B, the agent is identified
as an agent that binds and stabilizes PTP1B-OX and does not bind
PTP1B.
[0038] PTP is a key regulator of insulin and leptin signaling.
Consequently, PTP became a highly prized target in the
pharmaceutical industry for therapeutic intervention in diabetes
and obesity (Andersen et al., FASEB J. 18(1): 8, 2004; Tonks et al,
FEBS Lett. 3: 546, 2003). In addition, more recent studies suggest
that it may also be a therapeutic target in breast cancer (Tonks,
Cancer Cell 11(3): 214, 2007). Although there have been major
programs in industry focused on developing small molecule
inhibitors of PTP1B, these efforts have been frustrated by
technical challenges arising from the chemical properties of the
PTP active site. The susceptibility of PTPs to oxidation causes
problems in high throughput screens. In addition, the tendency of
potent inhibitors to be highly charged, for example
non-hydrolyzable pTyr mimetics, presents problems with respect to
bioavailability. Consequently new approaches to inhibition of PTP1B
are required to reinvigorate drug development efforts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1: Effect of individual scFvs on PTP1B activity.
[0040] FIG. 2: Screen for scFvs that stabilize the reversibly
oxidized form of PTP1B
[0041] FIG. 3: Effect of scFv45 intrabody on the tyrosine
phosphorylation status of the .beta.-subunit of the insulin
receptor.
[0042] FIG. 4: Effect of scFv45 intrabody expression on the
tyrosine phosphorylation status on IRS-1.
[0043] FIG. 5: Basic Antibody Structure and Subunit Composition.
The C region of an antibody is relatively conserved while the V
region is antigen-specific. The V region consists of alternating
framework (FW) and hyper variable complementarity-determining
regions (CDR). Antibody molecules contain discrete protein domains
or fragments (shown in the shaded area) that can be isolated by
protease digestion or produced by recombinant techniques. The
smaller Fv (fragment variable) is composed of the VL and VH regions
only. In scFvs (single chain Fvs), the two variable regions are
artificially joined with a neutral linker and expressed as a single
polypeptide chain.
[0044] FIG. 6: Aligned sequences of scFvs binding PTP1B-OX. The
displayed scFv fragments (from the N-terminus to the linker
sequence) include the N-terminal tag (MKKTAIAIAVALAGFATVAQAA, SEQ
ID NO: 150), the VL sequence and the linker sequence (GQSSRSS, SEQ
ID NO 20). The complementarity determining regions (CDRs) of the
light chain were identified as: CDR1: 29-50, CDR2: 60-83, and CDR3:
100-124.
[0045] FIG. 7: Aligned sequences of scFvs binding PTP1B-OX. The
displayed scFv fragments (from the linker sequence to the
C-terminus) include the linker sequence (GQSSRSS), SEQ ID NO 20),
the VH sequence and the C-terminal tag (GQAGQHHHHHHGAYPYDVPDYAS,
SEQ ID NO: 151). The complementarity determining regions (CDRs) of
the heavy chain were identified as: CDR1: 157-180, CDR2: 188-204,
and CDR3: 240-261.
[0046] FIG. 8: Reversible oxidation of PTP1B by H2O2.
[0047] FIGS. 9a-c: Interaction of scFvs and PTP1B. (a)
conformation-sensor scFv45 inhibits PTP1Breactivation by
selectively binding to PTP1B-OX in vitro. (b) scFv57 binds
specifically to PTP1B-OX in vitro. (c) specific interaction between
PTP1B-OX and scFvs in vitro.
[0048] FIG. 10: Intrabody expression in mammalian cells. Left:
Exemplary vector map of pcDNA3.2 with scFv45 insert. Right:
representative blot showing expression of scFv45 in HEK293T cells
as intrabody.
[0049] FIGS. 11a-b. Specific interaction of scFv45 and PTP1B-OX.
(a) Interaction between PTP1B-OX and intrabody45 in mammalian
cells. His-tagged scFv45 was pulled down with Ni-NTA agarose. (b)
Interaction between PTP1B-OX and intrabody45 in mammalian cells.
PTP was immunoprecipitated with anti-PTP1B antibody.
[0050] FIGS. 12a-12b. Screening of PTP1B-OX specific intrabodies in
mammalian cells. His-tagged scFvs were pulled down with Ni-NTA
agarose. Some scFvs show co-pulldown of PTP1B-OX in the presence of
H2O2 (scFv45, 136, 67, 102, and 106), but not in the presence of
NAC and TCEP.
[0051] FIG. 13: Intrabody 45 causes enhanced AKT activation in
response to insulin
[0052] FIGS. 14a-b: (a) Colocalization of PTP1B-OX and intrabody45
in Cos1 cells. (b) Intrabody45 colocalizes with PTP1B-OX in Cos1
cells in response to insulin.
DETAILED DESCRIPTION OF THE INVENTION
PTP1B-Inhibition as a Therapeutic Approach
[0053] Inhibition of PTP1B has been suggested as a strategy in the
treatment of a number of diseases. For example, inhibition of PTP1B
has been demonstrated to augment insulin action. Disruption of the
murine PTP1B gene ortholog in a knock-out mouse model results in
PTP1B-/-mice exhibiting enhanced insulin sensitivity, decreased
levels of circulating insulin and glucose, and resistance to weight
gain even on a high-fat diet, relative to control animals having at
least one functional PTP1B gene (Elchebly et al., Science 283: 1544
(1999)). Insulin receptor hyperphosphorylation has also been
detected in certain tissues of PTP1B deficient mice, consistent
with a PTP contribution to the physiologic regulation of insulin
and glucose metabolism (id.). This makes PTP1B an attractive target
for clinical interventions aimed at ameliorating the state of
insulin resistance common in Type II diabetes patients.
[0054] Additionally, PTP1B-deficient mice further exhibit decreased
adiposity (reduced fat cell mass but not fat cell number),
increased basal metabolic rate and energy expenditure, and enhanced
insulin-stimulated glucose utilization (Klaman et al., 2000 Mol.
Cell. Biol. 20: 5479).
[0055] Increased PTP1B activity has been correlated with impaired
glucose metabolism in other biological systems (e.g., McGuire et
al., Diabetes 40: 939 (1991); Myerovitch et al., J. Clin. Ifzvest.
84: 976 (1989); Sredy et al., Metabolism 44: 1074 (1995)),
including PTP1B involvement in biological signal transduction via
the insulin receptor (see, e.g., WO 99/46268 and references cited
therein).
[0056] Further, PTP acts as a negative regulator of signaling that
is initiated by several growth factor/hormone receptor PTKs,
including p210 Bcr-Abl (LaMontagne et al., Mol. Cell. Biol. 18:
2965-75 (1998); LaMontagne et al., Proc. Am. Acad. Sci. USA 95:
14094-99 (1998)), receptor tyrosine kinases, for example EGF
receptor, PDGF receptor, and insulin receptor (IR) (Tonks et al.,
Curr. Opin. Cell Biol. 13: 182-95 (2001)), and JAK family members,
for example Jak2 and others (Myers et al., J. Biol. Chem. 276:
47771-74 (2001)), as well as signaling events induced by cytokines
(Tonks and Neel, 2001).
[0057] Described herein is production of conformation-specific
antibodies that recognize oxidation-specific epitopes in PTP1B-OX
that are not found in the active, reduced enzyme. A double point
mutation in the PTP-loop of PTP1B, C215A/S216A, induces a stable
conformation (referred to as PTP1B-CASA) that is indistinguishable
in structure from the PTP1B-OX conformation that is induced by
H.sub.2O.sub.2. The mutant is useful as an antigen. Phage display
technology, which allows rapid and efficient screening of libraries
of high complexity (Burton et al, Phage Display: A Laboratory
Manual Cold Spring Harbor Laboratory, 3.1-3.18 2001; Hoogenboom et
al Immunology Today 21: 371 2000), was adapted and used in the work
described herein. Antibody molecules contain discrete protein
domains or fragments that can be separated by controlled protease
digestion or produced by recombinant techniques. The small Fv
(fragment variable), which is composed of the variable light
(V.sub.L) and variable heavy (V.sub.H) regions, were used to
generate the library. In a single chain variable fragment (scFv),
the two variable regions are artificially joined with a neutral
peptide linker. The recombinant antibody scFvs are presented on the
surface of bacteriophage and can be selected from combinatorial
libraries in vitro. In comparison with classical, animal-based
approaches, this has the major advantage of preserving the native
conformation of target antigens to allow the generation of
conformation specific antibodies. Use of scFvs as intracellular
antibodies (intrabodies) to target intracellular proteins has also
been reported (Biocca, Trends in Cell Biology, 5: 248 1995).
Varying biochemical conditions to include competitive molecules
during the selection steps of the phage display technique allows
enrichment of highly specific antibodies, for example selecting for
antibodies that recognize PTP1B-OX in the presence of excess native
PTP1B.
[0058] As described further herein, chickens were immunized with
purified PTP1B-CASA mutant protein and spleen and bone marrow were
harvested from immunized animals in which Applicant detected a
robust increase in serum antibody titer to the purified antigen.
Total RNA was isolated and first-strand cDNA was synthesized by
RT-PCR using an oligo (dT) primer. This first-strand cDNA was used
directly for PCR amplification of the V.sub.L and V.sub.H
sequences. Two scFv constructs were produced, one with a 7 residue
linker and one with a longer, 18 amino acid residue linker sequence
between the V.sub.L and V.sub.H domains. The shorter linker (7
residues) proved to be more effective. The scFv antibody construct
also encodes two expression tags at the C-terminus of the protein,
the 6.times.His tag for purification of soluble proteins and the HA
tag for immunodetection. Two PCR steps were performed to generate
scFv constructs. In the first, V.sub.H and V.sub.L segments were
amplified separately and in the second overlap PCR, V.sub.H and
V.sub.L were combined via the linker sequence to form full-length
scFv constructs, which were pooled. A modified version of the
pComb3XSS phagemid vector was used to construct the scFv phage
display library (Barbas et al, PNAS, 88: 7978 1991; Scott et al.,
Phage Display: A Laboratory Manual Cold Spring Harbor Laboratory,
2.1-2.19 2001). The library size (expressed as the transformation
efficiency of the ligated library construct) was determined to be
.about.10.sup.7. Phage particles displaying scFv on their surface
fused to the phage pIII coat protein were harvested from the
culture supernatant of phagemid-transformed bacterial cells. A
subtractive panning strategy was used to isolate
PTP1B-CASA-specific antibodies from the scFv library. PTP1B-CASA
was biotinylated at the N-terminus by expression in E. coli that
overexpressed biotin ligase (Smith et al., Nucleic Acid Res, 26(6):
1414 1998), and purified at homogeneity. This approach was used
because chemical biotinylation was observed to be inhibitory to
activity of reduced, active PTP1B. This biotinylated PTP1B-CASA
mutant was mixed with the library, together with 10-50.times. molar
excess of wild type enzyme, under reducing conditions, to direct
selection towards epitopes unique to the oxidized conformation of
PTP1B. The PTP1B-CASA-scFv-phage complex from this mixture was
isolated on streptavidin beads and phage particles with surface
exposed scFvs bound to PTP1B-CASA were then eluted and amplified
through four rounds of panning. A total of .about.400 individual
clones were selected randomly from the enriched scFv pools,
sequenced and found to sort into .about.100 distinct groups on the
basis of differences in their hypervariable regions. Phage
expressing these different scFvs were screened for their
specificity towards the CASA mutant by ELISA, in which recombinant
PTP1B-CASA or wildtype PTP1B was immobilized on the plate surface
and bound scFv was detected by recognizing the associated phage
with HRP-conjugated anti-phage antibody. Applicant did not find an
scFv that distinguished between reduced, active PTP and oxidized,
inactive PTP1B-OX using this approach. In this screen, the scFvs
are still fused to the surface of the phage particles, which may
interfere with recognition of the PTP1B-OX conformation in the
assay. In solid-phase ELISA the antigen may be partially denatured
due to immobilization on plastic surfaces, which may compromise
distinctive conformations. Therefore, Applicant utilized an amber
codon in the phagemid vector to express the scFv clones in a
non-suppressor bacterial strain to generate scFvs from which the
pIII phage protein tag had been removed. These were then subjected
to "in-solution" screening, based on a phosphtase enzyme activity
assay.
[0059] Applicant's hypothesis was that scFvs that are able to bind
to and stabilize the oxidized conformation of PTP1B in solution
would inhibit reactivation by reducing agent, but have no direct
inhibitory effect on phosphatase activity in assays in vitro.
Activity was measured using .sup.32P-labeled pTyr-Reduced
Carboxamidomethylated and Maleylated Lysozyme as substrate.
Applicant established conditions in which wild type PTP1B could be
reversibly oxidized in vitro. PTP1B was inactivated following
addition of H.sub.2O.sub.2. However, phosphatase activity was
completely restored upon the removal of H.sub.2O.sub.2 by a quick
buffer exchange and addition of the reducing agent TCEP. Purified
bacterially-expressed scFvs were incubated with PTP1B after
H.sub.2O.sub.2 treatment and the ability of an individual scFv to
stabilize the reversibly oxidized, inactive conformation of the
phosphatase was assessed by the ability of the antibody to inhibit
the reactivation of the enzyme by reducing agent. Results showed
that scFvs identified as described herein showed significant
inhibition of the reactivation of PTP1B-OX by reducing agent, but
did not exert any direct inhibitory effect on activity of reduced,
active PTP1B. Applicant validated this approach further, by testing
the effects of expressing one of the scFvs (scFv45) as a single
chain antibody fragment "intrabody" to PTP1B, using 293T cells as
the expression system. This intrabody was expressed transiently and
then tested for effects on insulin signaling, focusing initially on
the tyrosine phosphorylation status of the beta-subunit of the
insulin receptor and insulin receptor substrate (IRS-1). Results
indicated that for both substrates, expression of the intrabody had
no impact on the basal level of tyrosyl phosphorylation, but it
enhanced and extended the time course of insulin-induced
phosphorylation, consistent with Applicant's proposed mechanism of
action.
[0060] Applicant developed an alternative strategy that was based
on knowledge of the structure of cyclic sulphenamide form PTP1B-OX.
In light of Applicant's observation that upon PTP1B oxidation, Tyr
46 adopts a solvent-exposed position, Applicant generated rabbit
polyclonal antibodies to a peptide modeled on the sequence that
surrounds Tyr 46, using both phosphorylated and unphosphorylated
forms of the peptide as antigen. The effects of these
affinity-purified antibodies were tested in the assays in vitro
described above. Interestingly, the effects of the anti-peptide
antibody were similar to those of scFv45 in vitro, whereas the
anti-pTyr peptide antibody was without effect. Again, neither
antibody exerted a direct inhibitory effect on the activity of
PTP1B. Thus, antibodies produced by two distinct approaches
resulted in similar data.
[0061] Disclosed herein are methods and agents relating to the
surprising discovery that the effect of PTP1B on signaling can be
inhibited by stabilizing PTP1B in its inactive, reversibly oxidized
state (PTP1B-OX), without directly inhibiting activity of PTP1B in
its active, reduced state. When PTP1B is oxidized, a sulphenic acid
intermediate is produced and is rapidly converted into
sulphenyl-amide species, in which the sulfur atom of the catalytic
cysteine is covalently linked to the polypeptide backbone nitrogen
atom of the amino acid residue situated immediately C-terminal to
the cysteine. This creates a cyclic sulphenamide form, referred to
herein in the alternative and interchangeably as the OX form or as
the SN form (Salmeen et al., Nature 2003, PCT application
PCT/US2004/017710). Oxidation of PTP to the cyclic sulphenyl-amide
PTP1B-OX form is accompanied by conformational changes in the
catalytic site that effectively inhibit substrate binding and,
thus, result in inhibition of PTP1B activity (Salmeen et al.,
Nature 2003). This unusual protein modification protects the active
site cysteine residue of PTP1B from irreversible oxidation to
sulphinic acid and sulphonic acid and permits redox regulation of
the enzyme by promoting its reversible reduction by thiols.
[0062] Conventional strategies aimed at PTP1B signaling modulation
rely on the identification of agents that directly interfere with
the enzymatic activity of the reduced, active PTP1B, for example by
inhibitory agents that directly bind the catalytic pocket of the
active enzyme. In contrast, methods and compositions described
herein relate to modulating the stability of the PTP1B-OX pool in
order to modulate PTP1B signaling, for example by stabilizing
PTP1B-OX in order to inhibit (partially or completely) PTP1B
signaling. In a specific embodiment, the methods and compositions
result in stabilization of inactive PTP1B (PTP1B in oxidized form;
PTP1B-OX) and reduced (partially or complete) activity of PTP
(reduced activity of PTP1B, relative to PTP activity in the absence
of the method or composition described herein).
[0063] Compositions and methods described herein may find a variety
of uses, for instance, in screening for agents that modulate
PTP1-mediated regulation of signaling (for example antibody
screening, small molecule screening, drug screening) and
therapeutic applications in which the PTP1B-OX is stabilized,
reduction of reversibly oxidized, inactive PTP1B-OX to reduced,
active PTP1B is inhibited, and PTP1B-mediated regulation of
signaling is modulated.
[0064] Described herein are agents, for example antibodies,
antibody fragments, PTP1B-OX-binding polypeptides and small
molecules, that selectively bind PTP1B-OX and modulate its
stability (stabilize the inactive form), but do not bind to or
directly modulate the enzymatic activity of PTP1B in its reduced,
active state. Some aspects of this invention relate to methods of
identifying agents antibodies, antigen-binding fragments thereof,
polypeptides, small molecules) that bind and stabilize PTP1B-OX and
do not bind to or directly inhibit enzymatic activity of PTP1B in
its reduced, active state.
[0065] The term "directly inhibit enzymatic activity of PTP1B," as
used herein, refers to a substantial inhibition of enzymatic
activity of PTP in its reduced, active state. The terms "enzymatic
activity" and "catalytic activity" are used interchangeably herein.
The term "PTP1B", as used herein, and if not further qualified,
refers to the reduced, active form of PTP1B, which exhibits
tyrosine phosphatase activity (Salmeen et al., Nature 2003). It is
also referred to herein as "reduced, active PTP1B". For example, an
agent that binds the catalytic site of PTP1B in its reduced, active
conformation and inhibits enzyme/substrate interaction, resulting
in a substantial decrease of enzymatic activity, modulates
enzymatic activity of PTP1B in its reduced, active state in the
sense this term is used herein. As a further example, an agent, for
example an antibody, or antibody fragment thereof, that does not
bind the catalytic site of reduced, active PTP1B and/or does not
affect the catalytic activity of reduced, active PTP directly, is
not encompassed within the term "directly inhibit the enzymatic
activity of PTP1B." An agent may, for example, selectively bind and
stabilize PTP1B-OX, thus resulting in a decrease of reduced, active
PTP1B molecules, which in turn, may result in a decrease in PTP1B
signaling, without directly inhibiting the enzymatic activity of
PTP1B in its reduced, active state.
[0066] One way to determine whether an agent, for example an
antibody, or fragment thereof, modulates the enzymatic activity of
PTP1B in its reduced, active state, is to measure and compare the
enzymatic activity of PTP1B in the presence and in the absence of
the agent, for example in an in vitro phosphatase assay under
reducing conditions as described herein.
[0067] Some methods described herein are assays useful for
identifying compounds that modulate (e.g., inhibit, partially or
completely) reduction of reversibly oxidized, inactive PTP1B, for
example the inactive, reversibly oxidized cyclic sulphenamide form
of PTP1B. In some embodiments, agents identified by these assays
antagonize indirectly the effect of PTP1B on signaling (e.g.,
agents that inhibit the reduction of reversibly oxidized, inactive
PTP1B-OX to reduced, active PTP1B, or agents that decrease the pool
of reduced, active PTP1B, for example by binding and stabilizing
PTP1B-OX). A PTP1B modulating agent may be, for example, a
physiological substance or a natural or synthetic drug, for example
a naturally occurring or recombinantly produced antibody, or
antigen-binding fragment thereof, or an organic small molecule as
provided herein.
[0068] As used herein, the term "PTP" means a protein tyrosine
phosphatase enzyme capable of dephosphorylating a phosphorylated
tyrosine residue in a protein, polypeptide, or peptide. Such PTPs
are identified by their signature catalytic cysteine motif
H-C-(X).sub.5-R- (SEQ ID NO: 2), wherein the cysteine residue is
the catalytic cysteine and "X" can be any amino acid residue,
natural or unnatural.
[0069] The term PTP, as used herein, includes "classical" PTPs,
which dephosphorylate tyrosine residues and which have the
signature motif sequence H-C-S-(X).sub.4-R- (SEQ ID NO: 3), for
example, -H-C-S-X-G-X-G-R-X-G- (SEQ ID NO: 4), wherein "X" can be
any amino acid residue. Andersen et al. (Mol. Cell. Bio. 21:
7117-7136 (2001); FASEB J. 18: 8-30 (2004), incorporated herein by
reference) describe and illustrate this structural relationship
among classical protein tyrosine phosphatase domains. Such
classical PTPs are described, and GenBank reference numbers
provided therefore, in Andersen et al. (2001 Mol. Cell. Biol. 21:
7117) and herein.
