U.S. patent application number 15/706631 was filed with the patent office on 2018-02-08 for plasma kallikrein binding proteins.
This patent application is currently assigned to Dyax Corp.. The applicant listed for this patent is Dyax Corp.. Invention is credited to Daniel J. Sexton, Malini Viswanathan.
Application Number | 20180037666 15/706631 |
Document ID | / |
Family ID | 44306145 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180037666 |
Kind Code |
A1 |
Sexton; Daniel J. ; et
al. |
February 8, 2018 |
PLASMA KALLIKREIN BINDING PROTEINS
Abstract
Plasma kallikrein binding proteins and methods of using such
proteins are described.
Inventors: |
Sexton; Daniel J.; (Melrose,
MA) ; Viswanathan; Malini; (Burlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dyax Corp. |
Burlington |
MA |
US |
|
|
Assignee: |
Dyax Corp.
Burlington
MA
|
Family ID: |
44306145 |
Appl. No.: |
15/706631 |
Filed: |
September 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14339053 |
Jul 23, 2014 |
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15706631 |
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12985914 |
Jan 6, 2011 |
8822653 |
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14339053 |
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61292614 |
Jan 6, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/02 20180101; C07K
2317/76 20130101; A61K 49/0004 20130101; A61P 9/00 20180101; A61P
29/00 20180101; C07K 2317/92 20130101; A61P 1/00 20180101; A61P
11/00 20180101; A61P 19/02 20180101; A61P 31/04 20180101; A61P
17/02 20180101; A61P 25/04 20180101; A61P 43/00 20180101; A61K
2039/505 20130101; A61P 19/06 20180101; A61P 7/00 20180101; A61P
7/02 20180101; A61P 13/12 20180101; A61P 9/10 20180101; C07K 16/40
20130101; A61P 7/10 20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 49/00 20060101 A61K049/00 |
Claims
1. (canceled)
2. An isolated monoclonal antibody, which binds human plasma
kallikrein (pKal), wherein the monoclonal antibody binds an epitope
comprising one or more of residues Arg551, Gln553, Tyr555, Thr558,
and Arg560 of SEQ ID NO:2.
3. The isolated monoclonal antibody of claim 2, wherein the
antibody binds human plasma prekallikrein less effectively relative
to the human pKal by a factor of at least 10 fold.
4. The isolated monoclonal antibody of claim 3, which binds human
plasma prekallikrein less effectively relative to the human pKal by
a factor of at least 100 fold.
5. The isolated monoclonal antibody of claim 4, which binds human
plasma prekallikrein less effectively relative to the human pKal by
a factor of at least 1000 fold.
6. The isolated monoclonal antibody of claim 2, which competes with
epi-Kal2 for binding to the human pKal.
7. The isolated monoclonal antibody of claim 2, which competes with
AEBSF for binding to the human pKal.
8. The isolated monoclonal antibody of claim 2, which decreases the
catalytic activity of the human pKal as compared to the catalytic
activity of the human pKal in the absence of the monoclonal
antibody and under the same conditions.
9. The isolated monoclonal antibody of claim 8, wherein the
monoclonal antibody decreases the production of Factor XIIa or
bradykinin by at least 50% as compared to the production of Factor
XIIa or bradykinin in the absence of the monoclonal antibody and
under the same conditions.
10. The isolated monoclonal antibody of claim 9, wherein the
monoclonal antibody decreases the production of Factor XIIa or
bradykinin by at least 75% as compared to the production of Factor
XIIa or bradykinin in the absence of the monoclonal antibody and
under the same conditions.
11. The isolated monoclonal antibody of claim 10, wherein the
monoclonal antibody decreases the production of Factor XIIa or
bradykinin by at least 90% as compared to the production of Factor
XIIa or bradykinin in the absence of the monoclonal antibody and
under the same conditions.
12. The isolated monoclonal antibody of claim 2, which is a human
antibody.
13. The isolated monoclonal antibody of claim 2, which is an IgG
molecule.
14. The isolated monoclonal antibody of claim 13, which is an IgG1
molecule.
15. The isolated monoclonal antibody of claim 13, which is a
germlined antibody.
16. The isolated monoclonal antibody of claim 2, which is
substantially a full-length antibody.
17. The isolated monoclonal antibody of claim 2, which comprises an
antigen-binding fragment of a full-length antibody.
18. The isolated monoclonal antibody of claim 2, which comprises a
heavy chain variable region and a light chain variable region.
19. A pharmaceutical composition, comprising the monoclonal
antibody of claim 2 and a pharmaceutically acceptable carrier.
20. A method for treating hereditary angioedema, comprising
administering to a subject in need thereof an effective amount of
the monoclonal antibody of claim 2.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 14/339,053, filed on Jul. 23, 2014, which is a divisional of
U.S. application Ser. No. 12/985,914, filed on Jan. 6, 2011, issued
as U.S. Pat. No. 8,822,653, which claims priority to U.S.
Application Ser. No. 61/292,614, filed on Jan. 6, 2010. The
contents of each of the prior applications are incorporated by
reference in their entirety.
BACKGROUND
[0002] Plasma kallikrein is a serine protease. Prekallikrein is the
precursor of plasma kallikrein.
SUMMARY
[0003] Plasma kallikrein is a serine protease component of the
contact system and a potential drug target for different
inflammatory, cardiovascular, infectious (sepsis) and oncology
diseases (Sainz I. M. et al., Thromb Haemost 98, 77-83, 2007). The
contact system is activated by either factor XIIa upon exposure to
foreign or negatively charged surfaces or on endothelial cell
surfaces by prolylcarboxypeptidases (FIG. 1) (Sainz I. M. et al.,
Thromb Haemost 98, 77-83, 2007). Activation of the plasma
kallikrein amplifies intrinsic coagulation via its feedback
activation of factor XII and enhances inflammation via the
production of the proinflammatory nonapeptide bradykinin. As the
primary kininogenase in the circulation, plasma kallikrein is
largely responsible for the generation of bradykinin in the
vasculature. A genetic deficiency in the C1-inhibitor protein
(C1-INH), the major natural inhibitor of plasma kallikrein, leads
to hereditary angioedema (HAE). Patients with HAE suffer from acute
attacks of painful edema often precipitated by unknown triggers
(Zuraw B. L. et al., N Engl J Med 359, 1027-1036, 2008). Through
the use of pharmacological agents or genetic studies in animal
models, the plasma kallikrein-kinin system (plasma KKS) has been
implicated in various diseases.
[0004] Plasma kallikrein binding proteins (e.g., antibodies, e.g.,
inhibitory antibodies) are useful therapeutic agents for a variety
of diseases and conditions, e.g., diseases and conditions that
involve plasma kallikrein activity, due to their high potency,
specificity, and prolonged serum residency. High potency can
translate to efficacy and a low drug dosage, and high specificity
can reduce side effects due to the inhibition of related off target
serine proteases. In general, small molecule serine proteases are
not as specific as antibody inhibitors. Prolonged serum residency
can permit infrequent dosing.
[0005] In some aspects, the disclosure features an isolated protein
(e.g., antibody, e.g., human antibody) that binds to the active
form of plasma kallikrein (e.g., human plasma kallikrein and/or
mouse plasma kallikrein), and, e.g., does not bind preplasma
kallikrein (e.g., human preplasma kallikrein and/or mouse preplasma
kallikrein).
[0006] In some embodiments, the plasma kallikrein binding protein
binds the same epitope or competes for binding with a kallikrein
binding protein described herein. In some embodiments, the plasma
kallikrein binding protein binds the same epitope or competes for
binding with a protein (e.g., epi-Kal2) and/or a small molecule
(e.g., AEBSF) described herein and does not bind pre-plasma
kallikrein.
[0007] In some embodiments, the protein described herein is
selected from the group consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01,
X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04.
[0008] In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as X81-B01 and, e.g., does
not bind pre-plasma kallikrein.
[0009] In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as X67-D03 and, e.g., does
not bind pre-plasma kallikrein.
[0010] In some embodiments, the plasma kallikrein binding protein
competes with or binds to the same site as X101-A01 and, e.g., does
not bind pre-plasma kallikrein.
[0011] In some embodiments, the plasma kallikrein binding protein
competes with or binds to the same site as M162-A04 and, e.g., does
not bind pre-plasma kallikrein.
[0012] In some embodiments, the plasma kallikrein binding protein
competes with or binds to the same site as X63-G06 and, e.g., does
not bind pre-plasma kallikrein.
[0013] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or mouse
prekallikrein), but binds to the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or mouse plasma kallikrein).
[0014] In certain embodiments, the protein binds at or near the
active site of the catalytic domain of plasma kallikrein, or a
fragment thereof, or binds an epitope that overlaps with the active
site of plasma kallikrein and, e.g., does not bind pre-plasma
kallikrein.
[0015] In some embodiments, the protein binds to one or more amino
acids that form the catalytic triad of plasma kallikrein: His434,
Asp483, and/or Ser578 (numbering based on the human sequence) and,
e.g., does not bind pre-plasma kallikrein.
[0016] In some embodiments, the protein binds to one or more amino
acids of: Ser479, Tyr563, and/or Asp585 (numbering based on the
human sequence) and, e.g., does not bind pre-plasma kallikrein.
[0017] In some embodiments, the plasma kallikrein binding protein
binds one or more amino acids of: Arg551, Gln553, Tyr555, Thr558,
and/or Arg560 (numbering based on the human kallikrein sequence).
In other embodiments, the plasma kallikrein binding protein binds
two, three, four or five (i.e., all) amino acids of: Arg551,
Gln553, Tyr555, Thr558, and/or Arg560 (numbering based on the human
sequence) and, e.g., does not bind pre-plasma kallikrein.
[0018] In some embodiments, the plasma kallikrein binding protein
binds one or more amino acids of: S478, N481, S525, and K526
(numbering based on the human kallikrein sequence). In other
embodiments, the plasma kallikrein binding protein binds two, three
or four (i.e., all) amino acids of: S478, N481, S525, and K526
(numbering based on the human kallikrein sequence).
[0019] In some embodiments, the plasma kallikrein binding protein
decreases Factor XIIa and/or bradykinin production by greater than
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% as compared to a standard, e.g., the Factor XIIa
and/or bradykinin production under the same conditions but in the
absence of the protein.
[0020] In some embodiments, the plasma kallikrein binding protein
has an apparent inhibition constant (K.sub.i,app) of less than
1000, 500, 100, 10, 1, 0.5 or 0.2 nM.
[0021] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain.
[0022] In another embodiment, the HC and LC variable domain
sequences are components of different polypeptide chains. For
example, the plasma kallikrein binding protein is an IgG., e.g.,
IgG1, IgG2, IgG3, or IgG4. The plasma kallikrein binding protein
can be a soluble Fab (sFab).
[0023] In some embodiments, the plasma kallikrein binding protein
has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks or more, in vivo, e.g., in humans. In one embodiment, the
plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2,
IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
[0024] In some embodiments, the plasma kallikrein binding protein
is physically associated with a moiety that improves serum
residence time, e.g., a moiety described herein.
[0025] In other embodiments, the plasma kallikrein binding protein
includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion,
HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule that
comprises the antigen combining site of one of the binding proteins
herein. The VH and VL regions of these Fabs can be provided as IgG,
Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated scFv, PEGylated
Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1,
HSA::LC+VH::CH1, or other appropriate construction.
[0026] In one embodiment, the plasma kallikrein binding protein is
a human or humanized antibody or is non-immunogenic in a human. For
example, the protein includes one or more human antibody framework
regions, e.g., all human framework regions.
[0027] In one embodiment, the plasma kallikrein binding protein
includes a human Fc domain, or an Fc domain that is at least 95,
96, 97, 98, or 99% identical to a human Fc domain.
[0028] In one embodiment, the plasma kallikrein binding protein is
a primate or primatized antibody or is non-immunogenic in a human.
For example, the protein includes one or more primate antibody
framework regions, e.g., all primate framework regions.
[0029] In one embodiment, the plasma kallikrein binding protein
includes a primate Fc domain, or an Fc domain that is at least 95,
96, 97, 98, or 99% identical to a primate Fc domain. "Primate"
includes humans (Homo sapiens), chimpanzees (Pan troglodytes and
Pan paniscus (bonobos)), gorillas (Gorilla gorilla), gibons,
monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and
tarsiers.
[0030] In one embodiment, the plasma kallikrein binding protein
includes human framework regions, or framework regions that are at
least 95, 96, 97, 98, or 99% identical to human framework
regions.
[0031] In certain embodiments, the plasma kallikrein binding
protein includes no sequences from mice or rabbits (e.g., is not a
murine or rabbit antibody).
[0032] In certain embodiments, the plasma kallikrein binding
protein is capable of binding to a cell or tissue, e.g., that
expresses plasma kallikrein.
[0033] In one embodiment, the plasma kallikrein binding protein is
physically associated with a nanoparticle, and can be used to guide
a nanoparticle to a cell or tissue expressing plasma
kallikrein.
[0034] In some aspects, the disclosure features an isolated protein
(e.g., antibody, e.g., human antibody) that binds the same epitope
or competes for binding with a kallikrein binding protein described
herein.
[0035] In some embodiments, the protein binds the same epitope or
competes for binding with a protein (e.g., epi-Kal2) and/or a small
molecule (e.g., AEBSF) described herein.
[0036] In some embodiments, the isolated protein comprises a heavy
chain immunoglobulin variable domain sequence and a light chain
immunoglobulin variable domain sequence, wherein:
[0037] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein, and/or
the light chain immunoglobulin variable domain sequence comprises
one, two, or three (e.g., three) CDR regions from the light chain
variable domain of a protein described herein,
[0038] wherein the protein binds to plasma kallikrein.
[0039] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises one, two, or three (e.g., three) CDR
regions from the heavy chain variable domain of M162-A04, M160-G12,
M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,
X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,
X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0040] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0041] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or mouse plasma kallikrein).
[0042] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X81-B01
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X81-B01.
[0043] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X67-D03
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X67-D03.
[0044] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X63-G06
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X63-G06.
[0045] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from M162-A04
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from MJ162-A04.
[0046] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0047] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03,
X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04
(respectively).
[0048] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
X81-B01, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of X81-B01.
[0049] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
X67-D03, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of X67-D03.
[0050] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0051] In some embodiments, the protein comprises the heavy chain
of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,
X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04.
[0052] and/or the light chain of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0053] In some embodiments, the protein comprises the heavy chain
of X81-B01, and/or the light chain of X81-B01.
[0054] In some embodiments, the protein comprises the heavy chain
of X67-D03, and/or the light chain of X67-D03.
[0055] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or mouse
prekallikrein), but binds to the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or mouse plasma kallikrein).
[0056] In some embodiments, the plasma kallikrein binding protein
decreases Factor XIIa and/or bradykinin production by greater than
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% as compared to a standard, e.g., the Factor XIIa
and/or bradykinin production under the same conditions but in the
absence of the protein.
[0057] In some embodiments, the protein includes one or more of the
following characteristics: (a) a human CDR or human framework
region; (b) the HC immunoglobulin variable domain sequence
comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical
to a CDR of a HC variable domain described herein; (c) the LC
immunoglobulin variable domain sequence comprises one or more
(e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC
variable domain described herein; (d) the LC immunoglobulin
variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain
described herein (e.g., overall or in framework regions or CDRs);
(e) the HC immunoglobulin variable domain sequence is at least 85,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical
to a HC variable domain described herein (e.g., overall or in
framework regions or CDRs); (f) the protein binds an epitope bound
by a protein described herein, or competes for binding with a
protein described herein; (g) a primate CDR or primate framework
region; (h) the HC immunoglobulin variable domain sequence
comprises a CDR1 that differs by at least one amino acid but by no
more than 2 or 3 amino acids from the CDR1 of a HC variable domain
described herein; (i) the HC immunoglobulin variable domain
sequence comprises a CDR2 that differs by at least one amino acid
but by no more than 2, 3, 4, 5, 6, 7, or 8 amino acids from the
CDR2 of a HC variable domain described herein; (j) the HC
immunoglobulin variable domain sequence comprises a CDR3 that
differs by at least one amino acid but by no more than 2, 3, 4, 5,
or 6 amino acids from the CDR3 of a HC variable domain described
herein; (k) the LC immunoglobulin variable domain sequence
comprises a CDR1 that differs by at least one amino acid but by no
more than 2, 3, 4, or 5 amino acids from the CDR1 of a LC variable
domain described herein; (1) the LC immunoglobulin variable domain
sequence comprises a CDR2 that differs by at least one amino acid
but by no more than 2, 3, or 4 amino acids from the CDR2 of a LC
variable domain described herein; (m) the LC immunoglobulin
variable domain sequence comprises a CDR3 that differs by at least
one amino acid but by no more than 2, 3, 4, or 5 amino acids from
the CDR3 of a LC variable domain described herein; (n) the LC
immunoglobulin variable domain sequence differs by at least one
amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acids from a LC variable domain described herein (e.g., overall or
in framework regions or CDRs); and (o) the HC immunoglobulin
variable domain sequence differs by at least one amino acid but by
no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from a HC
variable domain described herein (e.g., overall or in framework
regions or CDRs).