[0070] For example, the PTP may be PTP1B (protein-coding DNA and
amino acid sequences of PTP1B are described, for example, under
GenBank accession NM.sub.--002827 (SEQ ID NO: 5), NP.sub.--002818
(SEQ ID NO: 6), BT006752 (SEQ ID NO: 7), AAP35398 (SEQ ID NO: 8),
M31724 (SEQ ID NO: 9), AAA60223 (SEQ ID NO: 10), M33689 (SEQ ID NO:
11), AAA60157 (SEQ ID NO: 12), BC015660 (SEQ ID NO: 13), AAH15660
(SEQ ID NO: 14), BC018164 (SEQ ID NO: 15), AAH18164 (SEQ ID NO:
16), AK316563 (SEQ ID NO: 17), BAG38152 (SEQ ID NO: 18), or
sequences relating to Unigene Cluster Hs. 417549 (UGID:223337) Homo
sapiens (human) PTPN1). The signature motif sequence of PTP1B is
generally described as H-C-S-(X).sub.4-R (SEQ ID NO: 3), for
example. As a non-limiting example of a mutant PTP1B mimicking the
conformation of reversibly oxidized PTP1B, a double mutant PTP1B,
in which two mutations have been introduced within the catalytic
motif (C215A and S216A), PTP1B-CASA is described herein (SEQ ID NO:
19).
[0071] The term "isolated", as used herein, refers to a molecule or
agent, for example PTP1B, PTP1B-OX, or PTP1B-CASA, that has been
removed from its source, biological environment or milieu (for
example by removing a protein from an intact cell source). A
"molecule" may be any chemical or biological molecule, whether
naturally occurring or non-naturally occurring/synthetic, such as
can be made by chemical synthetic methods, recombinant methods or
other non-naturally occurring method. A molecule can be, for
example, a protein, polypeptide or oligopeptide, a nucleic acid,
for example a oligonucleotide or polynucleotide, a molecule in a
complex of molecules, a chemical substance, for example a small
organic compound, whether naturally occurring or not or synthetic.
PTP1B, PTP1B-OX, mutant PTP1B, PTP1B-CASA, a scFv, an antibody, an
antigen binding antibody fragment, and a small organic compound are
non-limiting examples of molecules. Isolation may be accomplished,
for example, by any method suitable for removing or separating a
molecule from surrounding molecules or substances, for example
those naturally associated with a molecule, such as by using
chemical, physical, and/or enzymatic methodology well known in the
art of molecule isolation.
[0072] Some embodiments involve the use of an agent that binds to
PTP1B-OX or a mutant PTP1B, for example PTP1B-CASA. Such agents may
be used in methods described herein, including methods to identify
molecules that bind PTP1B-OX or bind a mutant PTP (for example,
PTP1B-CASA); methods to determine, quantitatively or qualitatively,
the binding of a candidate molecule to PTP1B, PTP1B-OX, or to
mutant PTP (for example PTP1B-CASA) and methods to modulate
PTP1B-mediated regulation of a signaling pathway.
[0073] Antibodies, antibody-fragments and PTP1B-OX-binding
polypeptides described herein typically comprise L-amino acid
residues. They can further comprise D-amino acid residues,
naturally-occurring amino acid residues (e.g., L-amino acid
residues), non-naturally occurring amino acid residues, modified
aminod acid residues, alone or in combination. A variety of peptide
bonds can be present, such as at least one psi[CH.sub.2NH] reduced
amide peptide bond, at least one psi[COCH2] ketomethylene peptide
bond, at least one psi[CH(CN)NH] (cyanomethylene)amino peptide
bond, at least one psi[CH2CH(OH)] hydroxyethylene peptide bond, at
least one psi[CH2O] peptide bond, and/or at least one psi[CH2S]
thiomethylene peptide bond.
[0074] An antibody, or antigen-binding antibody fragment, for
example as described herein, can be prepared by any of a variety of
methods well known in the art, including administering an antigen,
for example a specific peptide, a specific protein, a fragment of a
specific protein, a cell expressing a specific protein or a
fragment thereof to an animal to induce production of polyclonal
antibodies. The production of monoclonal antibodies is also well
known in the art.
[0075] As is well-known in the art, only a small portion of an
antibody molecule, the paratope, is involved in the binding of the
antibody to its epitope (FIG. 5, see, in general, Clark, W. R.
(1986), The Experimental Foundations of Modern Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab') fragment (or
F(ab')2 fragment), retains both of the antigen binding sites of an
intact antibody. Similarly, an antibody from which the Fc region
has been enzymatically cleaved, or which has been produced without
the Fc region, designated an Fab fragment, retains one of the
antigen binding sites of an intact antibody molecule. Fab fragments
consist of a covalently bound antibody light chain and a portion of
the antibody heavy chain denoted Fd. The Fd fragments are the major
determinant of antibody specificity (a single Fd fragment may be
associated with up to ten different light chains without altering
antibody specificity) and Fd fragments retain epitope-binding
ability in isolation.
[0076] F(ab') fragments derived from serum or cell culture by
purification of specific IgG followed by papain digestion show
similar binding compared to the complete IgG, while the Fc
component does not bind (FIG. 5).
[0077] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, W. R. (1986) The
Experimental Foundations of Modern Immunology Wiley & Sons,
Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,
Blackwell Scientific Publications, Oxford). In both the heavy chain
Fd fragment and the light chain of IgG immunoglobulins, there are
four framework regions (termed Fr or Fw regions) separated
respectively by three complementarity determining regions (CDR1
through CDR3). The CDRs, and in particular the CDR3 regions, and
more particularly the heavy chain CDR3, are largely responsible for
antibody specificity.
[0078] It is well-established in the art that the non-CDR regions
of a mammalian antibody may be replaced with similar regions of
nonspecific or heterospecific antibodies without removing the
epitopic specificity of the original antibody. For example, this is
the case in the development and use of "humanized" antibodies in
which non-human CDRs are covalently joined to human FR and/or
Fc/pFc' regions to produce a functional antibody. See, e.g., U.S.
Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762, and
5,859,205.
[0079] Fully human monoclonal antibodies also can be prepared by
immunizing mice transgenic for large portions of human
immunoglobulin heavy and light chain loci. Following immunization
of these mice (e.g., XenoMouse (Abgenix), HuMAb mice
(Medarex/GenPharm)), monoclonal antibodies can be prepared
according to standard hybridoma technology. These monoclonal
antibodies will have human immunoglobulin amino acid sequences and
therefore will not provoke human anti-mouse antibody (HAMA)
responses when administered to humans.
[0080] Thus, as will be apparent to one of ordinary skill in the
art, the present invention, at least in some aspects, also provides
for F(ab'), Fab, Fv, and Fd fragments; chimeric antibodies in which
the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3
regions have been replaced by homologous human or non-human
sequences; chimeric F(ab') fragment antibodies in which the FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; chimeric Fab
fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or
light chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced by
homologous human or non-human sequences. In some embodiments, the
present invention provides single chain antibodies (for example
scFv polypeptides), (single) domain antibodies (FIG. 5). In some
embodiments, the single chain antibodies or (single) domain
antibodies are intracellular antibodies, termed intrabodies.
[0081] Domain antibodies, camelid and camelized antibodies and
fragments thereof, are well known in the art (described, for
example, in patents and published patent applications of Ablynx NV
and Domantis) and can also be used in embodiments described
herein.
[0082] The term "conformation specific scFvs, as used herein,
refers to scFvs that bind PTP1B in a reversibly oxidized, cyclic
sulphenyl-amide conformation (PTP1B-OX or PTP1B-SN). They also bind
mutant PTP1B resembling PTP1B-OX, for example the double mutant
PTP1B-CASA.
[0083] Some methods described herein may be used for identifying
compounds or agents that bind to the oxidized cyclic
sulphenyl-amide form of PTPB1 and inhibit (partially or completely)
its reduction to its active cysteine thiol-containing state. Once
identified, such agents or compounds are useful for modulating the
flux of signal through a pathway that is regulated by PTP1B. In
some embodiments, such agents may be used to modulate a pathway
that is negatively regulated by PTP1B.
[0084] For example, a compound may be identified by crystallizing
isolated PTP1B, contacting the crystallized PTP1B with
H.sub.2O.sub.2, generating reversibly oxidized PTP1B-OX and then
soaking (or incubating, immersing, exposing, bathing, contacting or
otherwise maintaining) the crystal in a solution containing a
candidate compound and determining if the compound binds to the
PTP1B-OX, for example according to X-ray crystallography methods
described herein and known in the art (see, e.g., Salmeen et al.
Nature, 2003; Johnson and Blundell, Protein Crystallography
(Academic Press 1976); Blundell et al. Nat. Rev. Drug Discov. 1:
45-54 (2002)). Other methods for determining whether a compound
binds to a PTP-OX include isothermal titration calorimetry in the
solution state (Weber et al., Curr. Opin. Struct. Biol. 13: 115-21
(2003); Ward et al., Prog. Med. Cdaem. 38: 309-76 (2001); Cliff et
al., J. Mol. Recognit. 16: 383-91 (2003)) or surface plasmon
resonance (e.g., BIAcore, Biosensor, Piscataway, N.J.).
[0085] The function and advantage of these and other embodiments of
the present invention will be more fully understood from the
examples below. The following examples are intended to illustrate
the benefits of the present invention, but do not exemplify the
full scope of the invention.
EXAMPLES
Example 1
Development of antibodies selectively binding PTP1B-OX from a phage
display library
[0086] 1. Construction of scFv Phage Display Library
[0087] A phage display library displaying single chain variable
fragments (scFvs) fused to its surface protein pIII with a size of
.about.10.sup.7 was constructed from spleen and bone marrow of
chickens immunized with PTP1B-CASA (mutant structurally identical
to reversibly oxidized PTP1B). Construction of the library is
briefly described here.
[0088] 1.1. Chicken Immunizations: Chickens were immunized with
purified PTP1B-CASA and the blood from each immunized chicken was
titrated by ELISA to determine the presence of an antigen-specific
immune response. Detection of a strong serum antibody titer was an
early indication of the presence of an enriched pool of
PTP1B-CASA-binding immunoglobulin genes that make up the building
blocks of combinatorial antibody library.
[0089] 1.2. Total RNA Extraction: Spleen and bone marrow are the
major repository of plasma cells that secrete antibodies against
the antigen of interest and contain the highest levels of specific
mRNA. Therefore, spleen and bone marrow from immunized animals with
elevated immune response against PTP1B-CASA were harvested. Total
RNA was isolated from the spleen and bone marrow using TRI-Reagent
and isopropanol precipitation.
[0090] 1.3. First-strand cDNA Synthesis from the Total RNA:
First-strand cDNA was synthesized from the total RNA extracted from
both bone marrow and spleen by reverse transcription-polymerase
chain reaction (RT-PCR) using an oligo (dT) primer. This
first-strand cDNA was directly used for PCR amplification of the
V.sub.L and V.sub.H genes.
[0091] 1.4. Generation of Recombinant scFvs: A peptide linker of
varying length between the V.sub.H and the V.sub.L domains of
immunoglobulin molecule generates recombinant single chain Fvs
(scFvs) with improved folding and stability. An equimolar mixture
of first-strand cDNA derived from the spleen and bone marrow of the
PTP1B-CASA-immunized chickens was used to amplify V.sub.H and
V.sub.L genes for the construction of combinatorial scFv antibody
libraries. Two scFv constructs were produced, one with a 7 amino
acid linker sequence (GQSSRSS) (SEQ ID NO 20) and one with an 18
amino acid linker (GQSSRSSSGGGGSGGGGS) (SEQ ID NO 21). Two PCR
steps were performed in order to generate scFv constructs--in the
first PCR V.sub.H and V.sub.L gene segments were amplified
separately and in the second overlap PCR V.sub.H and V.sub.L were
combined via a linker sequence to form a full-length scFv
construct. The final scFv PCR products were pooled together,
ethanol precipitated and quantified.
[0092] 1.5. The Phagemid Vector: A modified version of the
pComb3XSS phagemid vector was used for the construction of the scFv
antibody libraries. This vector allows for a uniform directional
cloning strategy that utilizes a single restriction endonuclease,
Sfi I, based on asymmetry of the two sites. This vector contains
the amber codon, inserted between the 3' Sfi I restriction site and
the 5' end of phage gene III fragment. This allows for expression
of soluble antibody fragments in nonsuppressor strains of bacteria
without excising the gene III fragment. The pComb3XSS also contains
two peptide tags, the histidine (H6) tag for purification of
soluble proteins, and the hemagglutinin (HA) tag for
immunodetection. This phagemid was modified in such a way that it
has a reduced potential for recombination and deletion within the
vector. In addition the vector also contains an ampicillin
resistant gene for selection purposes.
[0093] 1.6. Construction of the scFv Library: Both the scFv PCR
products and phagemid were prepared for cloning by restriction
endonuclease digestion with Sfi I. The digested products were gel
purified and the cloning efficiency of the linearized vectors and
scFv inserts was tested using small scale ligations and found to be
in the range of 10.sup.7 to 10.sup.8 cfu/ng of DNA, with minimal
background (less than 5%). Library ligation was performed with a
2:1 molar ratio of insert:vector, 1.times. ligase buffer, 10 .mu.l
(1 U/.mu.l) T4 DNA ligase, in a 200-n1 reaction. The ligations were
incubated overnight at room temperature, ethanol precipitated and
resuspended in water. The ligated DNA was transformed into 300
.mu.l of electrocompetent XL-1 Blue cells. This E. coli is a male
strain that harbors F' pili, through which the filamentous
bacteriophage infect the bacteria. The F' factor containing XL-1
Blue cells are maintained by the selection pressure of
tetracycline. The library size (expressed as the transformation
efficiency of the ligated library construct) was determined by
plating the transformed cells on LB/carbenicillin plates. The size
of the library was found to be .about.10.sup.7. The culture was
then grown under the selection of carbenicillin to select for
transformed bacterial cells. To induce the production of functional
phage particles by the infected E. coli culture, preparation of a
filamentous helper phage VCSM13 (10.sup.12 PFU/ml) was added and
selected with kanamycin (there is a kanamycin resistant gene in the
genome of the helper phage). After overnight incubation at
37.degree. C. with shaking, the culture supernatant containing the
phage particles was harvested by centrifugation. The bacterial
pellet was used to purify phagemid DNA (the library DNA
preparation) using the Qiagen Maxiprep kit. The phage particles
displaying scFv fused to its pIII coat protein were finally
harvested from the supernatants by PEG precipitation.
Example 2
Subtractive Panning for Isolating PTP1B-CASA Specific Antibody
Fragments
[0094] A subtractive panning protocol was designed which will
select for scFvs specific for the PTP1B-CASA mutant while
eliminating the pools that recognize and detect the common epitopes
on both the oxidized and the reduced form of the enzyme. Addition
of molar excess reduced wild type PTP1B in the library in solution
will eliminate the scFvs that recognize the common structural
epitopes of the two forms of the enzyme. Specific antibodies can be
isolated with biotinylated PTP1B-CASA and subsequent capturing of
the antigen-antibody complex with streptavidin coated magnetic
beads. Different steps of the subtractive panning protocol are
briefly described below:
[0095] 2.1. PTP1B Biotinylation in vivo and Purification of
Biotinylated PTP1B: The biotinylation sequence is a unique peptide,
15 residues long (GLNDIFEAQKIEWHE) (SEQ ID NO 22), which is
recognized by biotin ligase. In the presence of ATP, the ligase
specifically attaches biotin to the lysine residue in this
sequence. This tag was fused to the N-terminal of PTP1B in pET19b
vector and the recombinant protein expressed in E. coli (BL21),
which was already transformed with the recombinant biotin ligase
(BirA) in pACYC184 (a ColEI compatible plasmid). Expression of
recombinant proteins in bacteria was induced with 1 mM IPTG in
presence of 50 .mu.M biotin in the growth medium for 4 hours at
37.degree. C. Biotin-protein ligase activates biotin to form
biotinyl 5' adenylate and transfers the biotin to biotin-accepting
proteins. Addition of biotin during the time of induction ensures
complete biotinylation of the tagged protein. Using an E. coli
strain that over-expresses biotin ligase, up to 95% biotinylation
of substrate proteins is possible. The in vivo biotinylated PTP1B
(both CASA and wild type) was purified in a two-step purification
scheme. In the first step the biotinylated recombinant protein was
separated by monomeric avidin column and in the second step the
protein was further purified by an anion exchange column (HiTrap Q
FF). The activity of the in vivo biotinylated protein was measured
by a pNPP assay and the catalytic activity of PTP1B was found to be
conserved by this specific modification. This implies that the
N-terminal biotinylation does not cause structural modification at
the catalytic site of the enzyme. The in vivo biotinylated protein
was used for panning.
[0096] 2.2. Subtractive Panning: The scFv library was mixed in
solution with 10-50 times molar excess of wild type PTP1B than the
biotinylated PTP1B-CASA under reducing conditions for 4 hours at
4.degree. C. to eliminate the pools of antibodies that recognize
the common epitopes on both the oxidized and the reduced form of
PTP1B. Biotinylated PTP1B-CASA was mixed to this solution and
incubated for another 4 hours at 4.degree. C. From this mixture,
scFv displaying phage bound to this biotinylated PTP1B-CASA were
captured by streptavidin coated magnetic beads. After magnetic
separation of the antibody-antigen complex captured on the beads,
non-specific binders were removed by repeated washing. Bound phage
displaying specific scFvs on their surface were eluted under acidic
(glycine-HCl, pH 2.2) condition, neutralized and amplified.
Amplified phage from the first round of panning were used for the
second round selection and a total of four rounds of panning were
performed accordingly under the same condition. The input phage
were preincubated with the streptavidin coated beads before each
round of panning to eliminate the bead- and streptavidin binding
phage. Input and output phage were estimated to determine whether
selective enrichment of specific scFv-displaying phage occurred.
The detailed protocol for the subtractive panning is described
below: [0097] 1. The streptavidin (SA) coated magnetic beads were
prewashed with 10 volumes of TBS containing 2% BSA and 0.2% NaN3 by
mixing and centrifuging at 12,000 rpm for 30 seconds. The washed
beads were resuspended in TBS containing 2% BSA, 5 mM DTT and 0.2%
NaN3 to make the final SA concentration to 10 mg/ml.
[0098] 2. The binding capacity of the beads is 5-10 .mu.g (30-60
pmole) of biotinylated antibody (IgG) per mg of SA beads.
Therefore, 25 .mu.l of SA beads at 10 mg/ml, should bind 7.5-15.0
pmole of biotinylated protein. Applicant used 400 ng of
biotinylated PTP1B-CASA (.about.10 pmoles). In the first and second
panning steps Applicant used 4 .mu.g (10.times. molar access) and
10 .mu.g (25.times. molar access) of untagged wild type PTP1B
(PTP1B-WT), respectively. In both the 3rd and 4th round of panning
we used 20 .mu.g of untagged wild type PTP1B, which is 50.times.
molar access of the biotinylated PTP1B-CASA. [0099] 3. The scFv
library was preadsorbed by SA beads in 400 .mu.l final volume in
1.5 ml microcentrifuge tube by mixing 2.times.10.sup.12 phage
particles in 400 .mu.l of 2% BSA in TBS plus 1% Tween 20, 10 mM DTT
and finally adding 25 .mu.l of prewashed SA beads and incubating
for 2 hours at 4.degree. C. [0100] 4. Recombinant 37-kDa wild type
PTP1B (4 .mu.g) was added in 400 .mu.l 1.times.TBS, 2% BSA, 0.5%
T20 and 5 mM DTT to 200 .mu.l of preadsorbed phage (10.sup.12 phage
particles) and incubated for 4 hours at 4.degree. C. The
biotinylated PTP1B-CASA (400 ng) was added in 400 .mu.l
1.times.TBS, 2% BSA, 0.5% T20 and 5 mM DTT to this mixture and
incubated at 4.degree. C. for 4 hours in a humidified box. [0101]
5. Pre-washed SA beads (25 .mu.l) were added to the phage/screening
molecule reaction in the tubes and incubated for 15 minutes at RT
on a rocking platform. [0102] 6. To block the additional
streptavidin binding sites on the beads 100 .mu.l of 1 mM biotin
(.about.0.1 mM final) was added in TBS containing 2% BSA and 5 mM
DTT to the solution and incubated for 5 minutes at RT. [0103] 7.
The supernatant was aspirated after the incubation and the beads
were washed 3.times. by resuspending the beads in 1 ml of TBS and
centrifuging at 12,000 rpm. [0104] 8. The phage particles
associated with the SA-biotinylated PTP1B-CASA complex were eluted
after adding 100 .mu.l of elution buffer (0.2 M glycine-HCl, pH
2.2, 1 mg/ml BSA), pipetting up and down to mix the beads in the
elution buffer and incubating for 10 minutes at RT. The beads were
held at the bottom of the tube with the magnet, the sup was
collected and transferred to a new microfuge tube and neutralized
with 20 .mu.l of neutralization buffer (1 M Tris base, pH 9.1).
[0105] 9. The eluates were added to infect 2-ml overnight culture
of XL-1 Blue strain of E. coli in SB medium and incubated at room
temperature for 15 minutes. To this 6 ml of prewarmed (at
37.degree. C.) SB medium (+1.6 .mu.l of 100 mg/ml carbenicillin+12
.mu.l of 5 mg/ml tetracycline) was added and the culture was
transferred to a 50-ml tube and shaken at 250 rpm for 1 hour at 250
rpm. Then 2.4 .mu.l of 100 mg/ml carbenicillin was added to the
culture and shaken for an additional hour at 250 rpm and 37.degree.
C. [0106] 10. Input and Output titering: [0107] a. Five p. 1 of the
infected culture was diluted in 500 .mu.l of SB medium and 10 and
100 .mu.l of this dilution were plated on LB/Amp or
LB/Carbenicillin plates for output tittering. [0108] b. One hundred
.mu.l of the prepared E. coli culture was infected with 2 .mu.l of
a 10-8 dilution of the phage preparation [1 .mu.l in 1000 .mu.l SB
(a 10.sup.-3 dilution); mix, 1 .mu.l of the 10.sup.-3 dilution in
1000 .mu.l of SB (a 10.sup.-6 dilution), mix and dilute 10 .mu.l of
the 10.sup.-6 dilution in 1000 .mu.l of SB (a 10.sup.-8 dilution)].
The infected culture was incubated for 15 minutes at RT and plated
on an LB/Amp plate. [0109] c. The output and input plates were
incubated at 37.degree. C. overnight. [0110] 11. The helper phage
VCSM13 (10.sup.12 pfu) was added to the 8-ml culture and
transferred to a 500-ml flask. To this 91 ml of prewarmed
(37.degree. C.) of SB medium (with 46 .mu.l of 100 mg/ml
carbenicillin and 184 .mu.l of 5 mg/ml tetracycline) was added and
finally the culture was shaken at 300 rpm for 2 hours at 37.degree.
C. Then 140 .mu.l of 50 mg/ml Kanamycin was added and shaking was
continued overnight at 300 rpm and 37.degree. C. [0111] 12. The
culture was centrifuged at 3000 g for 15 minutes at 4.degree. C.
The supernatant was transferred to a clean 500-ml centrifuge bottle
and 25 ml 5.times.PEG/NaCl (20% w/v PEG, 2.5 M NaCL) was added and
mixed and incubated on ice for 30 minutes.
[0112] 13. Phage particles were precipitated by centrifuging at
15,000 g for 15 minutes at 4.degree. C. The supernatant was drained
and the phage particles were resuspended in 2 ml of 1% BSA in
TBS.
[0113] The amplified phage particles form the first round of
panning were used for the second round and a total number of four
rounds of panning were performed. After each round of panning the
output phage titers were determined on LB+carbenicillin plates and
these output titer plates are the source of individual phage
particles expressing scFv fused to the surface protein PIII.
[0114] 2.3. Screening Phage Pools: The phage pools after each round
of panning were screened to assess whether the subtractive panning
experiment was successful and to select antibody pools displaying
the desired specificity and affinity. Most protocols for evaluating
phage display libraries recommend ELISA for isolation of positive
clones. However, protocols using antigens coated on plastic lead to
partial antigen denaturation, which is not an ideal experiment for
screening conformation specific antibodies. It has been reported in
some studies that scFvs directed against denatured proteins are
less efficient than those against unaltered protein conformations
in solution, in immunofluorescence and in vivo expression. So
screening against denatured antigen is not adapted to the selection
of conformation sensitive scFvs.
[0115] Selected pools of antibody-fragment displaying phage after
several rounds of panning were analyzed as a pool as well as
individually as separate clones. Even if a pool of selected phage
shows specificity to the reversibly oxidized form of PTP1B, it
still can have some high affinity individual scFvs which are
directed to the epitope(s) other than the altered active site and
may not be eliminated by the subtractive panning. We systematically
analyzed individual scFvs from initial selection after the
subtractive panning. Testing clones directly also gives us an
immediate idea of the specificity and applicability of the antibody
in a cellular context. Analysis of individual clones from a panned
pool can be done either with antibody fragment displaying phage
prepared from a single clone or with a single antibody fragment
prepared from IPTG induced culture as described later.
[0116] 2.4. Sequence Analysis of Individual scFv Clones: Individual
clones were sequenced from the subtractive panning steps. Analysis
of the scFv sequences made it possible to identify at a clonal
level different individual antibodies, which are of special
interest as the unique sequences of the antibodies might contribute
to the specificity to the individual clones. The sequences were
aligned with a chicken Ig VL+VH sequence (SEQ ID NO: 1) for
selecting functional scFv sequences and sorting them in groups
(FIG. 6, FIG. 7). In the displayed alignment the CDRs can be
identified. We have identified the CDR residues related to the
respective CDRs in the heavy and light chains. For the light chain:
CDR1: position 29-50, CDR2: position 60-83, CDR3: position 100-124.
For the heavy chain: CDR1: position 157-180, CDR2: position
188-204, CDR3: position 240-261. These CDRs are within alternate
framework regions (FR) which are more conserved in amino acid
composition.
[0117] Sequences were sorted into different groups on the basis of
their differences in the hypervariable regions hoping that
representative sequences of different groups would recognize
PTP1B-CASA mutant with different specificity or affinity. Selection
of functional scFv sequences was confirmed by checking whether the
sequences are of chicken origin. All the selected sequences in this
case aligned significantly with the light and heavy chains of
chicken IgG amino acid sequence except for the differences in the
hypervariable regions. The selected scFv sequences also contain the
6-His and HA tags at the C-terminal.
[0118] Individual clones selected randomly from the enriched scFv
pools from the subtractive panning steps were sequenced and aligned
for selecting functional scFv sequences. After initial ELISA
screening of 576 individual scFv clones 116 candidate PTP1B-binders
(SEQ ID NO: 34 to SEQ ID NO: 149) were shortlisted for a final and
definitive "in-solution" screening to isolate the conformation
sensor scFvs. The amino acid sequences provided in SEQ ID NO: 26 to
SEQ ID NO: 29 and SEQ ID NO: 34 to SEQ ID NO:149 represent scFv
sequences flanked by leader peptide and tag sequences. The
N-terminal 22 amino acids MKKTAIAIAVALAGFATVAQAA (SEQ ID NO: 150),
and the C-terminal 23 amino acids GQAGQHHHHHHGAYPYDVPDYAS (SEQ ID
NO: 151) are coded for by components of the phagemid pComb3XSS (SEQ
ID NO: 152) or the mammalian expression vector pcDNA3.2N5/GW/D-TOPO
(SEQ ID NO: 23) into which the scFvs were cloned. The sequences
present between these N- and C-terminal amino acids are the scFv
sequences. The sequences are displayed herein with the N-terminal
leader peptide, the C-terminal tag and the linker sequences
underlined. The linker sequence is used to join a variable light
(V.sub.L) chain and a variable heavy (V.sub.H) chain fragment to
produce a scFv.
[0119] The scFv sequences provided herein are of the general
pattern: NH.sub.2--V.sub.L-linker-V.sub.H--COOH. The N-terminal
leader peptide and the C-terminal tag as well as the linkers are
identified in the provided scFv sequences (SEQ ID NO: 26 to SEQ ID
NO: 29 and SEQ ID NO: 34 to SEQ ID NO: 149). The sequence between
the N-terminal leader peptide and the linker is the V.sub.L
fragment of the respective scFv, and the sequence between the
linker and the C-terminal tag is the V.sub.H fragment of the
respective scFv.
[0120] For example:
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSSAYGYGWYQ
QKSPGSAPVTVIYNNNKRPSNIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSED
SSTDAIFGAGTTLTVLGQSSRSSTVTLDESGGGLQAPGGALSLVCKASGFTFSSY
DMGWIRQAPGKGLEYVAGITDNGRYASYGSAVDGRATISRDNGQSSVRLQLNN
LRAEDTGTYYCARDDGSGWTGNSIDAWGHGTEIIVSSTSGQAGQHHHHHHGAY PYDVPDYAS
(SEQ ID NO: 78). In this sequence, the N-terminal leader peptide,
the C-terminal tag, and the linker sequence are underlined. The
scFv sequence is the sequence starting with "LTQ . . . " and ending
with " . . . STS" between the N-terminal leader peptide and the
C-terminal tag. Accordingly, the V.sub.L fragment of this scFv is
the sequence starting with "LTQ . . . " and ending with " . . .
TVL" between the N-terminal leader peptide and the linker, and the
V.sub.H fragment of this scFv is the sequence starting with "TVT .
. . " and ending with " . . . STS" between the linker and the
C-terminal tag. Where possible, the C-terminal tag, the N-terminal
leader peptide, the linker and the V.sub.L and V.sub.H fragments
are identified in a similar manner in the scFv sequences provided
herein.
[0121] Fifteen of the listed scFvs (scFv4, scFv28, scFv9, scFv10,
scFv29, scFv31, scFv11, scFv18, scFv63, scFv75, scFv79, scFv81,
scFv88, scFv89 and scFv91) display truncated amino acid sequences
because of incomplete DNA sequences from the sequencing results.
The alignments provided in FIG. 6 and FIG. 7 allow identification
of CDRs even in those sequences.
[0122] Antibody fragments or polypeptides can comprise a portion or
segment (continuous or discontinuous) of a scFv of any one of SEQ
ID NO: 26 to SEQ ID NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149, bind
PTP1B-OX and stabilize reversibly oxidized, inactive conformation.
For example, embodiments include antibodies, antibody fragments and
polypeptides whose amino acid composition is sufficiently similar
to all or a portion of a scFv of any one of SEQ ID NO: 26 to SEQ ID
NO: 29 or SEQ ID NO: 34 to SEQ ID NO: 149 that they bind reversibly
oxidized, inactive PTP1B-OX in such a manner that they inhibit its
reduction (reactivation by reducing agent to reduced, active
PTP1B).
[0123] As explained above, 15 of the listed scFvs display truncated
amino acid sequences because of incomplete DNA sequences from the
sequencing results. These clones, however, expressed well as
functional scFvs in E. coli and bound to PTP1B-CASA in ELISA.
Therefore, these scFvs were included in the shortlist of candidates
for screening the conformation sensor antibodies against PTP1B-OX.
In this screening Applicant included wild type PTP1B, reversibly
oxidized by H.sub.2O.sub.2, with the individual bacterially
expressed scFvs and then added .sup.32P-labeled reduced
carboxamidomethylated and maleylated lysozyme (RCML) as standard
PTP1B substrate with or without reducing agent. Conformation-sensor
scFvs that are able to bind and stabilize the oxidized conformation
in solution would inhibit the enzyme to revert back to its active
state when the reducing condition is restored.
Example 3
In Vitro Assessment of PTP1B-OX Stabilization by Selected Scfvs
[0124] 3. Screening Individual scFvs as Conformation-Sensor
Antibodies to PTP-OX: For purification of individual scFv as
functional antibody fragment, single scFv was expressed without
pIII fusion in a nonsuppressor strain of E. coli (e.g. TOP10F'
cells). After several rounds (2 to 4) of subtractive panning and
subsequent sequencing, individual clones were chosen for scFv
production by IPTG induction. The culture supernatants were then
screened directly by ELISA for antigen-specific binding as
described earlier.
[0125] 3.1. Characterization of Selected scFvs: After panning and
initial screening against PTP1B-CASA by ELISA, potential individual
scFv clones were selected and their ability to bind specifically to
oxidized PTP1B was confirmed by both in vitro and in vivo
assays.
[0126] 3.2. Expression and Purification of Soluble scFvs: Selected
scFv clones were expressed under IPTG induction in TOP10F' E. coli
and purified from the culture supernatant with Ni-NTA resin
exploiting the C-terminal His tag and subsequent elution with
imidazole.
[0127] TOP10F' E. coli cells were grown from single colonies in
Super Broth (SB) medium (1% MOPS, pH 7.0, 3% tryptone, 2% yeast
extract) at 37.degree. C. with shaking at 250 rpm overnight. This
overnight culture was diluted 1:50 in SB medium and incubated at
37.degree. C. at 250 rpm until the OD.sub.600 nm=0.8. This culture
was then shaken slowly at 100 rpm at 37.degree. C. for 15 minutes
to regenerate the sheared F' pili of the TOP10F' strain. This
TOP10F' cells were then infected with phage particles expressing
individual scFvs by adding 10.sup.12 phage particles/ml of
bacterial culture and incubating at room temperature for 15 minutes
with occasional gentle shaking. The infected culture was then
diluted 1:50 in SB medium+50 .mu.g/ml of Carbenicillin and
incubated at 37.degree. C. and 250 rpm until the OD.sub.600 nm=0.5.
The culture was induced with 1.5 mM IPTG at 30.degree. C. and 250
rpm for 16 hours. The cells were harvested and resuspended in Lysis
Buffer (50 mM NaH.sub.2PO.sub.4, pH 8.0, 500 mM NaCl, 10 mM
imidazole, 1 mM PMSF and EDTA free protease inhibitor cocktail).
Resuspended culture was incubated with lysozyme (0.5 mg/ml) for 30
minutes at 4.degree. C. The suspension was sonicated and
centrifuged at 50,000 g for 30 minutes at 4.degree. C. The
supernatant was used to purify the soluble scFv by using Ni-NTA
gravity columns. After adding the supernatant to the column it was
washed with 20 column volume of Wash Buffer (50 mM
NaH.sub.2PO.sub.4, pH 8.0, 500 mM NaCl, 20 mM imidazole). The bound
protein was finally eluted with the Elution Buffer (50 mM
NaH.sub.2PO.sub.4, pH 8.0, 500 mM NaCl, 250 mM imidazole).
[0128] 3.3. Expression and Purification of Recombinant PTP1B: The
catalytic domain (37 kDa) of wild type PTP1B (PTP1B_WT) or
PTP1B-CASA was constructed in a pET19b vector and expressed in BL21
E. coli. Single colonies harboring the expression plasmid were
grown in LB+Ampicillin (50 .mu.g/ml) overnight. The overnight
culture was diluted 1:50 in LB+Ampicillin (50 .mu.g/ml) at
37.degree. C. until OD.sub.600 nm=0.6. Expression of recombinant
PTP was induced with 0.3 mM IPTG at 37.degree. C., 250 rpm for 4
hours. Cells were harvested and resuspended in Lysis Buffer (25 mM
NaH.sub.2PO.sub.4, pH 6.5, 10 mM NaCl, 1 mM EDTA, 5 mM DTT, 1 mM
PMSF, 2 mM benzamidine and protease inhibitor cocktail). The
suspension was incubated with 0.5 mg/ml lysozyme at 4.degree. C.
for 30 minutes and sonicated to extract the soluble protein. The
sample was then centrifuged at 50,000 g at 4.degree. C. for 30
minutes and supernatant was collected for purification of the
recombinant PTP1B. The first purification step was done with a
cation exchange column (SP Sepharose HP) using 25 mM
NaH.sub.2PO.sub.4, pH 6.5, 10 mM NaCl, 1 mM EDTA, 5 mM DTT as the
binding buffer and the protein was eluted by a gradient elution
with 10-500 mM NaCl. Fractions containing PTP1B were pooled
together and used for a final purification step by an anion
exchange column (Q Sepharose HP) using 25 mM Tris-Hcl, pH 7.5, 1 mM
EDTA, 5 mM DTT as the binding buffer. The purified protein was
eluted by a gradient elution with 0-500 mM NaCl.
[0129] 3.4. Screening for the Conformation-Sensor scFvs: Reduced
carboxamidomethylated and maleylated lysozyme (RCML) was labeled
with .sup.32P using recombinant GST-FER kinase to stoichiometries
up to 0.8 (mol .sup.32P incorporated/mol of protein). This
.sup.32P-Labeled phospho-tyrosyl RCML was used as the substrate in
the in-solution phosphatase assay for screening the conformation
sensor scFvs. Recombinant PTP1B was reversibly oxidized and
transiently inactivated with H.sub.2O.sub.2 (5.times. molar excess
of the protein). The phosphatase activity was completely restored
upon the removal of H.sub.2O.sub.2 by a quick buffer exchange and
addition of reducing agent (TCEP/DTT). Purified bacterially
expressed scFvs (100.times. molar excess of PTP1B) were incubated
with PTP after H.sub.2O.sub.2 treatment and its removal and the
effect of individual scFv on stabilizing the reversibly oxidized
conformation was assessed by the phosphatase assay under reducing
condition.
Example 4
Expression of Intrabody in Mammalian Cells
[0130] Intracellular antibodies (or intrabodies) have been used
successfully for studying biological processes and for blocking
proteins inside cells. In the initial screening, four scFv
candidates that showed significant inhibition to the reactivation
process of transiently inactivated PTP1B (by stabilizing the
reversibly oxidized conformation of the enzyme). One of these scFvs
(scFv45) was cloned in pcDNA3.2/V5-GW/D-TOPO (SEQ ID NO: 23)
expression vector for transient expression of this single chain
antibody fragment in mammalian cells as intrabody to PTP1B. The
scFv45 sequence from the pCom3.times.SS phagemid construct was PCR
amplified and cloned directionally in the pcDNA3.2/V5-GW/D-TOPO
vector in such a way that the 5' and 3' Sfi I restriction sites
were retained in the mammalian construct. This particular intrabody
was transiently transfected in 293T cells using Fugene 6
transfection reagent. Transfected cells were harvested 24, 36 and
48 hours post transfection and lysed in RIPA buffer (25 mM HEPES,
pH 7.5, 150 mM NaCl, 0.25% Deoxychloate, 10% Glycerol, 25 mM NaF,
10 mM MgCl.sub.2, 1 mM EDTA, 1% Triton. X-100, 0.5 mM PMSF, 10 mM
Benzamidine, protease inhibitor cocktail). Soluble fractions from
the samples were used in Western Blot experiment using HRP
conjugated anti-HA antibody to detect the expressed scFv. Stable
expression of scFv 45 was shown at 24-48 hours post transfection as
the PTP1B-OX-specific intrabody.
[0131] In order to screen the isolated scFvs for those that inhibit
reactivation of PTP1B-OX by reducing agent, but have no direct
inhibitory effect on phosphatase activity in assays in vitro, the
effect of six scFvs (scFv20, scFv34, scFv45, scFv57, scFv64 and
scFv106) on PTP1B activity was determined. PTP1B activity was
measured using 32P-labeled pTyr-Reduced Carboxamidomethylated and
Maleylated Lysozyme as substrate (FIGS. 1 and 2). Addition of the
reducing agent TCEP alone had no substantial effect in PTP1B
activity was assessed. None of the above mentioned scFvs had a
substantial effect on PTP activity, indicating that no direct
inhibition of PTP activity was effected by any of these scFvs (FIG.
1).
[0132] In a different experiment, conditions were established in
which wild type PTP1B could be reversibly oxidized in vitro. PTP1B
was inactivated following addition of H.sub.2O.sub.2, however,
phosphatase activity was completely restored upon the removal of
H.sub.2O.sub.2 by a quick buffer exchange and addition of the
reducing agent TCEP (FIG. 2, "+H.sub.2O.sub.2"). Addition of the
reducing agent alone had no substantial effect on PTP1B activity
(FIGS. 1 and 2, "PTP1B"). The ability of individual scFvs to
stabilize the reversibly oxidized, inactive conformation of the
PTP1B was assessed by the ability of the scFv to inhibit the
reactivation of the enzyme by reducing agent (FIG. 2). As
illustrated in FIG. 2, scFv45, scFv57, scFv64, and scFv106 show a
substantial inhibition of the restoration of PTP1B activity after
reversible oxidation.
[0133] These results reflect the ability of these scFvs to
stabilize PTP1B-OX and inhibit the reduction of the OX form back to
an active, reduced form, while not directly inhibiting PTP
activity. Applicant identified scFvs that showed significant
inhibition of the reactivation of PTP1B-OX by reducing agent, but
did not exert any direct inhibitory effect on activity (See FIGS. 1
and 2).
[0134] In order to validate this approach further, Applicant tested
the effects of expressing one of these (scFv45) as a single chain
antibody fragment "intrabody" to PTP1B, using 293T cells as a
convenient expression system. This intrabody was expressed
transiently and then tested for effects on insulin signaling,
focusing initially on the tyrosine phosphorylation status of the
.beta.-subunit of the insulin receptor and IRS-1 (FIG. 2). Initial
indications are that for both substrates expression of the
intrabody had no impact on the basal level of tyrosyl
phosphorylation, but it enhanced and extended the time course of
insulin-induced phosphorylation, consistent with our proposed
mechanism of action.
[0135] 4.1 Role of Reversible PTP1B Oxidation in the Cellular
Signaling Response to Insulin: It was shown in Applicant's lab that
stimulation of cells with insulin caused rapid and transient
oxidation and inhibition of PTP and that this facilitates increased
phosphorylation of receptors. Some scFvs described herein can be
used to detect the intracellular reversibly oxidized PTP1B in
response to insulin. Since scFvs can also be expressed inside
mammalian cells, they may be used to understand the dynamics of PTP
redox regulation in vivo in response to insulin.
[0136] Once the scFv-PTP1B-OX complex is formed intracellularly,
some of the scFvs with high affinity will lock the enzyme in its
oxidized form. PTP1B, locked in its reversibly oxidized
conformation by the intrabody, should not be able to revert back to
its active conformation by thioredoxin or other cellular reducing
system. A high affinity intrabody, therefore, can inhibit the pool
of active PTP1B simply by keeping the enzyme in its inactive form
and hindering its reactivation. This provides a unique opportunity
to dissect the status of both upstream and downstream signaling
mechanism in response to insulin. For instance, the phosphorylation
of the tandem tyrosine residues (pYpY1162/1163) in the activation
loop of the .beta.-subunit of insulin receptor and the
phosphorylation of PKB/AKT is analyzed using phospho-specific
antibodies upon insulin stimulation in cells overexpressing
PTP1B.sub.ox-specific scFv. Insulin signaling events in this system
are also analyzed to verify whether locking PTP1B in its oxidized
conformation by intracellular scFv can prolong the signals
downstream of activated insulin receptor.
[0137] Intrabody scFv45 was transfected in 293T cells, serum
starved and insulin stimulated to investigate the physiological
relevance of this intrabody expression in mammalian cells in the
context of PTP1B mediated regulation of insulin signaling (or
insulin signaling mediated redox regulation of PTP1B activity).