[0058] In some embodiments, the protein has an apparent inhibition
constant (K.sub.i,app) of less than 1000, 500, 100, 10, 1, 0.5 or
0.2 nM.
[0059] In some embodiments, the antibody does not bind
prekallikrein (e.g., human prekallikrein and/or mouse
prekallikrein), but binds to the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or mouse plasma kallikrein).
[0060] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of antibodies
selected from the group consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0061] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0062] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0063] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from the group consisting of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0064] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0065] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0066] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0067] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs selected from the corresponding CDRs of the group of light
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0068] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0069] and one or more (e.g., 1, 2, or 3) light chain CDRs selected
from the corresponding CDRs of the group of light chains consisting
of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,
X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04 (respectively).
[0070] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of X81-B01.
[0071] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of X81-B01.
[0072] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of X81-B01.
[0073] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from X81-B01.
[0074] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
X81-B01.
[0075] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
X81-B01.
[0076] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the corresponding CDRs of the heavy chain of X81-B01.
[0077] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chain of X81-B01.
[0078] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the heavy chain of X81-B01 and one or more (e.g., 1, 2,
or 3) light chain CDRs from the corresponding CDRs of the light
chain of X81-B01.
[0079] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of X67-D03.
[0080] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of X67-D03.
[0081] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of X67-D03.
[0082] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from X67-D03.
[0083] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
X67-D03.
[0084] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
X67-D03.
[0085] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the corresponding CDRs of the heavy chain of X67-D03.
[0086] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chain of X67-D03.
[0087] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the heavy chain of X67-D03 and one or more (e.g., 1, 2,
or 3) light chain CDRs from the corresponding CDRs of the light
chain of X67-D03.
[0088] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain.
[0089] In some embodiments, the plasma kallikrein binding protein
has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks or more, in vivo, e.g., in humans. In one embodiment, the
plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2,
IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
[0090] In some embodiments, the plasma kallikrein binding protein
is physically associated with a moiety that improves serum
residence time, e.g., a moiety described herein.
[0091] In another embodiment, the HC and LC variable domain
sequences are components of different polypeptide chains. For
example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4.
The protein can be a soluble Fab (sFab).
[0092] In other embodiments, the protein includes a Fab2', scFv,
minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion,
Fab::HSA::Fab fusion, or other molecule that comprises the antigen
combining site of one of the binding proteins herein. The VH and VL
regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2',
scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2,
VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1,
or other appropriate construction.
[0093] In one embodiment, the protein is a human or humanized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more human antibody framework regions, e.g., all
human framework regions.
[0094] In one embodiment, the protein includes a human Fc domain,
or an Fc domain that is at least 95, 96, 97, 98, or 99% identical
to a human Fc domain.
[0095] In one embodiment, the protein is a primate or primatized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more primate antibody framework regions, e.g., all
primate framework regions.
[0096] In one embodiment, the protein includes a primate Fc domain,
or an Fc domain that is at least 95, 96, 97, 98, or 99% identical
to a primate Fc domain. "Primate" includes humans (Homo sapiens),
chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas
(Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia
madagascariensis), and tarsiers.
[0097] In one embodiment, the protein includes human framework
regions, or framework regions that are at least 95, 96, 97, 98, or
99% identical to human framework regions.
[0098] In certain embodiments, the protein includes no sequences
from mice or rabbits (e.g., is not a murine or rabbit
antibody).
[0099] In certain embodiments, the protein is capable of binding to
a cell or tissue, e.g., that expresses plasma kallikrein.
[0100] In one embodiment, protein is physically associated with a
nanoparticle, and can be used to guide a nanoparticle to a cell or
tissue expressing plasma kallikrein.
[0101] In some aspects, the disclosure features a pharmaceutical
composition comprising a kallikrein binding protein described
herein, e.g., including a pharmaceutically acceptable carrier. In
some embodiments, the composition can be at least 10, 20, 30, 50,
75, 85, 90, 95, 98, 99, or 99.9% free of other protein species. In
one embodiment, the pharmaceutical composition can be at least 10,
20, 30, 50, 75, 85, 90, 95, 98, 99, or 99.9% free of fragments of
the binding protein that do not binding plasma kallikrein (e.g.,
human plasma kallikrein) or bind plasma kallikrein (e.g., human
plasma kallikrein with a Ki, app of 5000 nM or greater.
[0102] In some aspects, the disclosure features a method of
treating or preventing a plasma kallikrein associated disorder in a
subject, the method comprising:
[0103] administering an isolated protein (e.g., antibody, e.g.,
human antibody) that binds plasma kallikrein (e.g., human plasma
kallikrein and/or mouse plasma kallikrein) and, e.g., does not bind
prekallikrein (e.g., human prekallikrein and/or mouse
prekallikrein) to the subject,
[0104] In some embodiments, the protein binds the same epitope or
competes for binding with a protein (e.g., epi-Kal2) and/or a small
molecule (e.g., AEBSF) described herein.
[0105] In some embodiments, the protein binds the same epitope or
competes for binding with a kallikrein binding protein described
herein.
[0106] In some embodiments, the plasma kallikrein associated
disorder is selected from the group consisting of rheumatoid
arthritis, gout, intestinal bowel disease, oral mucositis,
neuropathic pain, inflammatory pain, spinal stenosis-degenerative
spine disease, arterial or venous thrombosis, post operative ileus,
aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary
angioedema, cerebral edema, pulmonary embolism, stroke, clotting on
ventricular assistance devices or stents, head trauma or peri-tumor
brain edema, sepsis, acute middle cerebral artery (MCA) ischemic
event (stroke), restenosis (e.g., after angioplasty), systemic
lupus erythematosis nephritis, and burn injury. In some
embodiments, the plasma kallikrein binding protein reduces aberrant
clotting associated with the contact activation system (i.e.,
intrinsic activation system) by at least 10% as measured by e.g.,
an APTT clotting assay. In other embodiments, the plasma kallikrein
binding protein reduces aberrant clotting associated with the
contact activation system by at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 99%, or even 100% (i.e., no
detectable aberrant clotting).
[0107] In some embodiments, the plasma kallikrein binding protein
is administered in combination with another treatment for the
disorder.
[0108] In some embodiments, the protein described herein is
selected from the group consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0109] In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as X81-B01.
[0110] In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as X67-D03.
[0111] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or mouse
prekallikrein), but binds to the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or mouse plasma kallikrein).
[0112] In certain embodiments, the protein binds at or near the
active site of the catalytic domain of plasma kallikrein, or a
fragment thereof, or binds an epitope that overlaps with the active
site of plasma kallikrein.
[0113] In some embodiments, the protein binds to one or more amino
acids that form the catalytic triad of plasma kallikrein: His434,
Asp483, and/or Ser578 (numbering based on the human sequence).
[0114] In some embodiments, the protein binds to one or more amino
acids of Ser479, Tyr563, and/or Asp585 (numbering based on the
human sequence).
[0115] In other embodiments, the protein binds to one or more amino
acids of Arg551, Gln553, Tyr555, Thr558, and/or Arg560 (numbering
based on the human sequence). In some embodiments, the plasma
kallikrein binding protein binds one or more amino acids of: S478,
N481, S525, and K526 (numbering based on the human kallikrein
sequence).
[0116] In some embodiments, the plasma kallikrein binding protein
decreases Factor XIIa and/or bradykinin production by greater than
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% as compared to a standard, e.g., the Factor XIIa
and/or bradykinin production under the same conditions but in the
absence of the protein.
[0117] In some embodiments, the plasma kallikrein binding protein
has an apparent inhibition constant (K.sub.i,app) of less than
1000, 500, 100, 10, 5, 1, 0.5, or 0.2 nM.