Insulin stimulated 293T cells with or without the intrabody were
harvested in RIPA buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 0.25%
Deoxychloate, 10% Glycerol, 25 mM NaF, 10 mM MgCl.sub.2, 1 mM EDTA,
1% TritonX-100, 0.5 mM PMSF, 10 mM Benzamidine, protease inhibitor
cocktail, 1 mM sodium vanadate) and the cell lysates were examined
for total tyrosine phosphosphorylation pattern using
phospho-tyrosine specific antibodies (FIGS. 3 and 4). When cells in
which scFv45 was overexpressed were stimulated with insulin,
insulin receptor .beta. (IR.beta.) and insulin receptor substrate-1
(IRS-1) showed increased and prolonged tyrosine phosphorylation
(FIGS. 3 and 4). This result supports the hypothesis that the
intrabody is stabilizing the reversibly oxidized conformation of
PTP1B (induced by ROS produced in response to insulin stimulation)
in vivo and inhibits its reactivation by cellular reducing
machinery.
Example 5
Rabbit Polyclonal Antibody Against PTP1B-OX
[0138] In the catalytic cleft of PTP1B, there are two hydrogen
bonds that hold the active site together: the first one holds the
signature motif at the base of the active site cleft linking the
sulfur atom of the cysteine 215 with the serine 222. The second one
is between serine 216 and the invariant tyrosine 46 of the pTyr
loop that points down to the base of the active site and defines
the depth of the cleft. Upon controlled oxidation with
stoichiometric quantities of H.sub.2O.sub.2, formation of the
cyclic sulphenamide intermediate is induced in the PTP1B crystals.
One of the critical aspects about this oxidative modification of
the active site cysteine is that it induces profound structural
rearrangement at the active site cleft. The PTP loop, containing
the signature motif, and Tyr46 from the phosphotyrosine binding
loop, which are normally buried in the structure, flip out of the
active site to adopt solvent exposed positions due to the lack of
hydrogen bonding at the catalytic cleft. These conformational
changes are readily reversible, consistent with a mechanism for
reversible regulation of PTP function.
[0139] The crystal structure of the reversibly oxidized, inactive
form of PTP1B has been determined previously (Salmeen et al. Nature
2003). The surface representation of the crystal structure
indicates that the Tyr46 of the pTyr loop is popped open and
becomes solvent exposed as a result of the breaking of hydrogen
bonds in the reversibly oxidized conformation. A peptide sequence
(H.sub.2N-CNRYRDV-OH) (SEQ ID NO: 24) was designed on the basis of
the surface structure surrounding the solvent exposed Tyr46 of the
reversibly oxidized form of PTPB. This peptide was used as an
antigen to generate conformation specific rabbit polyclonal
antibodies that would recognize the oxidized form of PTP1B. As a
control measure to this approach another peptide
(H.sub.2N--CNRpYRDV-OH) (SEQ ID NO 25), in which the Tyr46 is
phosphorylated was used to generate rabbit polyclonal antibody
against the phosphorylated Tyr46. These two polyclonal antibodies
were used in the same RCML phosphatase assay as we have described
in section 3.4, to detect the ability to recognize the reversibly
oxidized conformation of PTP1B. The antibody generated against the
solvent exposed peptide sequence surrounding the Tyr46 has similar
inhibitory effect to the reactivation of reversibly oxidized PTP1B.
Hence, this polyclonal antibody is also able to stabilize the
reversibly oxidized conformation of PTP1B and acts as a
conformation sensor antibody.
Example 6
Interaction Between PTP1B-Ox and scFvs In Vitro
[0140] In their in vitro screening assay, Applicant have found
candidate PTP1B-OX specific scFvs (scFv45, scFv57, scFv64 and
scFv106) that inhibit reactivation of PTP1B-OX by reducing agent,
but have no direct inhibitory effect on phosphatase activity (FIG.
1 and FIG. 2). This result suggests that these candidate scFvs
inhibit the reactivation of PTP1B-OX by reducing agent by
specifically binding and stabilizing the reversibly oxidized,
inactive conformation of the enzyme.
[0141] In order to demonstrate direct interaction between PTP1B-OX
and candidate scFvs, Applicant performed an in vitro binding assay
using purified recombinant PTP1B (37 kDa) and purified scFvs under
both oxidizing and reducing conditions. They established conditions
in which PTP1B is reversibly oxidized by H.sub.2O.sub.2 in solution
(FIG. 8). An artificial protein substrate RCML, in which tyrosine
was phosphorylated with .sup.32P using recombinant GST-FER kinase
was used in this assay to measure the phosphatase activity.
Recombinant PTP (37 kDa) was incubated with increasing
concentration (50 .mu.M to 100 mM) of H.sub.2O.sub.2. A quick
buffer exchange was done to remove the H.sub.2O.sub.2 and reducing
agent (TCEP) was added to reactivate the enzyme. Results showed
that PTP1B is reversibly oxidized and transiently inactivated when
up to 500 .mu.M H.sub.2O.sub.2 was used and the activity of the
enzyme can be restored fully with the addition of reducing agent
under this condition (FIG. 8).
[0142] In the in vitro binding assay, purified PTP was reversibly
oxidized with .mu.M H.sub.2O.sub.2 followed by a quick buffer
exchange to remove H.sub.2O.sub.2 (FIG. 8). Purified scFv was
incubated in molar excess with PTP1B-OX or with PTP under reducing
condition (with 2 mM TCEP) in binding buffer (20 mM HEPES, pH 7.4,
300 mM NaCl, 0.05% BSA, 0.05% Tween-20 and 10 mM imidazole) for 2
hours at 4.degree. C. Ni-NTA agarose was added and incubated for
one hour at 4.degree. C. Protein complex bound to Ni-NTA agarose
beads was pulled down and washed (three times, 5 minutes each, at
4.degree. C.) with binding buffer containing 20 mM imidazole to
reduce non-specific binding to the beads. The protein complex was
eluted from the Ni-NTA agarose beads with 500 mM imidazole (in
binding buffer, pH 7.4) for 15 minutes at 4.degree. C. with gentle
shaking. The complex was separated by SDS-PAGE and PTP1B was
detected with anti-PTP1B antibody (FG6) and scFv was detected with
anti-HA antibody [anti-HA (3F10)-HRP, Roche] by immunoblotting.
Eight different scFvs (scFvs 20, 21, 24, 28, 45, 48, 57, and 105),
expressed and purified from bacterial cultures were tested by this
in vitro binding experiment. Among this group of scFvs, two (scFv45
and scFv 57) showed specific binding to PTP1B-OX, but not to PTP
under reducing condition (FIG. 9a and FIG. 9b). Under the
conditions used, scFvs 20, 21, 24, 28, 48, and 105 showed no
significant binding to PTP1B-OX or to PTP under reducing condition
(FIG. 9c).
Example 7
Interaction Between PTP1B-OX and scFvs in Mammalian Cells
[0143] One of the positive candidate scFvs (scFv45) was cloned in
pcDNA3.2/V5-GW/D-TOPO expression vector and transiently expressed
in mammalian cells as stable intracellular antibody (aka intrabody)
(FIG. 10). To detect PTP1B-OX and scFv45 interaction in mammalian
cells, scFv45 was overexpressed in 293T cells as described herein
(See Example 4) and 48 hours post transfection the cells were
serum-starved for 16 hours in growth medium (DMEM, low glucose)
without serum (FBS), then incubated with 1 mM H.sub.2O.sub.2 (in
growth medium without serum) for 5 minutes at 37.degree. C. or
treated with 20 mM NAC (in growth medium without serum) for 1 hour
at 37.degree. C. Cells treated with H.sub.2O.sub.2 were washed
twice with cold (4.degree. C.) PBS and lysed in lysis buffer (25 mM
HEPES, pH 7.4, 150 mM NaCl, 0.25% Deoxychloate, 1% TritonX-100, 25
mM NaF, 10 mM MgCl.sub.2, 1 mM EDTA, 10% Glycerol, 0.5 mM PMSF, 10
mM Benzamidine, protease inhibitor cocktail). Cells treated with
NAC were lysed with lysis buffer containing 2 mM TCEP to ensure a
post-lysis reducing environment. Interaction between PTP1B and
intrabody45 were tested by pulling down the protein complex from 1
mg of total cell lysate with Ni-NTA agarose or by
immunoprecipitating PTP with anti-PTP1B (FG6). For the pull down
experiment, Ni-NTA agarose beads were incubated with the lysates
(both oxidized and reduced) at 4.degree. C. Protein complex bound
to the Ni-NTA agarose beads was pulled down and washed three times;
first with lysis buffer containing 20 mM imidazole followed by two
more washes with wash buffer (PBS, pH 7.4, 20 mM imidazole, 0.05%
BSA, 0.05% Tween-20 and protease inhibitors). Applicant used 20 mM
imidazole in the wash buffer to reduce the non-specific binding of
proteins with the Ni-NTA agarose beads. The complex was eluted with
wash buffer containing 500 mM imidazole with gentle shaking at
4.degree. C. and the eluate was mixed with SDS sample buffer with
DTT. For the immunoprecipitation experiments, 1 mg of total cell
lystates (both oxidized and reduced) was incubated with anti-PTP1B
antibody (FG6) at 4.degree. C. The interacting protein complex was
immunoprecipitated after incubating the lysate-antibody mixture
with protein A/G Sepharose at 4.degree. C. After
immunoprecipitation, Sepharose beads were washed three times; first
with lysis buffer followed by two more washes with wash buffer
(PBS, pH 7.4, 0.05% BSA, 0.05% Tween-20 and protease inhibitors).
The Sepharose beads were then heated at 90.degree. C. in
2.times.SDS sample buffer with DTT. Proteins from the pulled down
or immunoprecipitated complex were separated by SDS-PAGE. PTP was
detected with anti-PTP1B antibody (FG6) and the intrabodies were
detected with anti-HA (3F10)-HRP antibody [anti-HA (3F10)-HRP,
Roche] by immunoblotting. In both the pull-down and
immunoprecipitation experiments, interaction between scFv45 and
PTP1B-OX was observed in the H.sub.2O.sub.2-treated cells but not
in the cells that were under reducing condition (FIG. 11a and FIG.
11b).
[0144] In the initial in vitro phosphatase screening assay using
reversibly oxidized (with H.sub.2O.sub.2) recombinant PTP1B,
Applicant found four different PTP1B-OX specific scFvs (scFv45,
scFv57, scFv64 and scFv106) from the initial pool of 12 different
scFvs. Two of these scFvs (scFv45 and scFv57) were shown to
interact with PTP1B-OX, but not with PTP1B under reducing condition
in the in vitro interaction assay. Applicant has also shown that
scFv45, expressed as an intrabody in mammalian cells, can bind to
endogenous PTP1B-OX but not endogenous PTP under reducing
conditions. Further assessment was carried out to identify
additional PTP1B-OX-specific intrabodies. Additional scFvs have
been subcloned in pcDNA3.2/V5-GW/D-TOPO mammalian expression vector
(FIG. 10) from their respective phagemid constructs. After
transient overexpression of these scFvs as intrabodies in 293T
cells, the interaction between endogenous PTP1B and these
intrabodies in both H.sub.2O.sub.2-treated cells and cells was
observed under reducing condition (treated with NAC and lysed with
lysis buffer containing TCEP). Using Ni-NTA pull down assay, as
described earlier for the mammalian cell lysates, Applicants
identified four additional scFvs (scFv57, scFv67, scFv102, scFv106)
that bound to endogenous PTP1B-OX strongly; five others (scFv 136,
scFv 61, scFv62, scFv64, scFv34 and scFv118) bound weakly to
PTP1B-OX (FIG. 12a and FIG. 12b). None of the 28 intrabodies,
however, showed any binding to endogenous PTP1B under reducing
conditions (FIG. 12a and FIG. 12b). All can be used in the methods
and compositions described herein. Additional scFvs, such as some
or all of the 137 selected individual scFvs from the library, can
be similarly tested by in vitro screening and/or by binding assay
in mammalian cells.
Role of Intrabody45 on the Cellular Signaling Response Downstream
of Insulin Receptor:
[0145] Insulin mediated phosphorylation and activation of IRK
causes increased downstream signaling. PTP1B down-regulates this
signal by regulating phosphorylation at the level of the receptor
and acts as brake in this signaling pathway by maintaining a
competitive fine-tuning in this intricate regulatory mechanism.
However, stimulation of cells with insulin causes rapid and
transient oxidation and inhibition of PTP1B and facilitates
increased phosphorylation of receptors. So, generation of ROS by
insulin accelerates the signaling by removing the foot off the
brake. This transient inactivation of PTP1B is reversible and the
brake is restored in the signaling pathway by dephosphorylating and
deactivating the insulin receptor by the reactivated PTP1B.
[0146] As described herein, Applicant has shown that intrabody45
expression in mammalian cells caused enhanced and prolonged
tyrosine phosphorylation of insulin receptor (3 subunit (IR.beta.)
and insulin receptor substrate-1 (IRS-1) (FIGS. 3 and 4). This
result supports the hypothesis that the intrabody stabilizes the
reversibly oxidized conformation of PTP1B (induced by ROS produced
in response to insulin stimulation) in vivo and inhibits its
reactivation by cellular reducing machinery.
[0147] Insulin induces activation of the insulin-receptor kinase
(IRK) through autophosphorylation. Recruitment of insulin-receptor
substrate (IRS) proteins induces activation of Phosphoinositide
3-kinase (PI3K). PI3K activation triggers downstream effectors,
such as phosphatidylinositol-dependent kinase 1 (PDK1) and protein
kinase B or AKT, leading to translocation of glucose transporter 4
(GLUT4) and glucose uptake in muscle, and inactivation of
glycogen-synthase kinase 3 (GSK3). PTP1B dephosphorylates
membrane-bound or endocytosed insulin receptors, causing their
deactivation and plays a negative inhibitory role in the signaling
events downstream of insulin receptor.
[0148] Applicant assessed the role of PTP1B-OX specific intrabody
on the downstream readout of insulin signaling by following the
phosphorylation (activation) status of AKT. Cells with or without
scFv45 overexpression were stimulated with insulin for different
time periods and phosphorylation of AKT activation loop at residue
Threonine 308 (T308) was observed with phospho-specific AKT
antibody [phospho-Akt (T308), Cell Signaling]. Cells overexpressing
scFv45 displayed enhanced and sustained AKT phosphorylation at
residue Threonine 308 (T308) (FIG. 13). This result suggests that
upon insulin stimulation, intrabody45 binds and stabilizes
endogenous PTP1B-OX, causing an enhanced and prolonged
phoshorylation of insulin receptor 13 subunit (IR.beta.) and
insulin receptor substrate 1 (IRS-1) and this signal is transmitted
downstream to cause an enhanced and prolonged activation of
AKT.
[0149] Colocalization of Intrabody 45 and PTP1B-OX under Insulin
Stimulation: In order to verify the interaction between PTP1B-OX
and scFv45 in mammalian cells, Applicant determined whether PTP and
scFv45 colocalized after insulin stimulation, using
immunofluorescence. Cos1 cells were grown on cover slips and
transfected with scFv45 using Fugene6 as the transfection reagent
according to manufacturer's instructions. The cells were serum
starved 24 hours post transfection, for 16 hours at 37.degree. C.
and then stimulated with 25 nM insulin (in growth medium without
serum) or left untreated. Following insulin treatment the cells
were washed 2.times. with PBS and fixed with 5% formalin (in PBS)
for 15 minutes at room temperature. The cells were washed with PBS
three times at room temperature. The fixed cells were permeabilized
with 0.5% Triton-X100 (in PBS) for 5 minutes at room temperature.
Cells were rinsed with Wash Buffer [PBS, pH 7.4 with 0.1% BSA, 0.2%
TritonX-100, 0.05% Tween-20 and 0.05% sodium azide] at room
temperature. The coverslips were blocked with 5% normal goat serum
in wash buffer for 1 hour at room temperature to reduce the
non-specific binding of the secondary antibodies. To detect
endogenous PTP1B, the coverslips were incubated with anti-PTP1B
antibody [rabbit polyclonal anti-PTP1B (H-135), Santa Cruz
Biotechnology]at RT for 1 hour. The cells were washed with the wash
buffer at room temperature to remove excess antibodies. A cocktail
of Alexa 594 conjugated anti-HA mouse monoclonal antibody [16B12,
Invitrogen, 21288] and goat anti-rabbit alexa 488 (Invitrogen,
A-11034) secondary antibody in the blocking buffer (5% normal goat
serum) was added to the coverslip and incubated at RT for 1 hour.
The coverslips were washed 3.times. with IF wash buffer and
incubated with DAPI (0.3 .mu.g/ml in PBS) for 10 minutes at room
temperature to stain the nucleus and washed again with IF wash
buffer to remove excess unbound DAPI. The cover slip was mounted on
glass slide with Vectasheild Mounting Medium (H-1000, Vector
Laboratories) and observed using confocal microscope (LSM 710,
Zeiss) with 63.times. objective lens and immersion oil. Strong
colocalization between PTP1B and intrabody45 was observed in cells,
following stimulation with insulin; colocalization between PTP1B
and intrabody45 was not significant in cells without insulin
stimulation (FIG. 14). To quantify colocalization of PTP1B and
intrabody45 from the merged images, 15 individual images were
analyzed by Zeiss (LSM 710) Colocalization Viewer Software. The
degree of colocalization of PTP1B and intrabody45 in each cell was
expressed as colocalization coefficients that measure relative
number of colocalizing pixels for the respective fluorophores for
PTP1B and intrabody45, as compared to the total number of pixels.
The numeric range for this colocalization method is set as 0-1,
where "0" indicates no colocalization and "1" indicates
colocalization of all pixels in a cell.