[0118] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain.
[0119] In some embodiments, the plasma kallikrein binding protein
has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks or more, in vivo, e.g., in humans. In one embodiment, the
plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2,
IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
[0120] In some embodiments, the plasma kallikrein binding protein
is physically associated with a moiety that improves serum
residence time, e.g., a moiety described herein.
[0121] In another embodiment, the HC and LC variable domain
sequences are components of different polypeptide chains. For
example, the plasma kallikrein binding protein is an IgG, e.g.,
IgG1, IgG2, IgG3, or IgG4. The plasma kallikrein binding protein
can be a soluble Fab (sFab).
[0122] In other implementations the plasma kallikrein binding
protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA
fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule
that comprises the antigen combining site of one of the binding
proteins herein. The VH and VL regions of these Fabs can be
provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated
scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC,
LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other appropriate
construction.
[0123] In one embodiment, the plasma kallikrein binding protein is
a human or humanized antibody or is non-immunogenic in a human. For
example, the protein includes one or more human antibody framework
regions, e.g., all human framework regions.
[0124] In one embodiment, the plasma kallikrein binding protein
includes a human Fc domain, or an Fc domain that is at least 95,
96, 97, 98, or 99% identical to a human Fc domain.
[0125] In one embodiment, the plasma kallikrein binding protein is
a primate or primatized antibody or is non-immunogenic in a human.
For example, the protein includes one or more primate antibody
framework regions, e.g., all primate framework regions.
[0126] In one embodiment, the plasma kallikrein binding protein
includes a primate Fc domain, or an Fc domain that is at least 95,
96, 97, 98, or 99% identical to a primate Fc domain. "Primate"
includes humans (Homo sapiens), chimpanzees (Pan troglodytes and
Pan paniscus (bonobos)), gorillas (Gorilla gorilla), gibons,
monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and
tarsiers.
[0127] In one embodiment, the plasma kallikrein binding protein
includes human framework regions, or framework regions that are at
least 95, 96, 97, 98, or 99% identical to human framework
regions.
[0128] In certain embodiments, the plasma kallikrein binding
protein includes no sequences from mice or rabbits (e.g., is not a
murine or rabbit antibody).
[0129] In certain embodiments, the plasma kallikrein binding
protein is capable of binding to a cell or tissue, e.g., that
expresses plasma kallikrein.
[0130] In one embodiment, the plasma kallikrein binding protein is
physically associated with a nanoparticle, and can be used to guide
a nanoparticle to a cell or tissue expressing plasma
kallikrein.
[0131] A method of treating or preventing a plasma kallikrein
associated disorder in a subject, the method comprising:
[0132] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0133] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0134] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0135] wherein the protein binds to plasma kallikrein (e.g., human
plasma kallikrein and/or mouse plasma kallikrein).
[0136] In some embodiments, the plasma kallikrein associated
disorder is selected from the group consisting of rheumatoid
arthritis, gout, intestinal bowel disease, oral mucositis,
neuropathic pain, inflammatory pain, spinal stenosis-degenerative
spine disease, arterial or venous thrombosis, post operative ileus,
aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary
angioedema, cerebral edema, pulmonary embolism, stroke, clotting of
ventrical assistance devices or stents, head trauma or peri-tumor
brain edema, sepsis, acute middle cerebral artery (MCA) ischemic
event (stroke), restenosis (e.g., after angioplasty), systemic
lupus erythematosis nephritis, and burn injury. In some
embodiments, the plasma kallikrein binding protein reduces aberrant
clotting associated with the contact activation system (i.e.,
intrinsic activation system) by at least 10% as measured by e.g.,
an APTT clotting assay (e.g., by at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, at least 99%, or even 100% (i.e., no
detectable aberrant clotting)).
[0137] In some embodiments, the protein is administered in
combination with another treatment for the disorder.
[0138] In some embodiments, the protein is administered in
combination with a second agent selected from the group consisting
of ecallantide, a C1 esterase inhibitor, aprotinin, a bradykinin B2
receptor inhibitor (e.g., icatibant).
[0139] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises one, two, or three (e.g., three) CDR
regions from the heavy chain variable domain of M162-A04, M160-G12,
M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,
X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,
X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or
[0140] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0141] In some embodiments, the protein inhibits plasma
kallikrein.
[0142] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X81-B01
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X81-B01.
[0143] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X67-D03
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X67-D03.
[0144] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0145] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
M162-A04, M160-G12, M142-H08 X63-G06, X101-A01, X81-B01, X67-D03,
X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0146] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
X81-B01, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of X81-B01.
[0147] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
X67-D03, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of X67-D03.
[0148] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0149] In some embodiments, the protein comprises the heavy chain
of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,
X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04, and/or the light chain of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0150] In some embodiments, the protein comprises the heavy chain
of X81-B01, and/or the light chain of X81-B01.
[0151] In some embodiments, the protein comprises the heavy chain
of X67-D03, and/or the light chain of X67-D03.
[0152] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0153] In some embodiments, the plasma kallikrein binding protein
decreases Factor XIIa and/or bradykinin production by greater than
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% as compared to a standard, e.g., the Factor XIIa
and/or bradykinin production under the same conditions but in the
absence of the protein.
[0154] In some embodiments, the protein includes one or more of the
following characteristics: (a) a human CDR or human framework
region; (b) the HC immunoglobulin variable domain sequence
comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical
to a CDR of a HC variable domain described herein; (c) the LC
immunoglobulin variable domain sequence comprises one or more
(e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC
variable domain described herein; (d) the LC immunoglobulin
variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain
described herein (e.g., overall or in framework regions or CDRs);
(e) the HC immunoglobulin variable domain sequence is at least 85,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical
to a HC variable domain described herein (e.g., overall or in
framework regions or CDRs); (f) the protein binds an epitope bound
by a protein described herein, or competes for binding with a
protein described herein; and (g) a primate CDR or primate
framework region.
[0155] In some embodiments, the protein has an apparent inhibition
constant (K.sub.i,app) of less than 1000, 500, 100, 10, 5, 1, 0.5
or 0.2 nM.
[0156] In some embodiments, the antibody does not bind
prekallikrein (e.g., human prekallikrein), but binds to the active
form of plasma kallikrein (e.g., human plasma kallikrein).
[0157] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of antibodies
selected from the group consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0158] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0159] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0160] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from the group consisting of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0161] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0162] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0163] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0164] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs selected from the corresponding CDRs of the group of light
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0165] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04 and one or more (e.g., 1, 2, or 3)
light chain CDRs selected from the corresponding CDRs of the group
of light chains consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0166] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of X81-B01.
[0167] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of X81-B01.
[0168] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of X81-B01.
[0169] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from X81-B01.
[0170] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
X81-B01.
[0171] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
X81-B01.
[0172] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the corresponding CDRs of the heavy chain of X81-B01.
[0173] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chain of X81-B01.
[0174] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the heavy chain of X81-B01 and one or more (e.g., 1, 2,
or 3) light chain CDRs from the corresponding CDRs of the light
chain of X81-B01.
[0175] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of X67-D03.
[0176] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of X67-D03.
[0177] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of X67-D03.
[0178] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from X67-D03.
[0179] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
X67-D03.
[0180] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
X67-D03.
[0181] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the corresponding CDRs of the heavy chain of X67-D03.
[0182] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chain of X67-D03.
[0183] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the heavy chain of X67-D03 and one or more (e.g., 1, 2,
or 3) light chain CDRs from the corresponding CDRs of the light
chain of X67-D03.
[0184] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain.
[0185] In some embodiments, the plasma kallikrein binding protein
has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks or more, in vivo, e.g., in humans. In one embodiment, the
plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2,
IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
[0186] In some embodiments, the plasma kallikrein binding protein
is physically associated with a moiety that improves serum
residence time, e.g., a moiety described herein.
[0187] In another embodiment, the HC and LC variable domain
sequences are components of different polypeptide chains. For
example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4.
The protein can be a soluble Fab (sFab).
[0188] In other implementations the protein includes a Fab2', scFv,
minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion,
Fab::HSA::Fab fusion, or other molecule that comprises the antigen
combining site of one of the binding proteins herein. The VH and VL
regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2',
scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2,
VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1,
or other appropriate construction.