TABLE-US-00001 Sequences of scFv45, scFv57, scFv64, and scFv106:
scFv45 (SEQ ID NO: 26) M K K T A I A I A V A L A G F A T V A Q A A
L T Q P S S V S A N P G G T V K I T C S G S S S A Y G Y G W Y Q Q K
S P G S A P V T V I Y N N N K R P S N I P S R F S G S K S G S T G T
L T I T G V Q A E D E A V Y F C G S E D S S T D A I F G A G T T L T
V L G Q S S R S S T V T L D E S G G G L Q A P G G A L S L V C K A S
G F T F S S Y D M G W I R Q A P G K G L E Y V A G I T D N G R Y A S
Y G S A V D G R A T I S R D N G Q S S V R L Q L N N L R A E D T G T
Y Y C A R D D G S G W T G N S I D A W G H G T E V I V S S T S G Q A
G Q H H H H H H G A Y P Y D V P D Y A S scFv57 (SEQ ID NO: 27) M K
K T A I A I A V A L A G F A T V A Q A A L T Q P S S V S A N P G A F
N K I T C S G S S S A Y G Y G W N Q Q K S P G S A P V T V I Y N N N
K R P S N I P S R F S G S K S G S T G T L T I T G D Q D E D E D F Y
F C G S E Y S S T D A I F G A G T T L T V L G Q S S R S S T V T L D
E S G G G L Q A P G G A L S L V C K A S G F T F S S Y D M G W I P Q
A P G K G L E Y V A G I T D N G I Y A S Y G S A V D G R A T I S R D
N R Q S S V K L Q L N N L K A D D T G T Y Y C A R D D G S G W T G N
S I D A W G H G T E V I V S S T S G Q A G Q H H H H H H G A Y P Y D
V P D Y A S scFv64 (SEQ ID NO: 28) M K K T A I A I A V A L A G F A
T V A Q A A L T Q P S S V S A N P G D P L K I T C S G D S S G Y G Y
G W Y Q Q K S P G S A P V T V I Y N N N K R P S D I P S R F S G S K
S G S T G T L T I T G V Q A E D E A V Y F C G S E D S N T D A V F G
A G T T L T V L G Q S S R S S T V T L D E S G G G L Q T P G G T L S
L A C K A S G F T F S G Y D M G W V R Q A P G K G L E Y V A G I T S
D G R Y A S Y G S A V D G R A A I W R D N G Q S T V R L Q L K N L R
T E D T A T Y Y C A R N D G S G W N G N N I D A W G H G T E V I V S
S T S G Q A G Q H H H H H H G A Y P Y D V P D Y A S scFv106 (SEQ ID
NO: 29) M K K T A I A I A V A L A G F A T V A Q A A L T Q P S S V S
A N P G E T V K I T C S G D S S D Y G Y G W Y Q Q K S P G S A P V T
V T Y S N N Q R P P N I P S R F S G S A S G S T A T L T I T G V Q V
E D E A V Y Y C G S E D S T T D A V F G A G T T L T V L G Q S S R S
S A M T L D E S G G G L Q T P G G A L S L V C K A S G F T F S S Y D
M G W V R Q A P G K G L E Y V A G I T N D G R Y A S Y G S A V D G R
A T I S R D N G Q S T V R L Q L N N L R A E D T G T Y Y C A R D D G
S G W T G N T I D T W G H G T E V I V S S T S G Q A G Q H H H H H H
G A Y P Y D V P D Y A S Sequences of the PTP1B-OX-Specific scFv
Antibodies: >scFv45 M K K T A I A I A V A L A G F A T V A Q A A
L T Q P S S V S A N P G G T V K I T C S G S S S A Y G Y G W Y Q Q K
S P G S A P V T V I Y N N N K R P S N I P S R F S G S K S G S T G T
L T I T G V Q A E D E A V Y F C G S E D S S T D A I F G A G T T L T
V L G Q S S R S S T V T L D E S G G G L Q A P G G A L S L V C K A S
G F T F S S Y D M G W I R Q A P G K G L E Y V A G I T D N G R Y A S
Y G S A V D G R A T I S R D N G Q S S V R L Q L N N L R A E D T G T
Y Y C A R D D G S G W T G N S I D A W G H G T E V I V S S T S G Q A
G Q H H H H H H G A Y P Y D V P D Y A S S . >scFv57 M K K T A I
A I A V A L A G F A T V A Q A A L T Q P S S V S A N P G G T V K I T
C S G S S S A Y G Y G W Y Q Q K S P G S A P V T V I Y N N N K R P S
N I P S R F S G S K S G S T G T L T I T G V Q A E D E A V Y F C G S
E D S S T D A I F G A G T T L T V L G Q S S R S S A V T L D E S G G
G L Q T P G G A L S L V C K A S G F T F S S Y D M G W V R Q A P G K
G L E Y V A G I T N D G R Y A S Y G S A V D G R A T I S R D N G Q S
T V R L Q L N N L R A E D T G T Y Y C A R D D G S G W T G N T I D T
W G H G T E V I V S S T S G Q A G Q H H H H H H G A Y P Y D V P D Y
A S S . >scFv64 M K K T A I A I A V A L A G F A T V A Q A A L T
Q P S S V S A N P G E T V K I T C S G D S S G Y G Y G W Y Q Q K S P
G S A P V T V I Y N N N K R P S D I P S R F S G S K S G S T G T L T
I T G V Q A E D E A V Y F C G S E D S N T D A V F G A G T T L T V L
G Q S S R S S T V T L D E S G G G L Q T P G G T L S L A C K A S G F
T F S G Y D M G W V R Q A P G K G L E Y V A G I T S D G R Y A S Y G
S A V D G R A A I W R D N G Q S T V R L Q L K N L R T E D T A T Y Y
C A R D D G S G W S G N N I D A W G H G T E V I V S S T S G Q A G Q
H H H H H H G A Y P Y D V P D Y A S S . >scFv106 M K K T A I A I
A V A L A G F A T V A Q A A L T Q P S S V S A N P G E T V K I T C S
G D S S D Y G Y G W Y Q Q K S P G S A P V T V T Y S N N Q R P P N I
P S R F S G S A S G S T A T L T I T G V Q V E D E A V Y Y C G S E D
S T T D A V F G A G T T L T V L G Q S S R S S A M T L D E S G G G L
Q T P G G A L S L V C K A S G F T F S S Y D M G W V R Q A P G K G L
E Y V A G I T N D G R Y A S Y G S A V D G R A T I S R D N G Q S T V
R L Q L N N L R A E D T G T Y Y C A R D D G S G W T G N T I D T W G
H G T E V I V S S T S G Q A G Q H H H H H H G A Y P Y D V P D Y A S
S . >scFv 136 M K K T A I A I A V A L A G F A T V A Q A A L T Q
P S S V S A N P G E T V K I T C S G G S Y S Y G W Y Q Q K S P G S A
P V T V I Y S S D K R P S D I P S R F S G S K S G S T S T L T I T G
V Q A E D E A V Y Y C G S R D S N Y V G I F G A G T T L T V L G Q S
S R S S T V T L D E S G G G L Q T P G G A L S L V C K A S G F T F S
S Y E M Q W V R Q A P G K G L E F V A A I S S D G S Y T N Y G A A V
Q G R A T I S R D N G Q S T V R L Q L S N L R A E D T A T Y Y C A R
S P G G Y T W W P G A A G G I D A W G H G T E V I V S S T S G Q A G
Q H H H H H H G A Y P Y D V P D Y A S S . >scFv 67 M K K T A I A
I A V A L A G F A T V A Q A A L T Q P S S V S A N P G G T V K I T C
S G S S S A Y G Y G W Y Q Q K S P G S A P V T V I Y N N N K R P S N
I P S R F S G S K S G S T G T L T I T G V Q A E D E A V Y F C G S E
D S S T D A I F G A G T T L T V L G Q S S R S S A V T L D E S G G G
L Q T P G G A L S L V C K A S G F T F S S Y D M G W V R Q A P G K G
L E Y V A G I T N D G R Y A S Y G S A V D G R A T I S R D N G Q S T
V R L Q L N N L R A E D T G T Y Y C A R D D G S G W T G N T I D T W
G H G T E V I V S S T S G Q A G Q H H H H H H G A Y P Y D V P D Y A
S S . >scFv 102 M K K T A I A I A V A L A G F A T V A Q A A L T
Q P S S V S A N P G E T V K I T C S G G S G S Y G W Y Q Q E S P G S
A P V T V I Y Y N D K R P S D I P S R F S G S A S G S T A T L T I A
G V R A E D E A V Y F C G S W D S S T S A G I F G A G T A L T V L G
Q S S R S S A V T L D E S G G G L Q T P G G G L S L V C K A S G F S
S S H G M G W M R Q A P G K G L E F V A G I R S D G S S T A Y G A A
V D G R A T I T R D D G Q S T V T L Q L N N L R A E D T A T Y F C A
K T N S Y N S A G I I D A W G H G T E V I V S S T S G Q A G Q H H H
H H H G A Y P Y D V P D Y A S S . >scFv 34 M K K T A I A I A V A
L A G F A T V A Q A A L T Q P S S V S A N L G G T V E I T C S G S S
G S Y G W Y Q Q K S P G S A P V T V I Y Y N D K R P S D I P S R F S
G S T S G S T A T L T I T G V Q A E D E A V Y F C G G Y D S N Y I G
I F G A G T T L T V L G Q S S R S S A V T L D E S G G G L Q T P R G
A L S L V C K A S G F T F S S Y S M A W V R Q A P G K G L E F V
A
G I Q N D G S I T D Y G S A V D G R A T I S R D D G Q S T V R L Q L
N N L R T E D T A T Y Y C A K T T V A D G V I G A Y G I D A W G H G
T E V I V S S T S G Q A G Q H H H H H H G A Y P Y D V P D Y A S S .
>scFv 61 M K K T A I A I A V A L A G F A T V A Q A A L T Q P S S
V S A N P G E T V K I T C S G G G S Y A G S Y Y Y G W Y Q Q K A P G
S A P V T L I Y D N T N R P S N I P S R F S G S L S G S T G T L T I
T G V R A E D E A V Y Y C G S F D S S T D G G Y A A I F G A G T T L
T V L G Q S S R S Y A V T L D E S G G G L Q T P G G G L S L V C K A
S E F T F S S Y A M E W V R Q A P G K G L E W V A Y I N S D G S S T
W Y A P A V K G R A T I S R D N G Q S T V R L Q L N S L R A E D T A
T Y Y C T R G S G G E N I D T W G H G T E V I V S S T S G Q A G Q H
H H H H H G A Y P Y D V P D Y A S S . >scFv 62 M K K T A I A I A
V A L A G F A T V A Q A A L T Q P S S V S A N P G E T V K I T C S G
G G S Y A G S Y Y Y G W Y Q Q K A P G S A P V T L I Y D N T N R P S
N I P S R F S G S L S G S T G T L T I T G V R A E D E A V Y Y C G S
F D S S T D G G Y A A I F G A G T T L T V L G Q S S R S S A V T L D
E S G G G L Q T P G G G L S L V C K A S E F T F S S Y A M E W V R Q
A P G K G L E W V A Y I N S D G S S T W Y A P A V K G R A T I S R D
N G Q S T V R L Q L N S L R A E D T A T Y Y C T R G S G G E N I D T
W G H G T E V I V S S T S G Q A G Q H H H H H H A A Y P Y D V P D Y
A S S . >scFv 118 M K K T A I A I A V A L A G F A T V A Q A A L
T Q P S S V S A N L G G T V E I T C S G G S G S Y G W Y Q Q K S P G
G A P V T V I Y Y N D K R P S D I P S R F S G S K S G S T A T L T I
T G V Q V E D E A V Y Y C G S Y D S S Y V G I F G V G T T L T V L G
Q S S R S S A V T L D E S G G G L Q T P R G A L S L V C K A S G F T
F S S Y S M A W V R Q A P G K G L E F V A G I Q N D G S I T D Y G S
A V D G R A T I S R D D G Q S T V R L Q L N N L R T E D T A T Y Y C
A K T T V A D G V I G A Y G I D A W G H G T E V I V S S T S G Q A G
Q H H H H H H G A Y P Y D V P D Y A S S .
Listing of sequences in sequence listing:
1: Ig V.sub.L+V.sub.H (Gallus)
[0150] 2: PTP signature catalytic motif (HCX.sub.5R) 3: PTP
signature catalytic motif (CSX.sub.4R) 4: PTP signature catalytic
motif (HCSX.sub.4R)
5: NM.sub.--002827
6: NP.sub.--002818
7: BT006752
8: AAP35398
9: M31724
10: AAA60223
11: M33689
12: AAA60157
13: BC015660
14: AAH15660
15: BC018164
16: AAH18164
17: AK316563
18: BAG38152
19: PTP1B-CASA
[0151] 20: 7 amino acid residue linker for scFv 21: 18 amino acid
residue linker for scFv 22: biotinylation sequence 23:
pcDNA3.2/V5-GW/D-TOPO 24: peptide surrounding Tyr46 25: peptide
surrounding Tyr46, Tyr46 phosphorylated 26: scFv45, aa 27: scFv57,
DNA 28: scFv64, aa 29: scFv106, aa 30: scFv45, DNA 31: scFv57, DNA
32: scFv64, DNA 33: scFv106, DNA 34-149: 116 scFvs (including
scFv45, scFv57, scFv64 and scFv106) 150: N-terminal 22 amino acids
of scFv sequences 151: C-terminal 23 amino acids of scFv sequences
152: pComb3XSS 153: scFv136 154: scFv139 155: scFv103 156: scFv138
157: scFv134 158: scFv137 159: scFv140
TABLE-US-00002 Updated scFv Sequences >scFv1
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGVGQWYGWYQQKSPGSAPVTLIYESNQR
PSNIPSRFSGSLSGSTATLTITGVQPEDEAVYFCGGYDGNSGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPGRALSLVCKASGFTFSSYDMGWVRQAPGKGLEWVAYINSGSGSSTYYGTAVKGRASISRDNGQSTVRL
QLNNLRVEDTGTYFCAKGASGYYSSSIGAGEIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv2
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGGGSYYGWYQQKSPGSAPVTVIYDNTNRP
SNIPSRFSGSKSGSTGTLTITGVQADDEAVYYCGSTDSSADGVFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPGGGLSLVCKGSGFTFSSFDMFWVRQAPGKGLEWVAGIRNDGSDTAYGAAVKGRATISKDNGQSTVRLQ
LNNLRAEDTGTYYCAKAAGYCYVYSCAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv3
MKKTAIAIAVALAGFATVAQAAVSTNPGDTVKITCSGGNSWYGWFQQKSPGSAPVTVIYGNDERPSDIPSR
FSGSESGSTATLTITGVRAEDEAVYYCGSGDNSGAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGA
LSLVCKASGFTFSSNGMAWVRQAPGKGLEWVAGISSSGSYTNYGSAVKGRATISRDNGQSTVRLQLNNLRA
EDTATYYCAKSSYAYYGFGAPFIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv4
MKKTAIAIAVALAGFATVAQAAVIYDNDKRPSDVPSRFSGSKSGPTATLTITGVQAEDEAVYFCGSRDNSY
VGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGALSLVCKASGFTFSSYDMFWVRQAPGKGLEFVAQI
NSAGSYTNYGSAVKGRATISRDDGQSTVRLQLNNLRAEDTGIYFCAKSASGYYYSGSDAGDIDAWGHGTEV
IVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv5
MKKTAIAIAVALAGFATVAQAAVSANPGGTVKITCSGSSGRYGWYQQKSPGSAPVTVIYYNDKRPSDIPSR
FSGSASGSTATLTITGVQAEDEAVYFCGSYEVNIHEGIFGAGTSLTVLGQSSRSSTVTLDESGGGLQTPGR
ALSLVCKASGFTFSSNGMYWVRQAPGKGLEWVAGISSSGSYTNYAPAVKGRATISRDNGQSTVRLQLNNLR
AEDTGTYYCAKGASSYSWDGGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv6
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGDTVKITCSGSIRYYGWYQQKSPGSAPVTLIYYDDKRP
SDIPSRFSGSASGSTATLTITGVQADDEAIYFCGTADSTSSGAGIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGALSLVCKGSGFTFSSFNMFWVRQAPGKGLEWVAGIYSSGGGETNYGAAVKGRATISRDNGQSTV
RLQLNNLRAEDTGTYYCAKESADVGCPFTAGCIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv7
MKKTAIAIAVALAGFATVAQAAVSASLEGTVEITCSGGSGSYGWFQQKAPGSAPVTLIYDNTNRPSNIPSR
FSGSKSGSTATLTITGVQADDEAIYYCGSWDSSTDAAFGAGTTLTVLGQSSRSSTVTLDESGGGLQTPGGG
LSLVCKASGFTFSDYGMGWVRQAPGKGLEFVAGIGNTGSYTYYGSAVKGRATISRDNGQSTVRLQLNNLRA
EDTGIYFCAKSTDYWTYAGTIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv8
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSSSYYGWYQQKSPGSAPVTLIYESNE
RPSNIPSRFSGSESGSTGTLTITGVRAEDEAVYYCGSADSSNAGIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGALSLVCKASGFTFSSFNMGWVRQAPGKGLEFVAGIDNTGSFTHYGAAVKGRATISRDDGQSTVR
LQLDNLRAEDTGTYYCAKASGYYYSGVNAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv9
MKKTAIAIAVALAGFATVAQAAVIYGNDERPSDIPSRFSGSESGSTATLTITGVRAEDEAVYYCGSGDNSG
AGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGALSLVCKASGFTFSSNGMAWVRQAPGKGLEWVAGI
SSSGSYTNYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCAKSSYAYYGFGAPFIDAWGHGTEVIV
SSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv10
MKKTAIAIAVALAGFATVAQAAVIYANTDRPSDIPSRFSGSKSGSTATLTITGVRAEDEAVYFCGSGDSST
GIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGTLSLVCKGSGFTFSSVNMFWVRQAPGKGLEWVAGIY
SSGSSTHYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGIYYCAKDAGCYTSGDTAGCIDAWGHGTEVIV
SSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv11
MKKTAIAIAVALAGFATVAQAAVIYYNDKRPSNIPSRFSGSGSGSTNTLTIAGVRAEDEAVYYCGNEDSSG
AAFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGGLSLVCKASGFTFSDYDMFWVRQAPSKGLEFVAAIT
SSGTGTKYGAAVKGRATISKDNGQRTVRLQLNSLGAEDTGTYYCARSDADSTTWSAGEIDAWGHGTEVIVS
STSGQAGQHHHHHHGAYPYDVPDYAS >scFv12
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGEAVKITCSGGGSSSYYGWYQQKSPGSAPVTVIYWDDE
RPSNIPSRFSGSTSGSTGTLTITGVQAEDEAVYFCGGYDSSGDGIFGAGTTLTVLGQSSRSSSGGGSSGGG
GSAVTLDESGGGLQTPGRALSLVCKASGFTFSGYNMGWVRQAPGKGLEWVGGISGSGRYTEYGAAVKGRAT
ISRDNGQSTVRLQLNNLRAEDTGTYFCAKAAVSDYCGGGCAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHG
AYPYDVPDYAS >scFv13
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGGDSSYGWYQQKSPGSAPVTLIYDNTNRPS
DIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGNADSSSTAAFGAGTTLTVLGQSSRSSTVTLDESGGGLQ
TPGGALSLVCKASGFTFSSYAMGWVRQAPGKGLEYVAAISSAGSTTNYGAAVKGRATISRDNGQSTVRLQL
NNLRAEDTATYFCAKAAGSGYYVWSAIAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv14
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGYSGYGWFRQKAPGSAPVTLIYANTNRP
SDIPSRFSGSASGSTGTLTITGVQADDEAVYFCGSADSTYGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPGGALSLVCRASGFTFSDYGMEWVRQAPGKGLEWVAGIDDDGSTTFYAPAVKGRATISRDDGQSTVRLQL
NNLRAEDTATYYCAKSAGRGWNVAGWIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv15
MKKTAIAIAVALAGFATVAQAALSRPRCQQTQEHTVKITCSGGVGQWYGWYQQKSPGSAPVTLIYESNQRP
SNIPSRFSGSLSGSTATLTITGVQPEDEAVYFCGGYDGNSGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPGRALSLVCKASGFTFSSYDMGWVRQAPGKGLEWVAYINSGSGSSTYYGTAVKGRASISRDNGQSTVRLQ
LNNLRVEDTGT YFCAKGASGYYSSSGQAGQHHHHHHGAYPYDVPDYAS >scFv16
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYD
NTNRPSDIPSRFSGSTSGSTNTLTITGVQADDEAVYFCGSVDSSSGVFGAGTTLTVLGQSSRSSAVTLDES
GGGLQTPGGALSLVCKASGFTFSSFDMFWVRQAPGKGLEYVAEISDTGSSTYYGAAVKGRATISRDNGQST
VRLQLNNLRAEDTGTYFCAKSHSGYGWSTAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv17
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGYSYYGWYQQKTPGSAPVTLIYDNTNRP
SDIPSRFSGSKSGSTATLTITGVQVEDEAMYFCGSYEGSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFTFSSYDMAWVRQAPGKGLEFVAGIDIGSYTGYGAAVKGRATISRDNGQSTVRLQ
LNNLRAEDTGTYYCAKAAGSYYYSGAAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv18
MKKTAIAIAVALAGFATVAQAAVIYYNDKRPSDIPSRFSGSKSGSTATLTITGVRAEDEAVYYCGSADSTD
AVFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGGLSLVCKASGFTFSDYDMFWVRQAPSKGLEFVAAIT
SSGTGTKYGAAVKGRATISKDNGQSTVRLQLNSLRAEDTGTYYCARSDADSTTWSAGEIDAWGHGTEVIVS
STSGQAGQHHHHHHGAYPYDVPDYAS >scFv19
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVEITCSGGSNNYGYGWYQQKSPGSAPVTLIYSNDNR
PSNIPSRFSGSTSGSTSTLTITGVQAEDEAVYYCGSYDSSNDSGIFGAGTTLTVLSQSSRSSAVTLDESGG
GLQTPGGGLSLVCKASGFTFSTFNMFWVRQAPGKGLEFVAGISITGGWTGYGAAVKGRATISRDNGQSTVR
LQLNNLRAEDTGTYYCAKPAAWSCYRGCGGEFDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv20
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGIVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRP
SNIPSRFSGSGSGSTNTLTIAGVRAEDEAVYYCGNEDSSGAAFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPGGGLSLVCKASGFTFSDYDMFWVRQAPSKGLEFVAAITSSGTGTKYGAAVKGRATISKDNGQRTVRLQL
NSLGAEDTGTYYCARSDADSTTWSAGEIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv21
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSGSYYGWYQQKSPGSAPVTVIYDNDKR
PSDVPSRFSGSKSGPTATLTITGVQAEDEAVYFCGSRDNSYVGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFTFSSYDMFWVRQAPGKGLEFVAQINSAGSYTNYGSAVKGRATISRDDGQSTVRL
QLNNLRAEDTGIYFCAKSASGYYYSGSDAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv22
MKKTAIAIAVALAGFATVAQAAVSANPGDTVKITCSGDSNNYGWYQQKSPGSAPVTVIYDNTNRPSNIPSR
FSGSKSGSTATLTITGVQADDEAVYFCGSFDSSTDIFGAGTTLTVLGQSSRSSTVTLDESGGGLQTPGRAL
SLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQLNNLRAE
DTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv23
MKKTAIAIAVALAGFATVAQAAVSANLGGTVEITCSGGGSYYGWYQQKSPGSAPVTVIYANTNGPSDIPSR
FSGSTSGSTATLTITGVQADDEAVYSCGSYDSSYVGIFGAGTTLTVLGQSSRSSTVTLDESGGGLQTPGGA
LSLVCKASGFTFNSYALEWVRQAPGKGLEWVAGISGDGSYRHYGSAVKGRATISRDSGQSTVRLQLNNLRA
EDTGTYYCAKSTGSGAGWGASNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv24
KKKTAIAIAVALAGFATVAQAAVSASPGDTVKITCSGGNSSYGYGWYQQKSPGSAPVSVIYYNDERPSDIP
SRFSGSASGSTATLTITGVQADDEAVYYCGNADSSTYAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTP
GGGLSLVCKASGFDFSTNAMGWVRQAPGKGLEWVAGISGSGSSTWYATAVKGRATISRDNGQSTVRLQLNN
LRAEDTGTYYCTKYVGDYYWYIDAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv25
MKKTAIAIAVALAGFATVAQAALTPPSSVSANLGAPVEITCSGSSGNYGWFQQKSPGSAPVTVIYSNDKRP
SDIPSRFSGSLSGSTGTLTITGVRAEDEAVYYCGSIDNTYVGTGAFGAGTTLTVLGQSSRSSTVTLDESGA
GLQTPGRALSLVCKGSGFTFSSFYMFWVRQAPGKGLEFVACISSSGSSTRYGVVVKGRATISRDNGQSTVR
LRLNNLRADDTGTYYCARGTSSGANTIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv26
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVGITCSGGGSYYGWYQQKSPGSAPVTLIYENDMRP
SNIPSRFSGSTSGSTSTLTITGVQAEDEAVYFCGSYDSSNYVGEFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFTFSSFDMFWVRQAPGKGLEYVAEISDTGSSTYYGAAVKGRATISRDNGQSTVRL
QLNNLRAEDTGTYFCAKSHSGYGWSTAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv27
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGSNSYYGWYQQKAPGSAPVTLIYDDTNRPS
DIPSRFSGSKSGSTATLTITGVQADDEAVYFCGGFDSSSDSGFGAGTTLTVLGQSSRSSTVTLDESGGGLQ
TPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQL
NNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYA
S
>scFv28
MKKTAIAIAVALAGFATVAQAAVIYSNDERPSDIPSRFSGSTSGSTSTLTITGVQADDEAVYFCGSADSST
YAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGALSLVCKASGFTFSSFNMFWVRQAPGRGLEFVAG
ITSSGGSTYYGTAVKGRATISRDNGQSTVRMQLNNLRAEDTGTYFCARGAYDYYFYWNYAGTIDAWGHGTE
VIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv29
MKKTAIAIAVALAGFATVAQAAVIYDNTNRPSNIPSRFSGSKSGSTGTLTITGVQADDEAVYYCGSTDSSA
DGVFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGGLSLVCKGSGFTFSSFDMFWVRQAPGKGLEWVAGI
RNDGSDTAYGAAVKGRATISKDNGQSTVRLQLNNLRAEDTGTYYCAKAAGYCYVYSCAGSIDAWGHGTEVI
VSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv30
MKKTAIAIAVALAGFATVAQAAVSANPGETVKITCSGGGGSYGWYQQKAPGSAPVTVIYDNTNRPSNIPSR
FSGSESGSTATLTITGVRAEDEAAYYCGSADSSDAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPRRA
LSLVCKASGFTFSDYGMAWVRQAPGKGLEWVAGIGSSGSYTDYGSAVKGRATISRDNGQSTVRLQLNNLRA
EDTATYYCAKDIGSVYGCGWWACSAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv31
MKKTAIAIAVALAGFATVAQAAVIYDNTNRPSNIPSRFSGSLSGSTNTLTITGVQAEDEAVYFCGGYDSST
DSGMFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGGLSLVCKGSGFTFSSYDMAWVRQEPSKGLEFVAS
ISNTGSDTSYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCAKSAGSYYWNAGGAGSIDTWGHGTE
VIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv32
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTFKITCSGSSGSYAWYQQKSPGSAPVTVIYWNDKRP
SNIPSRFSGALSGSTATLTITGVQAEDEAVYFCGSADSSGAIFGAGTTLTVLGQSSRSSTVTLDESGGGLQ
TPGGGLSLVCKGSGFAFSNYGMGWMRQAPGKGLEYVAGISTGSYTDYGPAVKGRATISRDNGQSTVRLQLN
NLRAEDAAIYFCAKTAGSGYGCGSGTDLGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv33
MKKTAIAIAVALAGFATVAQAAVSANPGDTVKITCSGGYSGYGWYQQKSPGSAPVTVIYSNNQRPSNIPSR
FSGSTSGSTNTLTITGVQVEDEAIYFCGGYDCSTGSVKASFGAGTTLTVLGQSSRSSAVTLDESGGGLQTP
GGTLSLVCKGSGFTFSSHGMGWVRQAPGKGLEWVAGIYSGSSTYYGAAVKGRATISRDNGQSTVRLQLNNL
RAEDTATYFCTRGGGAGRIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv34
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRP
SDIPSRFSGSTSGSTATLTITGVQAEDEAVYFCGGYDSNYIGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPRGALSLVCKASGFTFSSYSMAWVRQAPGKGLEFVAGIQNDGSITDYGSAVDGRATISRDDGQSTVRLQ
LNNLRTEDTATYYCAKTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv35
MKKTAIAIAVALAGFATVAQAAVSANPGDTVEITCSGSSGSYGWYQQKSPGSAPVTVIYANTNRPSDIPSR
FSGSKSGSTATLTITGVRAEDEAVYYCGGYDSSTDAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGG
GLSLVCKASGFTFSDYGMGWMRQAPGKGLEYVAGIDNTGSSTGYGAAVKGRATISRDNRQSTVRLQLNNLR
AEDTGIYFCAKTAGSGGGWWSDWIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv36
MKKTAIAIAVALAGFATVAQAALSRPRCQQTLGGTVKITCSGSNGSYDWCHQKTSAGAAAAVIIYDNNKTS
YIPSSLFCASSCSPATLLIIGVVADDDDVDYCGSANDNSSVVIVGATTTMIVRRSSSSSSAMMEDEGGGLL
TTRGGLLILCCAASGFIFSYYEMLWLHPAPGEVQDFVTIISGGGNYTYYGSAVDGGAIISRDDGKRMLMLQ
LNILEDDDTGFYFCADGASGYYYGGADAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv37
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGDTVKITCSGGGGYAGSYYYGWYQHNAAGGAPVTLIYD
NTITPSDIPSRFSGSTSGSTNALTINGVQADYAVYFCGSVNCSSGVFGAGTTLTVLCHSSTSSDVTLDHSR
GGLQTPGGSLSLVCNASGFTFSSFHMFWVRQAPGEGLEYVAEITDTGSSTYYGAAVKGRATISRDNGQSTV
RLQLNNLMADDTGTYFCAKSHSGYGWSTAGDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv38
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGSSGYGWYQQKSPGSAPVTVIYYNDKRP
SDIPSRFSGALSGSTATLTITGVQAEDEAVYFCGSGDSSTVAGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGTLSLVCKASGFDFSSYGMHWVRQEPGKGLEWVAGISRTGSFTYYGAAVKGRAAISRDNGQSTVRL
QLNNLRAEDTGTYYCAKGGSDCSGYRCDYSAGNIDGWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYA
S >scFv39
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSDYYGWYQQKSPGSAPVTLIYENDKR
PSNIPSRFSGSKSGSTATLTITGVQADDEAVYFCGNADTITGIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCKASGFTFSSYTMAWVRQAPGKGLEWVAGINDGGSYTNYGPAVQGRATISRDNGQSTVRLQ
LNNLRAEDTAIYYCAKSAGGYYYSGAAGTIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv40
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGIVKLTCSGGSGSCGWYQQKSPGSAPVTLIYDNDKRPS
DIPSRFSGSTSGSTHTLTITGVQAEDEAIYFCGSEDSSTYASGFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQ
LNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVPSTSGQAGQHHHHHHGAYPYDVPDY
AS >scFv41
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGGSSYYGWYQQKSPGSAPVTLIYENNNRPS
DIPSRFSGSASGSTATLTITGVQAEDGAVYFCGSEDSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQL
NNLGAEDTATYYCAKAAGNAYYYTAVTPAFAGSIDAWGHGTEVIVPSTSGQAGQHHHHHHGAYPYDVPDYA
S >scFv42
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVEITCSGGGSSSNYGWHQQKSPGSAPVTVIYDNTN
RPPNIPSRFSGSLSGSTGTLTITGVQAEDEAVYYCGGHDSSTYAGIFGAGTTLTVLGQSSRSSAVTLDESG
GGLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTV
RLQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDV
PDYAS >scFv43
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSSSYYGWYQQKSPDSAPVTLIYESNK
RPSNIPSRFSGSTSGSTSTLTITGVQADDEAVYFCGSADSSYVGIFGAGTTLTVLGQSSRSSAVTLDESGG
GLQTPGGALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVR
LQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVP
DYAS >scFv44
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGETVKITCSGGGSYAGCYYYSWYDHTAAGVVPVTLIDS
TIPSSYFRSRFCCSASGSINALTINEDPAYYAVYFCGSVDVFGGVFGASTTLTAPGSSSISSDETLDDSGS
GLRTPGRALNVFCFASGFFFMIFELFGVRQAPGWVLEYIADVSDTGNSTYYRAAVNVRAAISRNNGQMTLR
LLLNDHTADDTCTYFCGYCHSDYCWSTAGDIDAWSHVIDFIVSSTSGQAGQHHHHHHGAYPYDDPDYAS
>scFv45
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSSAYGYGWYQQKSPGSAPVTVIYNNNK
RPSNIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSEDSSTDAIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQAPGGALSLVCKASGFTFSSYDMGWIRQAPGKGLEYVAGITDNGRYASYGSAVDGRATISRDNGQSSVR
LQLNNLRAEDTGTYYCARDDGSGWTGNSIDAWGHGTEIIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv46
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGSSGSYGWYQQKSPGSAPVTVIYWNDKRP
SNIPSRFSGALSGSTATLTITGVQAEDEAVYFCGSADSSGAIFGAGTTLTVLGQSSRSSTVTLDESGGGLQ
TPGGGLSLVCKGSGFAFSNYGMGWMRQAPGKGLEYVAGISTGSYTDYGPAVKGRATISRDNGQSTVRLQLN
NLRAEDAAIYFCAKGHGTEIIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv47
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGGDGSYGWYQQKSPGSAPVTVIYDNTNRPS
DIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGNADSSGAAAFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPGGALSLGCEASGFTFSSYAMGWVRQAPGKGLEYVATISSAGSNTNYGAAVKGRATISRDNGQSTVRLQL
NNLEDDDTATYFCAEAAGNGYYVWSAIAGDIDAWGHGTDVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv48
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSEGGRSSYYGWYQQKAPGSAPVTVIYDSSS
RPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSTDSSTSAAIFGAGTTLTVLGQSSRSSTVTLDESG
GGLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTV
RLQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDV
PDYAS >scFv49
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVEITCSGGGGSYGWFQQKSPGSAPVTLIYNNNNRP
SDIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGTRDSSYAGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPGGALSLVCKGSGFTFSDYSMMWVRQAPGKGLEWVAGISSNSGTTRYGSAVKGRATISRDNGQSTVRLQ
LNNLRAEDTGTYYCAKTTGVNSYDVPAIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv50
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGDTVEITCSGGYSNYGWYQQKSPGSAPVTVIYGSTSRP
SDIPSRFSGSESGSTGTLTITGVQAEDEAVYFCGNADSSYVGLFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQ
LNNLRAEDTATYYCAKAAGSAYYYTAATPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDY
AS >scFv51
MKKTAIAIAVALAGFATVAQAALVSANPGETVKITCSGGGSNSAGSYYYGWYQQKPPGSAPVTVIHNNNKR
PSDIPSRFSGSKSGSTGTLTITGVQVDDEAVYYCGSRDSSYIGTFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGRALSLACKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRL
QLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPD
YAS >scFv52
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGDPVEITCSGSSGSYGWYQQKAPSSAPVTVIYSNDKRP
SDIPSRFSGSASGSTATLTITGVQAEDEAVYFCGSFDSSAGYGGIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVR
LQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVP
DYAS >scFv53
MKKTAIAIAVALAGFATVAQAALVSANPGEIVKITCSGNSSYYGWYQQKAPGSAPVTVIYDNNKRPSDIPS
RFSGSKSGSTGTLTITGVQAEDEAVYFCGNGATFGAGTTLTVLGQSSRSSTVTLDESGGGLQTPGRALSLV
CKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTA
TYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv54
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGSYSYGWYQQKSPGSAPVTVIYSSDKRP
SDIPSRFSGSKSGSTSTLTITGVQAEDEAVYYCGSRDSSYVGIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCEASGFTFSSYEMQWVRQAPGKGLEFVAAISSDGSYTNYGAAVQGRATISRDNGQSTVRLQ
LSNLRAEDTATYYCARSPGGYTWWPGAAGGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv55
MKKTAIAIAVALAGFATVAQAALVSANLGGTVEITCSGSSGNYGWFQQKSPGSAPVTVIYSNDKRPSDIPS
RFSGSLSGSTGTLTITGVRAEDEAVYYCGSIDNTYVGTGAFGAGTTLTVLGQSSRSSTVTLDESGGGLQTP
GRALSLVCKGSGFTFSSFYMFWVRQAPGKGLEFVASISSSGSSTRYGVVVKGRATISRDNGQSTVRLRLNN
LRAEDTGTYYCARGTSSGANTIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv56
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSSSYYGWYQQKSPGSAPVTLIYESNK
RPSGIPSRFSGSKSGSTHTLTITGVQAEDEAVYYCGAYDGSSYTGIFGAGTTLTVLGQSSRSSTVTLDESG
GGLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTV
RLQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDV
PDYA >scFv57
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSSAYGYGWYQQKSPGSAPVTVIYNNNKRPS-
NIPSR
FSGSKSGSTGTLTITGVQAEDEAVYFCGSEDSSTDAIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGALSL-
VCKAS
GFTFSSYDMGWVRQAPGKGLEYVAGITNDGRYASYGSAVDGRATISRDNGQSTVRLQLNNLRAEDTGTYYCARD-
DGSGW TGNTIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv58
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGDTVKITCSGGGSSSYYGWYQQRSPGSAPVTLIYSNDK
RPSDIPPRFSGSLSGSTATLTITGVQADDEAVYYCGGYDSSYVGLFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVYGRATISRDNGQSTVR
LQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVP
DYAS >scFv59
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGEIFKITCSGGGSYAGSYYYGWYQQKAPGSAPVTVIYD
NTNRPSNIPSRFSGSKSGSTATLTITGVRADDSAVYYCASTDSSSTGIFGAGTTLTVLGQSSRSSAVTLDE
SGGGLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQS
TVRLQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPY
DVPDYAS >scFv60
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGAIVKITCSEGGRSSYYGWYQQKAPGSAPVTVIYDSSS
RPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSTDSSTSAAIFGAGTTLTVLGQSSRSSTVTLDESG
GGLQTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTV
RLQLNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDV
PDYAS >scFv61
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYD
NTNRPSNIPSRFSGSLSGSTGTLTITGVRAEDEAVYYCGSFDSSTDGGYAAIFGAGTTLTVLGQSSRSYAV
TLDESGGGLQTPGGGLSLVCKASEFTFSSYAMEWVRQAPGKGLEWVAYINSDGSSTWYAPAVKGRATISRD
NGQSTVRLQLNSLRAEDTATYYCTRGSGGENIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv62
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYD
NTNRPSNIPSRFSGSLSGSTGTLTITGVRAEDEAVYYCGSFDSSTDGGYAAIFGAGTTLTVLGQSSRSSAV
TLDESGGGLQTPGGGLSLVCKASEFTFSSYAMEWVRQAPGKGLEWVAYINSDGSSTWYAPAVKGRATISRD
NGQSTVRLQLNSLRAEDTATYYCTRGSGGENIDTWGHGTEVIVSSTSGQAGQHHHHHHAAYPYDVPDYAS
>scFv63 (incomplete sequence information)
SNNYGWHQQKAPGSAPVTVIYDNTNRPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFG
AGTTLTVLGQSSRSSAVTLDESGGGLQTPGGTLSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGR
STGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAQAYGDSNIDRMGPRDR
>scFv64
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGDPLKITCSGDSSGYGYGWYQQKSPGSAPVTVIYNNNK
RPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSEDSNTDAVFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGTLSLACKASGFTFSGYDMGWVRQAPGKGLEYVAGITSDGRYASYGSAVDGRAAIWRDNGQSTVR
LQLKNLRTEDTATYYCARNDGSGWNGNNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv65
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGSSGSYGWYQQKSPGSAPVTLIYESDKRPS
DIPSRFSGSKSGSTGTLTITGVQADDEAVYFCGGYDSSAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQT
PGGALSLVCKASGFDFSSYGMGWMRQAPGKGLEFVAAIRKDGSYTAYGAAVDGRATISRDDGQSTVRLQLG
NLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv66
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVEITCSGSSGDYGYSWHQQKSPGSAPVTVIYESTKR
PSNIPSRFSGSTSGSTGTLTITGVQVEDEAVYFCGGYDGSTDAIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQMPGGGLSLVCKASGFDFSSSEMQWVRQAPGKGLQWVGIISSSGSTYYGSAVKGRATISRDNGQSAVRLQ
LNNLRAEDTGTYYCTKTTAYAHDIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv67
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSSAYGYGWYQQKSPGSAPVTVIYNNNK
RPSNIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSEDSSTDAIFGAGTTLTVLGQSSRSSAVTLDESGG
GLQTPGGALSLVCKASGFTFSSYDMGWVRQAPGKGLEYVAGITNDGRYASYGSAVDGRATISRDNGQSTVR
LQLNNLRAEDTGTYYCARDDGSGWTGNTIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv68
MKKTAIAIAVALAGFATVAQAALTQPSSVSASPGETVKITCSGGSGSYGWYRHKSPGSAPVTVIYYNDKRP
SDIPSRFSGSKSGSTSTLTITGVQAEDEADYYCGSYNSNAGYVGIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGGLSLVCKASGFTFSSYGMGWMRQAPGKGLEFVAGIRKDGRSTAYGAAVDGRATISRDDGQSTLR
LQLGNLRAEDTGTYFCAKTNSYDSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv69
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSSNNYGWHQQKAPGSAPVTVIYDNTN
RPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGTLSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGRSTGYGSAVKGRATISRDNGQSTVRL
QLNNLRAEDTGTYYCAKAYGDSNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv70
MKKTAIAIAVALAGFATVAQAALTQPSSVSASLGGIVEITCSGSSGTYGWYQQKSPGSAPVTVIYQNGKRP
SNIPSRFSGSKSGSTATLTITGVQADDEAVYFCGGYDSSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFDFSSYGMGWMRQAPGKGLEFVAAIRKDGSYTAYGAAVDGRATISRDDGQSTVRL
QLGNLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv71
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSSNNYGWHQQKAPGSAPVTVIYDNTN
RPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGTLSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGRSTGYGSAVKGRATISRDNGQSTVRL
QLNNLRAEDTGTYYCAKALWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv72
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGSSGSYGWYQQKSPGSAPVSLIYSNDKRP
SDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGGWDSYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPGGGLSLVCKASGFSSSHGMGWMRQAPGKGLEFVAGIRSDGSSTAYGAAVDGRATITRDDGQSTVTLQLN
NLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv73
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGSSGSYGWYQQKSPGSAPVTLIYESDKRPS
DIPSRFSGSKSGSTGTLTITGVQADDEAVYFCGGYDSSAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLHT
PGGALSLVCKASGFDFSSYGMGWMRQAPGKGLEFVAAIRKDGSYTAYGAAVDGRATISRDDGQSTVRLQLG
NLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv74
MKKTAIAIAVALAGFATVAQAALTQPSSVSASPGETVKITCSGGSGSYGWYQQKSPGSAPVTVIYYNDKRP