[0189] In one embodiment, the protein is a human or humanized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more human antibody framework regions, e.g., all
human framework regions.
[0190] In one embodiment, the protein includes a human Fc domain,
or an Fc domain that is at least 95, 96, 97, 98, or 99% identical
to a human Fc domain.
[0191] In one embodiment, the protein is a primate or primatized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more primate antibody framework regions, e.g., all
primate framework regions.
[0192] In one embodiment, the protein includes a primate Fc domain,
or an Fc domain that is at least 95, 96, 97, 98, or 99% identical
to a primate Fc domain. "Primate" includes humans (Homo sapiens),
chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas
(Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia
madagascariensis), and tarsiers.
[0193] In one embodiment, the protein includes human framework
regions, or framework regions that are at least 95, 96, 97, 98, or
99% identical to human framework regions.
[0194] In certain embodiments, the protein includes no sequences
from mice or rabbits (e.g., is not a murine or rabbit
antibody).
[0195] In certain embodiments, the protein is capable of binding to
a cell or tissue, e.g., that expresses plasma kallikrein.
[0196] In one embodiment, protein is physically associated with a
nanoparticle, and can be used to guide a nanoparticle to a cell or
tissue expressing plasma kallikrein.
[0197] In some aspects, the disclosure features a method of
promoting wound healing in a subject, the method comprising:
[0198] administering an isolated protein (e.g., antibody, e.g.,
human antibody) that binds plasma kallikrein (e.g., human plasma
kallikrein and/or mouse plasma kallikrein) and, e.g., does not bind
prekallikrein (e.g., human prekallikrein and/or mouse
prekallikrein) to the subject.
[0199] In some embodiments, the protein binds the same epitope or
competes for binding with a kallikrein binding protein described
herein. In some embodiments, the protein binds the same epitope or
competes for binding with a protein (e.g., epi-Kal2) and/or a small
molecule (e.g., AEBSF) described herein.
[0200] In some embodiments, the plasma kallikrein binding protein
is administered in combination with another treatment for wound
healing.
[0201] In some embodiments, the protein described herein is
selected from the group consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0202] In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as X81-B01.
[0203] In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as X67-D03.
[0204] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein), but binds
to the active form of plasma kallikrein (e.g., human plasma
kallikrein).
[0205] In certain embodiments, the protein binds at or near the
active site of the catalytic domain of plasma kallikrein, or a
fragment thereof, or binds an epitope that overlaps with the active
site of plasma kallikrein.
[0206] In some embodiments, the protein binds to one or more amino
acids that form the catalytic triad of plasma kallikrein: His434,
Asp483, and/or Ser578 (numbering based on the human sequence). In
other embodiments, the protein binds to one or more amino acids
that form a region for substrate recognition: Arg551, Gln553,
Tyr555, Thr558, and/or Arg560 (numbering based on the human
sequence). In some embodiments, the plasma kallikrein binding
protein binds one or more amino acids of: S478, N481, S525, and
K526 (numbering based on the human kallikrein sequence).
[0207] In some embodiments, the protein binds to one or more amino
acids of Ser479, Tyr563, and/or Asp585 (numbering based on the
human sequence).
[0208] In some embodiments, the plasma kallikrein binding protein
decreases Factor XIIa and/or bradykinin production by greater than
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% as compared to a standard, e.g., the Factor XIIa
and/or bradykinin production under the same conditions but in the
absence of the protein.
[0209] In some embodiments, the plasma kallikrein binding protein
has an apparent inhibition constant (K.sub.i,app) of less than
1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.
[0210] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain.
[0211] In some embodiments, the plasma kallikrein binding protein
has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks or more, in vivo, e.g., in humans. In one embodiment, the
plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2,
IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
[0212] In some embodiments, the plasma kallikrein binding protein
is physically associated with a moiety that improves serum
residence time, e.g., a moiety described herein.
[0213] In another embodiment, the HC and LC variable domain
sequences are components of different polypeptide chains. For
example, the plasma kallikrein binding protein is an IgG, e.g.,
IgG1, IgG2, IgG3, or IgG4. The plasma kallikrein binding protein
can be a soluble Fab (sFab).
[0214] In other implementations the plasma kallikrein binding
protein includes a Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA
fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule
that comprises the antigen combining site of one of the binding
proteins herein. The VH and VL regions of these Fabs can be
provided as IgG, Fab, Fab2, Fab2', scFv, PEGylated Fab, PEGylated
scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC,
LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other appropriate
construction.
[0215] In one embodiment, the plasma kallikrein binding protein is
a human or humanized antibody or is non-immunogenic in a human. For
example, the protein includes one or more human antibody framework
regions, e.g., all human framework regions.
[0216] In one embodiment, the plasma kallikrein binding protein
includes a human Fc domain, or an Fc domain that is at least 95,
96, 97, 98, or 99% identical to a human Fc domain.
[0217] In one embodiment, the plasma kallikrein binding protein is
a primate or primatized antibody or is non-immunogenic in a human.
For example, the protein includes one or more primate antibody
framework regions, e.g., all primate framework regions.
[0218] In one embodiment, the plasma kallikrein binding protein
includes a primate Fc domain, or an Fc domain that is at least 95,
96, 97, 98, or 99% identical to a primate Fc domain. "Primate"
includes humans (Homo sapiens), chimpanzees (Pan troglodytes and
Pan paniscus (bonobos)), gorillas (Gorilla gorilla), gibons,
monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), and
tarsiers.
[0219] In one embodiment, the plasma kallikrein binding protein
includes human framework regions, or framework regions that are at
least 95, 96, 97, 98, or 99% identical to human framework
regions.
[0220] In certain embodiments, the plasma kallikrein binding
protein includes no sequences from mice or rabbits (e.g., is not a
murine or rabbit antibody).
[0221] In certain embodiments, the protein is capable of binding to
a cell or tissue, e.g., that expresses plasma kallikrein.
[0222] In one embodiment, the plasma kallikrein binding protein is
physically associated with a nanoparticle, and can be used to guide
a nanoparticle to a cell or tissue expressing plasma
kallikrein.
[0223] In some aspects, the disclosure features a method promoting
wound healing in a subject, the method comprising:
[0224] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0225] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0226] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein, wherein
the protein binds to plasma kallikrein.
[0227] In some embodiments, the protein is administered in
combination with another treatment for wound healing.
[0228] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises one, two, or three (e.g., three) CDR
regions from the heavy chain variable domain of M162-A04, M160-G12,
M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,
X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,
X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or
[0229] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0230] In some embodiments, the protein inhibits plasma
kallikrein.
[0231] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X81-B01
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X81-B01.
[0232] In some embodiments, the one, two, or three (e.g., three)
CDR regions from the heavy chain variable domain are from X67-D03
and/or the one, two, or three (e.g., three) CDR regions from the
light chain variable domain are from X67-D03.
[0233] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0234] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03,
X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04
(respectively).
[0235] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
X81-B01, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of X81-B01.
[0236] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of
X67-D03, and/or the light chain immunoglobulin variable domain
sequence comprises the light chain variable domain of X67-D03.
[0237] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0238] In some embodiments, the protein comprises the heavy chain
of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,
X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04, and/or the light chain of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0239] In some embodiments, the protein comprises the heavy chain
of X81-B01, and/or the light chain of X81-B01.
[0240] In some embodiments, the protein comprises the heavy chain
of X67-D03, and/or the light chain of X67-D03.
[0241] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0242] In some embodiments, the plasma kallikrein binding protein
decreases Factor XIIa and/or bradykinin production by greater than
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% as compared to a standard, e.g., the Factor XIIa
and/or bradykinin production under the same conditions but in the
absence of the protein.
[0243] In some embodiments, the protein includes one or more of the
following characteristics: (a) a human CDR or human framework
region; (b) the HC immunoglobulin variable domain sequence
comprises one or more (e.g., 1, 2, or 3) CDRs that are at least 85,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical
to a CDR of a HC variable domain described herein; (c) the LC
immunoglobulin variable domain sequence comprises one or more
(e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC
variable domain described herein; (d) the LC immunoglobulin
variable domain sequence is at least 85, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100% identical to a LC variable domain
described herein (e.g., overall or in framework regions or CDRs);
(e) the HC immunoglobulin variable domain sequence is at least 85,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical
to a HC variable domain described herein (e.g., overall or in
framework regions or CDRs); (f) the protein binds an epitope bound
by a protein described herein, or competes for binding with a
protein described herein; and (g) a primate CDR or primate
framework region.