SDIPSRFSGSKSGSTSTLTITGVQAEDEAVYYCGSYDSSAGYVGIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGGLSLVCKASGFTFSSYGMGWMRQAPGKGLEFVAGIRKDGSSTAYGAAVDGRATISRDDGQSTLR
LQLGNLRAEDTGTYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv75 (incomplete sequence information)
RAPVTLIYNNNNRPSDIPPRFSGSKSGSTGTLAITGVQAEDEAVYFCGGYEGSTSTGIFGAGTTLTVLGQS
SRSSAVTLDESGGGLQTPGGALSLVCKASGFDFSSYGMGWMRQAPGKGLEFVAAIKKDGSYTAYGAAVDGR
ATISRDDGQSTVRLQLGNLRAEDTAP >scFv77
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGDPFKITCSGGGSSNNYGWHQQKAPGSAPVTVIYDNTN
RPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGTLSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGRSTGYGSAVKGRATISRDNGQSTVRL
QLNNLRAEDTGTYYCAKAYGDSNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv79 (incomplete sequence information)
LSRPRCQQTWGGTVKITCSGSSGGYGWYRHKSPGTAPVPLIYNNDNRPSDIPSRFSGSKSGSTSTLTITGV
QVQDEDDYFCGGYNKNTYADIFGAGTTLTVLRQSSTSSAVTMDDYGGGLLTTGGALILLCWASGFFTFHGL
DWMRQAPATGLEFVAGIRSDGDSTAYGAAVDGHATVSRDNGQSTMRLQLNILRAEDDATYFCA
>scFv80
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGPVKITCSGGSGSYGWYQQKSPGSAPVTVIYYNDQRPS
DIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGGYDSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQ
TPRGALSLVCKASGFTFSSYSMAWVRQAPGKGLEFVAGIQNDGSITDYGSAVDGRATISRDDGQSTVRLQL
NNLRTEDTATYYCAKTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv81 (incomplete sequence information)
ALTQPSSVSANPGGTVKITCSGSSSAYGYGWYQQKSPGSAPVTVIYNNNKRPSNIPSRFSGSKSGSTGTLT
ITGVQAEDEAVYFCGSEDSSTDAIFGAGTTLTVLGQSSRSSTVTLDESGGGLQAPGGALSLVCKASGFTFS
SYDMGWIRQAPGKGLEYVAGITDNGTYASYGS >scFv82
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGAFKITCSGGGGSYGWYQQKSPGSAPVTLIYYNDKRPS
DIPSRFSGSKSGSTATLTITGVQANDEAVYFCGSYEGSTYSGIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGGLSLVCKASGFSSSHGMGWMRQAPGKGLEFVAGIRSDGSSTAYGAAVDGRATITRDDGQSTVTLQL
NNLRAEDTATYFCAKNTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv83
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGPFEITCSGSSGSYGWYQQKSPGSAPVTLIYNNNNRP
SDIPPRFSGSKSGSTGTLAITGVQAEDEAVYFCGGYEGSTSTGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFDFSSYGMGWMRQAPGKGLEFVAAIRKDGSYTAYGAAVDGRATISRDDGQSTVRL
QLGNLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv84
MKKTAIAIAVALAGFATVAQAALKITCSGSSGSYAWYQQKSPGSAPVTLIYESDKRPSDIPSRFSGSKSGS
TGTLTITGVQADDEAVYFCGGYDSSAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGALSLVCKASG
FDFSSYGMGWMRQAPGKGLEFVAAIRKDGSYTAYGAAVDGRATISRDDGQSTVRLQLGNLRAEDTATYFCA
KTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv85
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPRTLLKITCSGSSSAYGYGWYQQKSPGSAPVTVIYNNNK
RPSNIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSEDSSTDAIFGAGTTLTVLGQSSRSSAVTLDESGG
GLQTPGGALSLVCKASGFTFSSYDMGWVRQAPGKGLEYVAGITNDGRYASYGSAVDGRATISRDNGQSTVR
LQLNNLRAEDTGTYYCARNDGSGWTGNTIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv86
MKKTAIAIAVALAGFATVAQAALGPDSAVLGVSKPGEALVKTTCSGGGGSYGWYQQKSPGSAPVTVIYYND
KRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSYDSSTDTGIFGAGTTLTVLGQSSRSSTVTLDES
GGGLQTPGGALSLVCKASGFIFSSHGMGWMRQAPGKGLEFVAAISKDGTATYYGPAVKGRATISRDDGQTT
VRLQLNNLRAEDTATYFCAKTKYYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv87
MKKTAIAIAVALAGFATVAQAALSRPRVSANPGDPVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTVIYDN
NQRPSNIPSRFSGSLSGSTGTLTITGVRAEDEAVYYCGSFDSSTDSGYAAIFGAGTTLTVLGQSSRSSAVT
LDESGGGLQTPGGGLSLVCKASGFTFSSYAMEWVRQAPGKGLEWVAYINSDGSSTWYATAVXGRATISRDN
GQSTVRLQLNNLRGEDTATYFCAKTKYYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYA
S >scFv88 (incomplete sequence information)
GSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSTSGSTATLTITGVQAEDEAVYFCGGYDSNYIG
IFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPRGALSLVCKASGFTFSSYSMAWVRQAPGKGLEFVAGIQN
DGSITDYGSAVDGRATISRDDGQSTVRLQLNNLRTEDTATYYCAKTTVADGVI >scFv89
(incomplete sequence information)
ALTQPSSVSANPGDTVKITCSGDSSDYGYGWYQQKSPGSAPVTVTYSNNQRPSDIPSRFSGSASGSTATLT
ITGVQVEDEAVYYCGSEDSTTDAVFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGALSLVCKASGFTFS
SYDMGWVRQAPGKGLEYVAGITNDGRYASYGSAVDGRATISRDNGQSTVRLQLNNPQG
>scFv90
MKKTAIAIAVALAGFATVAQAALSRPRCQQTWGGTVKITCSGSSGSYGWYQQKSPGSAPVTVIYQNDKRPS
DIPSRFSGSTSGSTATLTITGVQADDEAVYFCGGYDSSAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQT
PGGALSLVCKASGFSSSHGMGWMRQAPGKGLEFVAGIRSDGSSTAYGAAVDGRATISRDDGQSTVRLQLNN
LRAEDTATYFCAKTNSYNSAGIIDAWGPGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv91 (incomplete sequence information)
IYDNTNRPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFGAGTTLTVLGQSSRSSAVTL
DESGGGLQTPGGTLSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGRSTGYGSAVKGRATISRDNG
QSTVRLQLNNLRAEDTGTYYCAKAYGDS >scFv92
MKKTAIAIAVALAGFATVAQAALTQPSSVSASLGTFLEITCSGSSGTYGWYQQKSPGSAPVTVIYQNGKRP
SNIPSRFSGSKSGSTATLTITGVQADDEAVYFCGGYDSSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFDFSSYGMGWMRQAPGKGLEFVAAIRKDGSYTAYGAAVDGRATISRDDGQSTVRL
QLGNLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv93
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGALFKITCSGGGSSNNYGWHQQKAPGSAPVTVIYDNTN
RPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFGAGTTLTVLGQSSRSSTVTLDESGGG
LQTPGGALSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGGSTGYGSAVKGRATISRDNGQSTVRL
QLNNLRAEDTGTYYCAKAYGDSNIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv95
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVEITCSGGGGSYGWFQQKSPGSAPVTVIYESTKRP
SNIPSRFSGSGSGSTSTLTITGVRAEDEAVYYCGGYDGSSDAIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCKASGFTFSSHDMGWVRQAPGKGLEYVAGITDDGRYASYGPAVDGRATISRDNGQSTVRLQ
LKNLRAEDTATYYCARDDGSGWSGDTIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv96
MKKTAIAIAVALAGFATVAQAALPGVRHRDPGGPDSAVLGVSKPRRNDKITCSGGGSYAGSYYYGWYQQKA
PGSAPVTLIYDNTNRPSNIPSRFSGSLSGSTGTLTITGVRAEDEAVYYCGSFDSSTDGGYAAIFGAGTTLT
VLGQSSRSSAVTLDESGGGLQTPGGGLSLVCKASEFTFSSYAMEWVRQAPGKGLEWVAYINSDGSSTWYAP
AVKGRATISRDNGQSTVRLQLNSLRAEDTATYYCTRGSGGENIDTWGHGTEVIVSSTSGQAGQHHHHHHGA
YPYDVPDYAS >scFv97
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGSYNAYGWYQQKSPAGAPVTLIYDNTNR
PSNIPSRFSGSKSGSTHTLTITGVQADDEAVYFCGGYDSNADDGIFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGTLSLVCKASGFTFSSYAMNWMRQAPGKGLEWVAGIYSDGRYTNYGAAVKGRATISRDNGQSSVR
LQLNNLRAEDTATYYCTKSADSDYGCDNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv98
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSYVGSYYYGWYQQKSPVSAPVTLIYE
STKRPSNIPSRFSGSTSGSMGTLTITGVQAEDEAVYFCGSFDSSSSSVSDTADIFGAGTTLTVLGQSSRSS
TVTLDESGGGLQTPGGALSLVCKASGFTFNSYALEWVRQAPGKGLEWVAGISGDGSFTHYGSAVKGRATIS
RDNGQSTVRLHLNNLRAEDTATYYCAKSTGSGAGWGASNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPY
DVPDYAS >scFv100
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGSSDAYGWYQQKSPGSAPVTLIYDNTNRP
PDIPSRFSGALSGSTSTLTITGVRAEDEAVYYCGSADITYIGIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGGLSLVCKASGFTFSSHTMQWVRQAPGKGLEWVAEISADGSYTTYYGAAVKGRATISRDNGQSTVRL
QLNNLRAEDTATYFCAKSGYGGAGWGAGLIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv102
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGSGSYGWYQQESPGSAPVTVIYYNDKRP
SDIPSRFSGSASGSTATLTIAGVRAEDEAVYFCGSWDSSTSAGIFGAGTALTVLGQSSRSSAVTLDESGGG
LQTPGGGLSLVCKASGFSSSHGMGWMRQAPGKGLEFVAGIRSDGSSTAYGAAVDGRATITRDDGQSTVTLQ
LNNLRAEDTATYFCAKTNSYNSAGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv104
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVEITCSGSGGSYGYYGWYQQKAPGSAPVTVIYDNT
NRPSNIPSRFSGSASGSTGTLTITGVRAEDEAVYFCGGYDSSNTDAFGAGTTLTVLGQSSRSSTVTLDESG
GGLQTPGRALSLVCKASGFTFSSYTMGWVRQAPGKGLEFVAGIGNTGRYTGYGSAVKGRATISRDNGQSTV
RLQLNNLRAEDTGTYYCTKCAYGYYYSWGNIAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDY
AS >scFv105
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGEAVKITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRP
SDIPSRFSGSTSGSTSTLTITGVQAEDEAVYFCGGYDSNYLGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPRGALSLVCKASGFTFSSYSMAWVRQAPGKGLEFVAGIQNDGSITDYGSAVDGRATISRDDGQSTVRLQ
LNNLRTEDTATYYCAKTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv106
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGDSSDYGYGWYQQKSPGSAPVTVTYSNNQ
RPPNIPSRFSGSASGSTATLTITGVQVEDEAVYYCGSEDSTTDAVFGAGTTLTVLGQSSRSSAMTLDESGG
GLQTPGGALSLVCKASGFTFSSYDMGWVRQAPGKGLEYVAGITNDGRYASYGSAVDGRATISRDNGQSTVR
LQLNNLRAEDTGTYYCARDDGSGWTGNTIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv108
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYD
NTNRPSNIPSRFSGSLSGSPGTLAITGVRAEDEAVYYCGSFHSSTDGGYAAIFGAGTTLTVLGQTSRSSAV
TLDESGGGLQTPGGGLSLLCKASEFTSISYAMEWVRQAPGKGLEWVAYINSDGSSTWHAPAVKGRATISRD
NGQSTVRLQLNSLRAEDTATYYCTICSGGENIYTCCHGTEVIVSSTSGQDGQHHHHHHGAYPYDVPDYAS
>scFv110
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKLTCSGGSSYGYSWHQQKSPGSAPVTVIYSNDKR
PSDIPSRFSGSASGSTATLTITGVQVEDEAVYFCGSYDSSSIAGIFGAGTTLTVLGQSSRFSTVTLDESGG
GLQTPGGGLSLVCKASGFTFSSYGMAWVRQAPGKGLEWLAGIYRDDDSTYYAPAVKGRATISRDNGQSTVR
LQLNNLRTEDTATYYCAKESASGGWNAGWIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv111
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKLTCSGDSSDYGYGWYQQKSPGSAPVTVIYNNNK
RPSDIPSRFSGSKSGSTGTLTISGVQAEDEAVYFCGSEDSNTDAIFGAGTTLTVLGQSSRSSAVTLDESGG
GLQTPGGALSLVCEASGFTFSSYDMGWIRQAPGKGLEYVAGITSNGRYASYGSAVDGRATISRDNGQSTVR
LQLNNLRAEDTGTYYCARDDGSGWTGNTIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv112
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGGSGSYGWYQQKSPGSAPVTVIYYNDQRP
SDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGGYDSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPRGALSLVCKASGFTFSSYSMAWVRQAPGKGLEFVAGIQNDGSITDYGSAVDGRATISRDDGQSTVRLQ
LNNLRTEDTATYYCAKTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv113
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGDSSGYGYGWYQQKSPGSAPVTVIYNNNK
RPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYFCGSEDSNTDAVFGAGTTLTVLGQSSRSSTVTLDESGG
GLQTPGGTLSLACKASGFTFSGYDMGWVRQAPGKGLEYVAGITSDGRYASYGSAVDGRAAIWRDNGQSTVR
LQLKNLRTEDTATYYCARDDGSGWSGNNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv114
MKKTAIAIAVALAGFATVAQAALTQPSSVSASPGETVKITCSGGSGSYGWYQQKSPGSAPVTVIYYNDKRPSDI-
PSRFS
GSKSGSTSTLTITGVQAEDEAVYYCGSYDSSAGYVGIFGAGTTLTVLGQSSRSSTVTLDESGGGLQTPGGGLSL-
VCKAS
GFTFSSYGMGWMRQAPGKGLEFVAGIRKDGSSTAYGAAVDGRATISRDDGQSTLRLQLGNLRAEDTGTYFCAKT-
NSYNS AGIIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS >scFv115
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVEITCSGSSGSYGWYQQKPPGSAPVTVIYYNDKRP
SDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGGYGSTYLGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QMPGGGLSLVCKASGFTFSSYAMGWMRQAPGKGLEFVAGILNDGSITDYGSAVKGRATISRDDGQSTVRLQ
LSNLRTEDTATYYCAKTTVGDGVIGAYAIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv117
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYD
NTNRPSNIPSRFSGSLSGSTGTLTITGVRAEDEAVYYCGSFDSSTDSGYAAIFGAGTTLTVLGQSSRSSAV
TLDESGGGLQTPGGALSLVCRASGITFSTYAMEWVRQAPGKGLEFVAVVNAAGSTYYGAAVKGRATISRDN
GQSTVRLQLNNLRAEDTGTYYCTRGSGGENIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv118
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVEITCSGGSGSYGWYQQKSPGGAPVTVIYYNDKRP
SDIPSRFSGSKSGSTATLTITGVQVEDEAVYYCGSYDSSYVGIFGVGTTLTVLGQSSRSSAVTLDESGGGL
QTPRGALSLVCKASGFTFSSYSMAWVRQAPGKGLEFVAGIQNDGSITDYGSAVDGRATISRDDGQSTVRLQ
LNNLRTEDTATYYCAKTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv119
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGSSGSYGWYQQKSPGSAPVTVIYRNDKRP
SNIPSRFSGALSGSTATLTITGVQAEDEAVYFCGSADSSGAIFGAGTTLTALGQSSRSSTVTLEESGGGLH
TPGGGLILLCKGSGVSFCNYGMGWMRRDPGGGLEYVAGISTGSYTYYGPAVKGRGTVSRDNGQSTMRLQLN
HLRAEDETIYFCARTDASSHGCGSGTDLGSIDAWGHGTEVLLSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv120
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKLTCSGDSSDYGYGWYQQKSPGSAPVTVIYNNNK
RPSDIPSRFSGSKSGSTGTLTISGVQAEDEAVYFCGSEDSNSDAIFGAGTTLTVLGQSSRSSAVTLDEYGG
GLQTPGGALSLVCEASGFTFSSYDMLRIPHAPGKGLEYVAGLTSNGRYASYGSAVDGRATISRDNGQSTWR
LHLNNLGAEDTGPYYCAGYDGSGWTGNTIEAWGHRTEVLVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv121
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGGSSYYGWYQQKSPGSAPVTLIYENNNRP
SDIPSRFSGSASGSTATLTITGVQAEDGAVYFCGSEDSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQ
LNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDY
AS >scFv123
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGGSSYYGWYQQKSPGSAPVTLIYENNNRP
SDIPSRFSGSASGSTAPLTITGVQAEDGAVYFCGSEDSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
QTPGRALSLVCKASGFTFSSFNMGWVRQAPGKGLEYVASISSSGSYTAYGSAVKGRATISRDNGQSTVRLQ
LNNLRAEDTATYYCAKAAGSAYYYTAVTPAFAGSIDACGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDY
AS >scFv124
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGGSGSYGWYQQKSPGSAPVTVIYYNDQRP
SDIPSRFSGSKSGSTGTLTITGVHAEDEAVYYCGGYNSTYVGIFGAGTTLTVLGQSSRSSAVTLDESGGGL
HTPRGALSLICKASGFTFSSYSMAWVQQAPGKGLEFVPGILNDGSITDYGSADDGRATISRDDGQSTVRLH
LINLRTEDTATYYCAKTTVADGVIGAYGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv131
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVEITCSGGSSSYYGWYQQKSPGSAPVTVIYWNDKR
PSDIPSRFSGSESGSPATLTITGVRAEDEAVYFCGSGDSSGTGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGGLSLVCKASGFSFSDYTMNWVRQAPGKGLEWVGQISSDNGRYTTYGAAVKGRATISRDDGQSTVR
LQLNNLKAEDTATYYCAKESDGDYNGGAGLIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv134
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGSSGSYGWYQQKSPGSAPVTVIYWNDKRP
SNIPSRFSGALSGSTATLTITGVQAEDEAVYFCGSADSSGAIFGAGTTLTVLGQSSRSSTVTLDESGGGLQ
TPGGGLSLVCKGSGFAFSNYGMGWMRQAPGKGLEYVAGISTGSYTDYGPAVKGRATISRDNGQSTVRLQLN
NLRAEDAAIYFCAKTAGSGYGCGSGTDLGSIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
Additional scF1, Sequences (6) >scFv103
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVEITCSGGGGSYGWFQQKSPGSAPVTVIYESTKRP
SNIPSRFSGSGSGSTSTLTITGVRAEDEAVYYCGGYDGSSDAIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCKASGFTFSSHDMGWVRQAPGKGLEYVAGITDDGRYASYGPAVDGRATISRDNGQSTVRLQ
LKNLRAEDTATYYCARDDGSGWSGDTIDTWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv136
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGSYSYGWYQQKSPGSAPVTVIYSSDKRP
SDIPSRFSGSKSGSTSTLTITGVQAEDEAVYYCGSRDSNYVGIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCKASGFTFSSYEMQWVRQAPGKGLEFVAAISSDGSYTNYGAAVQGRATISRDNGQSTVRLQ
LSNLRAEDTATYYCARSPGGYTWWPGAAGGIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv137
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGETVKITCSGGGSSNNYGWEQQKAPGSAPVTVIYDNTN
RPSNIPSRFSGSKSSSTHTLTITGVQAEDEAVYYCESADSSSSIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGTLSLVCKASGFTFSSFNMFWVRQAPGKGLEFVAGIGNTGRSTGYGSAVKGRATISRDNGQSTVRL
QLNNLRAEDTGTYYCAKAYGDSNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv138
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVEITCSGGGSYYGWYQQKSPGSAPVTVIYANTNGP
SDIPSRFSGSTSGSTATLTITGVQADDEAVYSCGSYDSSYVGIFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCKASGFTFNSYALEWVRQAPGKGLEWVAGISGDGSYRHYGSAVKGRATISRDSGQSTVRLQ
LNNLRAEDTGTYYCAKSTGSGAGWGASNIDAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv139
MKKTAIAIAVALAGFATVAQAALTQPSSVSANLGGTVKITCSGGDSSYGWYQQKSPGSAPVTLIYDNTNRP
SDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGNADSSSTAAFGAGTTLTVLGQSSRSSTVTLDESGGGL
QTPGGALSLVCKASGFTFSSYAMGWVRQAPGKGLEYVAAISSAGSTTNYGAAVKGRATISSDNGQSTVRLQ
LNNLRAEDTATYFCAKTAGSGYYVWSAIAGDIYAWGHGTEVIVSSTSGQAGQHHHHHHGAYPYDVPDYAS
>scFv140
MKKTAIAIAVALAGFATVAQAALTQPSSVSANPGGTVKITCSGSSGSYYGWYQQKSPGSAPVTVIYDNDKR
PSDVPSRFSGSKSGPTATLTITGVQAEDEAVYFCGSRDNSYVGIFGAGTTLTVLGQSSRSSAVTLDESGGG
LQTPGGALSLVCKASGFTFSSYDMFWVRQAPGKGLEFVAQINSAGSYTNYGSAVKGRATISRDDGQSTVRL
QLNNLRAEDTGIYFCAKSASGYYYSGSDAGDIDAWGTGPKSSSPSTSGPGRPAPSPSPWRIPVRRSGLRFL
ERWARDQLSCTKWLI. (lacks the C-terminal 6His and HA tag) scFv45
atgaaaaagacagctatcgcgattgcagtggcactggctggtttcgctaccgtggcccag M K K
T A I A I A V A L A G F A T V A Q
gcggccctgactcagccgtcetcggtgtcagcgaacccgggaggaaccgtcaagatcacc A A L
T Q P S S V S A N P G G T V K I T
tgctccgggagtagcagtgcetatggttatggctggtatcagcagaagtcocctggcagt C S G
S S S A Y G Y G W Y Q Q K S P G S
gcccctgtcactgtgacctataacaacaotaagagaccctcaaacacccctCcacgattc A P V
T V I Y N N H K R P S N I P S R F
tccggttccaaacccggccccacgggcacattaaccatcaccggggtccaagccgcggac S G S
K S G S T G T L T I T G V Q A E D
gaggccgtctatttctgtgggagtgaogocagcagcactgatgctacatttggggccggg E A V
Y F C G S E D S S T D A I F G A G
acaaccctgaccgtcctaggtcagtectctagatcttccaccgCgacgttggacgagtcc T T L
T V L G Q S S R S S T V T L D E S
gggggcggcctccaggcgcccggaggagcgctcagcctcgtctgcaaggcctccgggttc G G G
L Q A P G G A L S L V C K A S G F
accctcagcagttacgacacgggttggatacgacaggcgcccggcaaggggctggaatac T F S
S Y D M G W I R Q A P G K C L B Y