[0244] In some embodiments, the protein has an apparent inhibition
constant (K.sub.i,app) of less than 1000, 500, 100, 5, 1, 0.5 or
0.2 nM.
[0245] In some embodiments, the antibody does not bind
prekallikrein (e.g., human prekallikrein and/or murine
prekallikrein), but binds to the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0246] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of antibodies
selected from the group consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0247] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0248] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0249] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from the group consisting of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0250] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0251] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0252] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0253] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs selected from the corresponding CDRs of the group of light
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0254] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04 and one or more (e.g., 1, 2, or 3)
light chain CDRs selected from the corresponding CDRs of the group
of light chains consisting of M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).
[0255] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of X81-B01.
[0256] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of X81-B01.
[0257] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of X81-B01.
[0258] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from X81-B01.
[0259] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
X81-B01.
[0260] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
X81-B01.
[0261] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the corresponding CDRs of the heavy chain of X81-B01.
[0262] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chain of X81-B01.
[0263] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the heavy chain of X81-B01 and one or more (e.g., 1, 2,
or 3) light chain CDRs from the corresponding CDRs of the light
chain of X81-B01.
[0264] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of X67-D03.
[0265] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of X67-D03.
[0266] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of X67-D03.
[0267] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from X67-D03.
[0268] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
X67-D03.
[0269] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
X67-D03.
[0270] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the corresponding CDRs of the heavy chain of X67-D03.
[0271] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs from the corresponding CDRs of the light chain of X67-D03.
[0272] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs from the heavy chain of X67-D03 and one or more (e.g., 1, 2,
or 3) light chain CDRs from the corresponding CDRs of the light
chain of X67-D03.
[0273] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain.
[0274] In some embodiments, the plasma kallikrein binding protein
has a serum residence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks or more, in vivo, e.g., in humans. In one embodiment, the
plasma kallikrein binding protein is an IgG, e.g., an IgG1, IgG2,
IgG3 or IgG4, that has a serum residence time of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks or more in vivo, e.g., in humans.
[0275] In some embodiments, the plasma kallikrein binding protein
is physically associated with a moiety that improves serum
residence time, e.g., a moiety described herein.
[0276] In another embodiment, the HC and LC variable domain
sequences are components of different polypeptide chains. For
example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4.
The protein can be a soluble Fab (sFab).
[0277] In other implementations the protein includes a Fab2', scFv,
minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion,
Fab::HSA::Fab fusion, or other molecule that comprises the antigen
combining site of one of the binding proteins herein. The VH and VL
regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2',
scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2,
VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1,
or other appropriate construction.
[0278] In one embodiment, the protein is a human or humanized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more human antibody framework regions, e.g., all
human framework regions.
[0279] In one embodiment, the protein includes a human Fc domain,
or an Fc domain that is at least 95, 96, 97, 98, or 99% identical
to a human Fc domain.
[0280] In one embodiment, the protein is a primate or primatized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more primate antibody framework regions, e.g., all
primate framework regions.
[0281] In one embodiment, the protein includes a primate Fc domain,
or an Fc domain that is at least 95, 96, 97, 98, or 99% identical
to a primate Fc domain. "Primate" includes humans (Homo sapiens),
chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas
(Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes (Daubentonia
madagascariensis), and tarsiers.
[0282] In one embodiment, the protein includes human framework
regions, or framework regions that are at least 95, 96, 97, 98, or
99% identical to human framework regions.
[0283] In certain embodiments, the protein includes no sequences
from mice or rabbits (e.g., is not a murine or rabbit
antibody).
[0284] In certain embodiments, the protein is capable of binding to
a cell or tissue, e.g., that expresses plasma kallikrein.
[0285] In one embodiment, protein is physically associated with a
nanoparticle, and can be used to guide a nanoparticle to a cell or
tissue expressing plasma kallikrein.
[0286] In some aspects, the disclosure features a method of
treating or preventing rheumatoid arthritis in a subject, the
method comprising:
[0287] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0288] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0289] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0290] wherein the protein binds to plasma kallikrein.
[0291] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0292] In some embodiments, the protein is administered in
combination with another treatment for rheumatoid arthritis.
[0293] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0294] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0295] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0296] In some aspects, the disclosure features a method of
treating or preventing gout in a subject, the method
comprising:
[0297] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0298] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0299] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein, wherein
the protein binds to plasma kallikrein.
[0300] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0301] In some embodiments, the protein is administered in
combination with another treatment for gout.
[0302] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0303] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0304] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0305] In some aspects, the disclosure features a method of
treating or preventing intestinal bowel disease in a subject, the
method comprising:
[0306] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0307] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0308] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0309] wherein the protein binds to plasma kallikrein.
[0310] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0311] In some embodiments, the protein is administered in
combination with another treatment for intestinal bowel
disease.
[0312] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0313] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0314] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0315] In some aspects, the disclosure features a method of
treating or preventing oral mucositis in a subject, the method
comprising:
[0316] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0317] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0318] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0319] wherein the protein binds to plasma kallikrein.
[0320] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0321] In some embodiments, the protein is administered in
combination with another treatment for oral mucositis.
[0322] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0323] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0324] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0325] In some aspects, the disclosure features a method of
treating or preventing neuropathic pain in a subject, the method
comprising:
[0326] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0327] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0328] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein, wherein
the protein binds to plasma kallikrein.
[0329] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0330] In some embodiments, the protein is administered in
combination with another treatment for neuropathic pain.
[0331] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0332] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0333] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0334] In some aspects, the disclosure features a method of
treating or preventing inflammatory pain in a subject, the method
comprising:
[0335] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0336] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0337] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0338] wherein the protein binds to plasma kallikrein.
[0339] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0340] In some embodiments, the protein is administered in
combination with another treatment for inflammatory pain.
[0341] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0342] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0343] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0344] In some aspects, the disclosure features a method of
treating or preventing spinal stenosis-degenerative spine disease
in a subject, the method comprising:
[0345] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0346] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0347] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0348] wherein the protein binds to plasma kallikrein.
[0349] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0350] In some embodiments, the protein is administered in
combination with another treatment for spinal stenosis-degenerative
spine disease.
[0351] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0352] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0353] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0354] In some aspects, the disclosure features a method of
treating or preventing arterial or venous thrombosis in a subject,
the method comprising:
[0355] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0356] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0357] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0358] wherein the protein binds to plasma kallikrein.
[0359] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0360] In some embodiments, the protein is administered in
combination with another treatment for arterial or venous
thrombosis.
[0361] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0362] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0363] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0364] In some aspects, the disclosure features a method of
treating or preventing post operative ileus in a subject, the
method comprising:
[0365] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0366] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0367] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0368] wherein the protein binds to plasma kallikrein.
[0369] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0370] In some embodiments, the protein is administered in
combination with another treatment for post operative ileus.
[0371] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0372] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0373] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0374] In some aspects, the disclosure features a method of
treating or preventing aortic aneurysm in a subject, the method
comprising:
[0375] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0376] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0377] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0378] wherein the protein binds to plasma kallikrein.
[0379] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0380] In some embodiments, the protein is administered in
combination with another treatment for aortic aneurysm.
[0381] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0382] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0383] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0384] In some aspects, the disclosure features a method of
treating or preventing osteoarthritis in a subject, the method
comprising:
[0385] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0386] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0387] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0388] wherein the protein binds to plasma kallikrein.
[0389] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0390] In some embodiments, the protein is administered in
combination with another treatment for osteoarthritis.
[0391] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0392] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0393] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0394] In some aspects, the disclosure features a method of
treating or preventing vasculitis in a subject, the method
comprising:
[0395] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0396] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0397] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0398] wherein the protein binds to plasma kallikrein.
[0399] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0400] In some embodiments, the protein is administered in
combination with another treatment for vasculitis.
[0401] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0402] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0403] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0404] In some aspects, the disclosure features a method of
treating or preventing head trauma or peri-tumor brain edema in a
subject, the method comprising:
[0405] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0406] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0407] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0408] wherein the protein binds to plasma kallikrein.
[0409] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0410] In some embodiments, the protein is administered in
combination with another treatment for head trauma or peri-tumor
brain edema.