gttgcgggcattaccgataatggtagatacgcaccatatgggtcggcggtggatggccgt V A G
I T D N G R Y A S Y C S A V D G R
gccaccatctcgagggaeaacgggcagagctcagtgaggctgcagctgoacaacctcagg A T I
S R D N G Q S S V R L Q L N N L R
gctgaggacaecggcacctaccactgcgccagagatgacggtagtggttggaccggtaat A E D
T G T Y Y C A R D D G S G W T G N
agtatcgacgcatggggccacgggaccgaagtcatcgtctcctccactagtggccaggcc S I D
A W G H G T E V I V S S T S G Q A
ggccagcaccatcaccatcaccatggcgcatacccgcacgacgttecggaccacgettct G Q H
H H H H H C A Y P Y D V P D Y A S tag - scFv57
atgaaaaagacagatatcgcgattgcagtggcactggccggttccgceaccgtggcccag M K K
T A I A I A V A L A G F A T V A Q
gcggccctgactcagccgtcctcggtgccagcgaacccgggaggaaccgtcaagatcacc A A L
T Q P S S V S A N P G G T V K I T
tgctccgggagtagcagtgcctatggttatggctggtatcagcagaagtcacctggcagt C S G
S S S A Y G Y G W Y Q Q K S P G S
gcccctgteactgtgatctataacaacaataagagacccccaaacatccctccacgactc A P V
T V I Y N N N K R P S N I P S R F
tccggttccaaatccggctccacgggcacatcaaccatcactggggtccaagccgoggac S G S
K S G S T G T L T I T G V Q A E D
goggctgtctattcctgtgggagtgaagacagcagcactgacgctatatctggggccggg E A V
Y F C G S E D S S T D A I F G A G
acaaccctgaccgtcccoggccagtcccctagatctcccgccgcgacgtcggacgagtcc T T L
T V L G Q S S R S S A V T L D E S
gggggcggcctccagacgcccggaggagcgctcagcctcgtctgcaaggcctccgggttc G G G
L Q T P G G A L S L V C K A S G F
accttcagcagttacgacatgggttgggtgcgacaggcgcccggcaagggactggaatac T F S
S Y D M G W V R Q A P G K G L E Y
gtcgcgggtattaccaatgatggtagatacgcatcatacgggtcggcggtggatggccgt V A G
I T N D G R Y A S Y G S A V D G R
gccaccatctcgagggacaacgggcagagcacagtgaggccgcagctgaacaacctcagg A T I
S R D N G Q S T V R L Q L N N L R
gctgaggacaccggcacccactactgcgccagagatgatggtagtggttggactggtaat A E D
T G T Y Y C A R D D G S G W T G N
actatcgacacatggggccacgggaccgaagccaccgtctcccccactagtggccaggcc T I D
T W G H G T E V I V S S T S G Q A
ggccagcaccaccaccatcaccauggcgcatacccgtacgacgctccggactacgcttct
G Q H H H H H H G A Y P Y D V P D Y A S tag - scFv64
acgaaaaagacagctatcgcgattgcagtggcactggctggtttcgctaccgtggcccag M K K
T A I A I A V A L A C F A T V A Q
gcggccctgacccagccgtcctcggcgtcagcgaacccgggagaaaccgtcaagatcacc A A L
T Q P S S V S A N P Q E T V K I T
cgccccggggatagcagtggctatggctatggctggtatcagcagaagtcacctggcagt C S G
D S S G Y G Y G W Y Q Q K S P C S
gcccctgccactgtgacccataacaacaataagagaccctcggacatcccttcacgattc A P V
T V I Y N N N K R P S D I P S R F
tccggctccaaatccggccccacgggcacattaaccatcactggggtccaagccgaggac S G S
K S G S T G T L T I T G V Q A E D
gaggctgtctatttccgtgggagtgaagacagcaacactgatgctgtattcggggccggg E A V
Y F C G S E D S N T D A V F G A G
acaaccctgaccgtcccaggtcagtcctctagatcttccaccgcgacgttggacgagtcc T T L
T V L G Q S S R S S T V T L D E S
gggggcggcctccagacgcccggaggaacgctcagcctcgcctgcaaggcctccgggttc G G G
L Q T P G G T L S L A C K A S G F
accttcagtggctacgacatgggctgggtgcgacaggcacccggcaaggggctggagtac T F S
G Y D M G W V R Q A P C K G L E Y
gttgcgggtatcaccagcgatggtagatacgcatcatacgggtcggcggtggatggccgc V A G
I T S D G R Y A S Y C S A V D G R
gccgccatctggagggacaacgggcagagcacagtgaggctgcagctgaaaaacctcagg A A I
W R D N G Q S T V R L Q L K N L R
actgaggacaccgccacccactaccgcgccagagatgatggtagtggctggagtggtaat T E D
T A T Y Y C A R D D G S G W S G N
aatatcgacgcatggggccacgggaccgaagtcatcgtctcctccactagcggccaggcc N I D
A W G H G T E V I V S S T S G Q A
ggccagcaccatcaccatcaccatggcgcatacccgtacgacgttccggactacgettct G Q H
H H H H H G A Y P Y D V P D Y A S tag - scFv106
atgaaaaagacagctatagcgattgcagtggcactggctggtttcgccaccgtggcccag M K K
T A I A I A V A L A G F A T V A Q
gcggccctgactcagccgtcctcggtgtcagcgaacccaggagaaaccgtcaagatcacc A A L
T Q P S S V S A N P G E T V K I T
tgctccggggatagcagtgactatggttatggctggtatcagcagaagtcacctggcagt C S G
D S S D Y G Y G W Y Q Q K S P G S
gcccctgtcactgtgacctatagcaacaaccagagacccccgaacatcccttcacgattc A P V
T V T Y S N N Q R P P N I P S R F
tccggttccgcatccggctccacagccacattaaccetcactggggtccaagtcgaggac S G S
A S G S T A T L T I T G V Q V E D
gaggctgtctattcctgtgggagtgaagccagtcccactgatgctgtatttggggccggg E A V
Y Y C G S E D S T T D A V F G A C
acaaccctgaccgccctaggccagtcctctagatcctccgccatgacgttggacgagtcc T T L
T V L G Q S S R S S A H T L D E S
gggggcggcctccagacgcccggaggagcgctcagccccgtctgcaaggcctccgggttc G G G
L Q T P G G A L S L V C K A S G F
accttcagcagttacgacatgggctgggtgcgacaggcgcccggcaaggggctggaatac T F S
S Y D M G W V R Q A P G K G L E Y
gttgcgggtattaccaatgacggtagatacgcatcatacgggtcggcggtggatggccgt V A G
I T N D G R Y A S Y G S A V D G R
gccaccatctcgagggacaacgggcagagcacagtgaggctgcagctgaacaacctcagg A T I
S R D N G Q S T V R L Q L N N L R
gctgaggacaccggcacctactactgcgccagagatgatggtagtggLtggactggtaat A E D
T G T Y Y C A R D D G S G K T G N
actatcgacacatggggccacgggaccgaagtcatcgtctcctccactagcggccaggcc T I D
T W G H G T E V I V S S T S G Q A
ggccagcaccotcoccatcaccatggcgcatacccgtacgacgttccggactacgcttct G Q H
H H H H H G A Y P Y D V P D Y A S tag - scFv136
atgaaaaagacagccatcgcgattgcagtggcactggctggttccgctaccgcggcccag M K K
T A I A I A V A L A G F A T V A Q
gcggccccgactcagccgtccccggcgtcagcaaacccaggagaaaccgtcaagaccacc A A L
T Q P S S V S A N P G E T V K I T
tgccccgggggcagccacagccatggctggcatcagcagaagccocctggcagcgccccc C S G
G S Y S Y G W Y Q Q K S P G S A P
gtcaccgtgacctatagcagcgacaagagacceccggacatcccttcacgatcctccggt V T V
I Y S S D K R P S D I P S R F S C
tccaaatccggctceacaagcacantaaccaceactggggtccaagccgaggacgaggct S K S
G S T S T L T I T G V Q A E D E A
gcccactaccgtgggagcagggacagcaactatgttggtacatccggggccgggacaacc V Y Y
C G S R D S N Y V G I F G A C T T
ctgaccgtcctaggtcagtcctctagatcttccaccgtgacgttggacgagtccgggggc L T V
L G Q S S R S S T V T L D E S G G
ggcctccagacgcecggaggagcgctcagcctcgcctgcaaggcccccggacceaccttc G L Q
T P G G A L S L V C K A S G F T F
agcagttatgagacgcagcgggtgcgacaggcgcccggcaaggggccggagctcgtcgca S S Y
E M Q W V R Q A P G K G L E F V A
gccatcagcagcgacggcagccacacaaactacggggcggcggcgcagggccgcgccacc A I S
S D G S Y T N Y G A A V Q G R A T
atctcgagggacaacgggcagagcacagtgaggctgcagctgagcaacctcagggctgag I S R
D N G G S T V R L Q L S N L R A B
gacoccgccacctactactgcgccagaagtcctggtggttacacttggtggcctggagct D T A
T Y Y C A R S P G G Y T W W P G A
gctggcggtatcgacgcatggggccacgggaecgaagtcatcgtctcctccactagtggc A G G
I D A K C H G T E V I V S S T S G
caggccggccagcaccatcaccatcaccatggcgcatacccgtacgacgttccggactac Q A G
Q H H H H H H C A Y P Y D V P D Y gctccctag A S - scPv67
acgaaaaagacagccatcgcgactgcagtggcaccggccggtcccgctaccgtggcccag M K K
T A I A I A V A I A G F A T V A Q
gcggccctgactcagccgtccccggtgccagcgaacccgggaggaaccgtcaagatcacc A A L
T Q P S S V S A N P G G T V K I T
tgccccgggagtagcagcgcccatggccacggctggcatcagcagaagtcacctggcagc C S G
S S S A Y G Y G W Y Q Q K S P G S
gcccccgtcaccgcgatctataacaacaacaagagaccctcaaacaccccctcacgattc A P V
T V I Y N N N K R P S N I P S P F
tccggttccaaatccggctccacgggcacattaaccatcactggggtccaagccgaggac S G S
K S G S T G T L T I T G V Q A E D
gaggctgtctatttctgtgggagtgaagacagcagcactgatgctatatttggggccggg E A V
Y F C G S E D S S T D A I F G A G
acaaccctgaccgtcctaggtcagtcctctagatcttccgccgtgacgttggacgagtcc T T L
T V L G Q S S R S S A V T L D E S
gggggcggcctccagacgcccggaggagcgctcagccccgcctgcaaggcctccgggttc G G G
L Q T P G G A L S L V C K A S G F
accttcagcagttacgacacgggttgggtgcgacaggcgcccggcaagggactggaatac T F S
S Y D H C W V R Q A P G K G L E Y
gctgcgggtattaccaacgacggcagacacgcaccacacgggccggcggtggatggccgt V A G
I T N D G R Y A S Y G S A V D G R
gccaccaccccgagggacaacgggcagagcacagcgaggccgcagccgaacaacctcagg A T I
S R D N G Q S T V R L Q L N H L R
gccgaggacaccggcacccaccaccgcgccagagacaacggcagcggtcggaccggtaat A E D
T G T Y Y C A R D D G S G W T G N
accatcgacacatggggccacgggaccgaagccaccgtcccccccactagcggccaggcc T I D
T W G H G T E V I V S S T S G Q A
ggccagcaccaccaccaccaccacggcgcacacccgcacgacgccccggaccacgcctct G Q H
H H H H H G A Y P Y D V P D Y A S Tag -
scFv102
atgaaaaagacagctatcgcgattgcagtggcactggctggtttcgctaccgtggcccag M K K
T A I A I A V A L A G F A T V A Q
gcggccccgactcagccgtcctcggcgtcagcgaacccgggagaaaccgtcaagatcacc A A L
T Q P S S V S A M P G E T V K I T
tgctccgggggtagtggcagctatggctggtatcagcaggagtcacctggcagcgctcct C S C
C S G S Y G W Y Q Q E S P G S A P
gtcactgtgatctactacaacgacaagagaccctcggacacccccccacgoctctccggc V T V
I Y V N D K R P S D I P S P F S G
tccgcatccggctccacagccacattaaccatcgctggggtccgagccgaggacgaggct S A S
G S T A T L T I A G V R A E D E A
gtctacttctgtgggagccgggatagcagcactagtgctggtatatttggggccgggaca V Y F
C G S W D S S T S A G I F G A G T
gccctgaccgtcccaggccagtcctctagaccttccgccgcgacgtcggacgagtccggg A L T
V L G Q S S R S S A V T L D E S G
ggcggcctccagacgcccggaggagggctcagcctcgcctgcaaggcccccggctccagc G G L
Q T P G G G L S L V C K A S G F S
agcagccatggcatgggctggatgcgccaggcacctggcaagggccctgaattcgtcgcg S S H
G M G K M R Q A P G K G L E F V A
ggtattagaagtgatggcagtagcacagcatacggggcggcggtggatggccgcgccacc G I R
S D G S S T A Y G A A V D G R A T
accacaagggacgatgggcagagcacagtgacactgcagctgaacaacctcagggctgag I T R
D D G Q S T V T L Q L N N L R A E
gacaccgccacctacctctgcgccaaaactaatagutacaatagcgctggcataatcgac D T A
T Y F C A K T N S Y N S A G I I D
gcatggggccacgggaccgaagtcatcgtctcctccactagtggccaggccggccagcac A W G
H G T E V I V S S T S G Q A G Q H
catcaccatcaccacggcgcatacccgtacgocgttccggactacgcttcttag H H H H H G
A Y P Y D V P D Y A S - ScPv34
atgaaaaagacagctatcgcgattgcagtggcactggctggtttcgccaccgtggcccag M K K
T A I A I A V A L A G F A T V A Q
gcggccctgactcagccgtcctcggtgtcagcaaacctgggaggaaccgtcgagaccacc A A L
T Q P S S V S A M L G G T V E I T
tgctccgggagtagtggcagccatggctggtatcagcagaagtcacctggcagtgcccct C S C
S S G S Y G W Y Q Q K S P G S A P
gccaccgcgatctattacaacgacaagagacccccggacatcccttcacgattccccggt V T V
I Y Y N D K R P S D I P S R F S G
tccacatccggctccacagccacattaaccaccaccggggtccaagccgaggacgaggct S T S
G S T A T L T I T G V Q A E D E A
gcccatttccgtggtggccacgacagcaactatatcggtatatttggggccgggacaacc V Y F
C G G Y D S N Y I G I F G A G T T
ccgaccgtcctaggtcagccctccagatcttccgccgcgacgttggacgagtccgggggc L T V
L G Q S S R S S A V T L D E S G G
ggcctccagacgceccgaggagcgctcagcctcgtccgcaaggcctccgggttcaccttc G L Q
T P R G A L S L V C K A S G F T F
agcagttacagcatggcctgggtgcgacaggcgcccggcaaggggctggagttcgtcgcg S S Y
S M A W V R Q A P G K G L E F V A
ggtattcagaatgatggtagtatcacagattacgggtcggcggtggatggccgtgccacc G I Q
N D G S I T D Y G S A V D G R A T
acctcgagggacgacgggcagagcacagtgaggctgcagctgaacaacctcaggactgag I S R
D D G Q S T V R L Q L N N L P T E
gacaccgccocctactactgcgccaaaactactgttgctgatggtgtcatcggtgcttat D T A
T Y Y C A K T T V A D G V I G A Y
ggcatcgacgcatggggccacgggaccgaagtcatcgccccctccaccagtggccaggcc G I D
A W G H G T E V I V S S T S G Q A
ggccagcaccatcaccatcaccatggcgcacacccgtacgacgttccggaccacgcttct G Q H
H H H H H G A Y P Y D V P D Y A S tag - scFv61
acgaaaaagacagccaccgcgattgcagtggcaccggctggtttcgccaccgtggcccag M K K
T A I A I A V A L A C F A T V A Q
gcggccctgactcagccgccctcggtgccagcaaacccaggagaaaccgccaagatcacc A A L
T Q P S S V S A H P C E T V K I T
cgctccgggggtggcagctacgctggaagttactatcatggctggtaccagcagaaggca C S C
G G S Y A G S Y Y Y G W Y Q Q K A
cctggcagcgcccctgccactctgatctatgacaacaccaacagaccctcgaacacccct P G S
A P V T L I Y D N T N R P S N I P
tcacgactccccggttccctatccggctccacgggcacattaaccatcactggggtccga S R F
S C S L S C S T G T L T I T G V R
gccgaggacgaggctgcctattactgtgggagctccgacagcagcaccgacggcggatac A E D
E A V Y Y C G S F D S S T D G G Y
gccgccatacttggggccgggacaaccccgaccgtcctaggtcagccctccagaccccac A A I
F G A G T T L T V L G Q S S R S Y
gccgtgacgttggacgagtccgggggcggcccccagacgcccggaggagggctcagcctc A V T
L D E S G G G L Q T P G G G L S L
gtctgcaaggcctccgagttcaccctcagcagttatgccatggagcgggtgcgccaggca V C K
A S E F T F S S Y A M E W V P Q A
cccggcaaggggctggagcgggtcgcctatattaacagcgatggtagtcgcacatggtac P G K
G L E W V A Y I N S D C S S T W Y
gcacctgcggtgaagggccgcgccaccacctcgagggacaacgggcagagcacagtgagg A P A
V K G P A T I S P D N C Q S T V R
ctgcagctgaacagcctcagggctgaagacaccgccacctaccaccgcaccagaggttct L Q L
N S L P A E D T A T Y Y C T P G S
ggtggtgaaaatatagacacatggggccacgggaccgaagtcatcgtctcctctactagt G G E
N I D T W C H G T E V I V S S T S
ggccaggccggccagcaccatcaccatcaccatggcgcatacccgcacgacgttcccgac G Q A
G Q H H H H H H C A Y P Y D V P D cacgctccttag Y A S - scFv62
atgaaaaagacagctaccgcgactgcagtggcaccggctggcttcgccaccgtggcccag M K K
T A T A I A V A L A G F A T V A Q
gcggccctgactcagccgcccccggtgtcagcaaacccaggagaaaccgccaagaccacc A A L
T Q P S S V S A N P Q E T V K I T
tgctccgggggtggcagctatgctggaagccactatcacggctggtaccagcagaaggca C S G
G G S Y A G S Y Y Y C W Y Q Q K A
cctggcagtgccectgtcaccccgatctacgacaacaccaacagaccctcgaacatccct P G S
A P V T L I Y D N T H R P S N I P
ccacgactctccggtcccccatccggctccacgggcacattaaccatcactggggtccga S R F
S G S L S G S T G T L T I T G V R
gccgaggacgaggccgtctattactgtgggagctccgacagcagcactgacggtggatat A E D
E A V Y Y C G S F D S S T D G G Y
gctgccatattcggggccgggacaaccctgaccgtcctaggtcagtcctctaaatcttcc A A I
F G A G T T L T V L G Q S S R S S
gccgcgacgctggacgagcccgggggcggcccccagacgcccggaggagggctcagcccc A V T
L D E S G C C L Q T P G G G L S L
gtctgcaaggcctccgagttcaccttcagcagttatgccatggagtgggtgcgccaggca V C K
A S E F T F S S Y A H E W V R Q A
cccggcaaggggctggagtgggtcgcctatattaacagcgatggtagtagcacatggtac P G K
G L E W V A Y I N S D G S S T W Y
gcacctgcggtgaagggccgcgccaccatctcgagggacaacgggcagagcacagtgagg A P A
V K G R A T X S R D N G Q S T V R
ctgcagccgaacagcctcagggccgaggacaccgccacccaccaccgcaccagaggctcc L Q L
N S L R A E D T A T Y Y C T R G S
ggcggcgaaaatatagacacatggggccacgggaccgaagtcatcgccccctccactagt G G E
N I D T W C H C T E V I V S S T S
ggccaggccggccagcaccatcaccatcaccatgccgcatacccgtacgacgttccagac G Q A
G Q H H H H H H A A Y P Y D V P D tacgcttcttag Y A S - scFv118
atgaaaaagacagctatcgcgatcgcagtggcaccggctggtttcgctaccgtggcccag M K K
T A I A I A V A L A C F A T V A Q
gcggccccgactcagccgccctcggtgtcagcaaacctgggaggaaccgtcgagatcacc A A L
T Q P S S V S A N L G G T V E I T
tgctccgggggtagcggcagctatggctggtatcagcagaagtcacccggcggcgcccct
C S G G S G S Y G W Y Q Q K S P G G A P
gtcactgtgacctattacaacgacaagagaccctcggacacccccccacgattctccggt V T V
I Y Y N D K R P S D I P S R F S G
tccaaatccggctccacagccacaccaaccatcactggggtccaagtcgaggacgaggct S K S
G S T A T L T I T G V Q V E D E A
gtctactactgtgggagctacgacagcagctatgctggtatatctggggtcgggacaacc V Y Y
C G S Y D S S Y V G I F G V G T T
ctgaccgccccaggccagccctctagaccccccgccgtgacgtcggacgagtccgggggc L T V
L G Q S S R S S A V T L D E S C G
ggcccccagacgccccgaggagcgctcagccccgtctgcaoggcctccgggtccaccttc G L Q
T P P G A L S L V C K A S G F T F
agcagtcacagcatggcccgggtgcgacaggcgcccggcaaggggctggagttcgtcgcg S S Y
S M A W V R Q A P G K C L E F V A
ggcactcagaatgatggcagtatcacagattacgggtcggcggtggacggccgtgccacc G I Q
N D G S I T D Y G S A V D G R A T
atctcgagggacgacgggcagagcacagtgaggccgcagctgaacaaccccaggactgag I S R
D D G Q S T V R L Q L N N L R T E
gacaccgccacctaccaccgcgccaaaaccactgttgctgatggtgttatcggtgcttat D T A
T Y Y C A K T T V A D G V I G A Y
ggcatcgacgcatggggccacgggaccgaagccaccgtctcctccactagtggccaggcc G I D
A W G H G T E V I V S S T S G Q A
ggccagcaccatcaccatcaccatggcgcatacccgcacgacgttccggactacgcttct G Q H
H H H H H G A Y P Y D V P D Y A S tag -
[0152] 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. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed.
[0153] The indefinite articles "a" and "an", as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0154] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined (elements that are conjunctively present in
some cases and disjunctively present in other cases). Other
elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified unless clearly
indicated to the contrary. Thus, as a non-limiting example, a
reference to "A and/or B", when used in conjunction with open-ended
language such as "comprising" can refer, in some embodiments, to A
without B (optionally including elements other than B); in some
embodiments, to B without A (optionally including elements other
than A); and in some embodiments, to both A and B (optionally
including other elements); etc.
TABLE-US-00003 ##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029##
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039##
##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044##
##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054##
##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059##
##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065##
REFERENCES
[0155] All publications, patents and sequence database entries
mentioned herein, including those items listed below, are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
* * * * *