[0411] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0412] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0413] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0414] In some aspects, the disclosure features a method of
treating or preventing sepsis in a subject, the method
comprising:
[0415] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0416] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0417] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0418] wherein the protein binds to plasma kallikrein.
[0419] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0420] In some embodiments, the protein is administered in
combination with another treatment for sepsis.
[0421] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0422] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0423] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0424] In some aspects, the disclosure features a method of
treating or preventing acute middle cerebral artery (MCA) ischemic
event (stroke) in a subject, the method comprising:
[0425] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0426] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0427] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0428] wherein the protein binds to plasma kallikrein.
[0429] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0430] In some embodiments, the protein is administered in
combination with another treatment for acute middle cerebral artery
(MCA) ischemic event (stroke).
[0431] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0432] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0433] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0434] In some aspects, the disclosure features a method of
treating or preventing restenosis (e.g., after angioplasty) in a
subject, the method comprising:
[0435] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0436] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0437] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0438] wherein the protein binds to plasma kallikrein.
[0439] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0440] In some embodiments, the protein is administered in
combination with another treatment for restenosis (e.g., after
angioplasty).
[0441] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0442] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0443] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0444] In some aspects, the disclosure features a method of
treating or preventing systemic lupus erythematosis nephritis in a
subject, the method comprising:
[0445] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0446] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0447] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0448] wherein the protein binds to plasma kallikrein.
[0449] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0450] In some embodiments, the protein is administered in
combination with another treatment for systemic lupus erythematosis
nephritis.
[0451] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0452] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0453] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0454] In some aspects, the disclosure features a method of
treating or preventing burn injury in a subject, the method
comprising:
[0455] administering an isolated protein (e.g., antibody, e.g.,
human antibody) comprising a heavy chain immunoglobulin variable
domain sequence and a light chain immunoglobulin variable domain
sequence to the subject, wherein:
[0456] the heavy chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
heavy chain variable domain of a protein described herein,
and/or
[0457] the light chain immunoglobulin variable domain sequence
comprises one, two, or three (e.g., three) CDR regions from the
light chain variable domain of a protein described herein,
[0458] wherein the protein binds to plasma kallikrein.
[0459] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0460] In some embodiments, the protein is administered in
combination with another treatment for burn injury.
[0461] In some embodiments, the protein inhibits plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0462] In some embodiments, the heavy chain immunoglobulin variable
domain sequence comprises the heavy chain variable domain of a
protein described herein, and/or the light chain immunoglobulin
variable domain sequence comprises the light chain variable domain
of a protein described herein.
[0463] In some embodiments, the protein comprises the heavy chain
of a protein described herein, and/or the light chain of a protein
described herein.
[0464] In some aspects, the disclosure features a method of
detecting plasma kallikrein in a sample, the method comprising:
contacting the sample with a plasma kallikrein binding protein
(e.g., a plasma kallikrein binding protein described herein); and
detecting an interaction between the protein and the plasma
kallikrein, if present.
[0465] In some embodiments, the protein includes a detectable
label.
[0466] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein). In some embodiments, the plasma kallikrein binding
protein binds prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein) and the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0467] In some aspects, the disclosure features a method of
detecting plasma kallikrein in a subject, the method comprising:
administering a plasma kallikrein binding protein (e.g., a plasma
kallikrein binding protein described herein) to a subject; and
detecting an interaction between the protein and the plasma
kallikrein in the subject, if present. For example, the detecting
comprises imaging the subject.
[0468] In some embodiments, the protein further includes a
detectable label.
[0469] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein). In some embodiments, the plasma kallikrein binding
protein binds prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein) and the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0470] In some aspects, the disclosure features a method of
modulating plasma kallikrein activity, e.g., in a method of
treating or preventing a plasma kallikrein associated disorder. The
method includes: contacting plasma kallikrein with a plasma
kallikrein binding protein (e.g., a plasma kallikrein binding
protein described herein) (e.g., in a human subject), thereby
modulating plasma kallikrein activity.
[0471] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0472] In some embodiments, the plasma kallikrein associated
disorder is selected from the group consisting of rheumatoid
arthritis, gout, intestinal bowel disease, oral mucositis,
neuropathic pain, inflammatory pain, spinal stenosis-degenerative
spine disease, arterial or venous thrombosis, post operative ileus,
aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary
angioedema, cerebral edema, pulmonary embolism, stroke, clotting
induced by ventricular assistance devices or stents, head trauma or
peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA)
ischemic event (stroke), restenosis (e.g., after angioplasty),
systemic lupus erythematosis nephritis/vasculitis, and burn
injury.
[0473] In some embodiments, the plasma kallikrein binding protein
reduces aberrant clotting associated with the contact activation
system (i.e., intrinsic activation system) by at least 10% as
measured by e.g., an APTT clotting assay. In other embodiments, the
plasma kallikrein binding protein reduces aberrant clotting
associated with the contact activation system by at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or even
100% (i.e., no detectable aberrant clotting).
[0474] In some aspects, the disclosure features a method of
treating a plasma kallikrein associated disorder, the method
comprising administering, to a subject, a plasma kallikrein binding
protein (e.g., a plasma kallikrein binding protein described
herein) in an amount sufficient to treat a plasma kallikrein
associated disorder in the subject. The method can further include
providing to the subject a second therapy that is therapy for the
plasma kallikrein associated disorder, e.g., as described
herein.
[0475] In some embodiments, the plasma kallikrein associated
disorder is selected from the group consisting of rheumatoid
arthritis, gout, intestinal bowel disease, oral mucositis,
neuropathic pain, inflammatory pain, spinal stenosis-degenerative
spine disease, arterial or venous thrombosis, post operative ileus,
aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary
angioedema, cerebral edema, pulmonary embolism, stroke, clotting
induced by ventricular assistance devices or stents, head trauma or
peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA)
ischemic event (stroke), restenosis (e.g., after angioplasty),
systemic lupus erythematosis nephritis/vasculitis, and burn
injury.
[0476] In some aspects, the disclosure features a method of imaging
a subject. The method includes administering a plasma kallikrein
binding protein (e.g., a plasma kallikrein binding protein
described herein) to the subject, and e.g., detecting an
interaction between the protein and the plasma kallikrein in the
subject, if present.
[0477] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein). In some embodiments, the plasma kallikrein binding
protein binds prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein) and the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0478] In some embodiments, the protein does not inhibit plasma
kallikrein activity.
[0479] In some embodiments, the protein inhibits plasma kallikrein
activity (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0480] In some embodiments, the plasma kallikrein binding protein
may include a detectable label (e.g., a radionuclide or an
MRI-detectable label).
[0481] In some embodiments, the subject has or is suspected of
having a plasma kallikrein associated disorder. The method is
useful, e.g., for diagnosis of a plasma kallikrein associated
disorder.
[0482] In some embodiments, the plasma kallikrein associated
disorder is selected from the group consisting of rheumatoid
arthritis, gout, intestinal bowel disease, oral mucositis,
neuropathic pain, inflammatory pain, spinal stenosis-degenerative
spine disease, arterial or venous thrombosis, post operative ileus,
aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary
angioedema, cerebral edema, pulmonary embolism, stroke, clotting
induced by ventricular assistance devices or stents, head trauma or
peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA)
ischemic event (stroke), restenosis (e.g., after angioplasty),
systemic lupus erythematosis nephritis, and burn injury.
[0483] In some embodiments, the plasma kallikrein binding protein
reduces aberrant clotting associated with the contact activation
system (i.e., intrinsic activation system) by at least 10% as
measured by e.g., an APTT clotting assay (e.g., by at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or even
100% (i.e., no detectable aberrant clotting)).
[0484] In some aspects, the disclosure features a method of imaging
plasma kallikrein, e.g., in a subject or sample (e.g., biopsy
sample). The method includes administering a plasma kallikrein
binding protein (e.g., a plasma kallikrein binding protein
described herein), e.g., to the subject or the sample, and
detecting an interaction between the protein and the plasma
kallikrein, if present.
[0485] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein). In some embodiments, the plasma kallikrein binding
protein binds prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein) and the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0486] In some embodiments, the protein does not inhibit plasma
kallikrein activity.
[0487] In some embodiments, the protein inhibits plasma kallikrein
activity (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0488] In some embodiments, the plasma kallikrein binding protein
may include a detectable label (e.g., a radionuclide or an
MRI-detectable label).
[0489] In some embodiments, the subject has or is suspected of
having a plasma kallikrein associated disorder. The method is
useful, e.g., for diagnosis of a plasma kallikrein associated
disorder.
[0490] In some embodiments, the plasma kallikrein associated
disorder is selected from the group consisting of rheumatoid
arthritis, gout, intestinal bowel disease, oral mucositis,
neuropathic pain, inflammatory pain, spinal stenosis-degenerative
spine disease, arterial or venous thrombosis, post operative ileus,
aortic aneurysm, osteoarthritis, vasculitis, edema, hereditary
angioedema, cerebral edema, pulmonary embolism, stroke, clotting
induced by ventricular assistance devices or stents, head trauma or
peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA)
ischemic event (stroke), restenosis (e.g., after angioplasty),
systemic lupus erythematosis nephritis, and burn injury.
[0491] In some embodiments, the plasma kallikrein binding protein
reduces aberrant clotting associated with the contact activation
system (i.e., intrinsic activation system) by at least 10% as
measured by e.g., an APTT clotting assay (e.g., by at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or even
100% (i.e., no detectable aberrant clotting)).
[0492] In one aspect, the disclosure features the use of a plasma
kallikrein binding protein described herein for the treatment of a
disorder described herein, e.g., rheumatoid arthritis, gout,
intestinal bowel disease, oral mucositis, neuropathic pain,
inflammatory pain, spinal stenosis-degenerative spine disease,
arterial or venous thrombosis, post operative ileus, aortic
aneurysm, osteoarthritis, vasculitis, edema, hereditary angioedema,
cerebral edema, pulmonary embolism, stroke, clotting induced by
ventricular assistance devices or stents, head trauma or peri-tumor
brain edema, sepsis, acute middle cerebral artery (MCA) ischemic
event (stroke), restenosis (e.g., after angioplasty), systemic
lupus erythematosis nephritis, or burn injury; or to promote wound
healing.
[0493] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein). In some embodiments, the plasma kallikrein binding
protein binds prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein) and the active form of plasma kallikrein
(e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0494] In one aspect, the disclosure features the use of a plasma
kallikrein binding protein described herein for the manufacture of
a medicament for the treatment of a disorder described herein,
e.g., rheumatoid arthritis, gout, intestinal bowel disease, oral
mucositis, neuropathic pain, inflammatory pain, spinal
stenosis-degenerative spine disease, arterial or venous thrombosis,
post operative ileus, aortic aneurysm, osteoarthritis, vasculitis,
edema, hereditary angioedema, cerebral edema, pulmonary embolism,
stroke, clotting induced by ventricular assistance devices or
stents, head trauma or peri-tumor brain edema, sepsis, acute middle
cerebral artery (MCA) ischemic event (stroke), restenosis (e.g.,
after angioplasty), systemic lupus erythematosis nephritis, or burn
injury; or for the manufacture of a medicament for wound
healing.
[0495] In some embodiments, the plasma kallikrein binding protein
reduces aberrant clotting associated with the contact activation
system (i.e., intrinsic activation system) by at least 10% as
measured by e.g., an APTT clotting assay (e.g., by at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or even
100% (i.e., no detectable aberrant clotting)).
[0496] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein).
[0497] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0498] The contents of all cited references including literature
references, issued patents, published or non-published patent
applications cited throughout this application as well as those
listed below are hereby expressly incorporated by reference in
their entireties. In case of conflict, the present application,
including any definitions herein, will control.
BRIEF DESCRIPTION OF DRAWINGS
[0499] FIG. 1 is a schematic representation of the role of plasma
kallikrein (pKal) in intrinsic coagulation pathway and
inflammation.
[0500] FIG. 2 depicts the effect of M162-A04 on carrageenan-induced
rat paw edema. Paw swelling was measured by water displacement.
[0501] FIG. 3 depicts the effect of M162-A04 on carrageenan-induced
thermal hyperalgesia. Pain latency was measured by the Hargreaves
method after carrageenan injection.
[0502] FIG. 4 depicts the alignment of the light chain DNA sequence
of nongermlined (X63-G06) (SEQ ID NO:1991) and germlined, codon
optimized (X81-B01) (SEQ ID NO:1992) versions of the same antibody
discovered using ROLIC affinity maturation. Positions indicated
with an asterisk (*) are conserved, whereas blank spaces correspond
to bases changed in X81-B01 due to either codon optimization or
germlining.
[0503] FIG. 5 depicts the alignment of the light chain amino acid
sequence of nongermlined (X63-G06) (SEQ ID NO:1993) and germlined,
codon optimized (X81-B01) (SEQ ID NO:1994) versions of the same
antibody discovered using ROLIC affinity maturation. Positions
indicated with an asterisk (*) are conserved, whereas blank spaces
correspond to amino acids changed in X81-B01 due to germlining. A
total of 11 amino acids differ between the nongermlined (X63-G06)
and germlined, codon optimized antibody (X81-B01).
[0504] FIG. 6 depicts the alignment of the heavy chain DNA sequence
of nongermlined (X63-G06) (SEQ ID NO:1996) and germlined, codon
optimized (X81-B01) (SEQ ID NO:1995) versions of the same antibody
discovered using ROLIC affinity maturation. Positions indicated
with an asterisk (*) are conserved, whereas blank spaces correspond
to DNA bases changed in X81-B01 due to codon optimization.
[0505] FIG. 7 depicts the alignment of the heavy chain amino acid
sequence of nongermlined (X63-G06) (SEQ ID NO:1998) and germlined,
codon optimized (X81-B01) (SEQ ID NO:1997) versions of the same
antibody discovered using ROLIC affinity maturation. Positions
indicated with an asterisk (*) are conserved. The two antibodies
have the same amino acid sequence in the heavy chain.
[0506] FIG. 8A depicts the EPI-KAL2 competition for X81-B01 binding
pKal. X81-B01 (IgG) was captured on an anti-human Fc fragment
specific surface of a CM5 BIACORE.RTM. chip. pKal (100 nM) was
flowed over the surface in the presence (lower sensorgram in the
figure) or absence of 1 .mu.M EPI-KAL2 (upper sensorgram in the
figure).
[0507] FIG. 8B depicts the EPI-KAL2 competition for X67-D03 binding
pKal. X67-D03 (IgG) was captured on an anti-human Fc fragment
specific surface of a CM5 Biacore chip. pKal (100 nM) was flowed
over the surface in the presence (lower sensorgram in the figure)
or absence of 1 .mu.M EPI-KAL2 (upper sensorgram in the
figure).
[0508] FIG. 9 depicts the results of CLIPS epitope mapping for
antibodies listed in Table 12.
[0509] FIGS. 10A-10C depict ClustalW alignment of pKal sequences
from different species (SEQ ID NOs:1999-2006). Positions indicated
by a "*" are conserved positions between, whereas positions
indicated ":" indicate conservative substitutions between species.
Positions indicated by a "." have nonconservative substitutions in
some species. Stretches of amino acids indicated by the symbol "@"
were shown to be highly solvent exposed by solvent accessible
surface area calculation. Stretches of amino acids indicated by a
"+" were identified as potential epitopes of antibodies listed in
Table 12. Amino acids highlighted in grey were found by solvent
accessible surface area calculation to be buried when complexed
with a Kunitz domain active site inhibitor. The underlined
positions are the amino acids that form the catalytic triad
(His434, Asp483, and Ser578, numbering based on the human
sequence).
[0510] FIGS. 11A and 11B depict a Biacore competition analysis with
epi-kal2, as described herein in Example 12, for (i) DX-2922, and
(ii) M6-D09 antibodies.
[0511] FIG. 12 depicts a Biacore competition analysis with AEBSF,
as described herein in Example 12, for (i) DX-2911, and (ii) M6-D09
antibodies.
[0512] FIG. 13 depicts a Biocore analysis showing that DX-2922
binds to plasma kallikrein that bound to high molecular weight
kininogen (HMWK).
[0513] FIG. 14 depicts a graph showing dose dependent inhibition of
edema by X101-A01 in carrageenan-induced paw edema (CPE) in
rats.
DETAILED DESCRIPTION
Definitions
[0514] For convenience, before further description of the present
invention, certain terms employed in the specification, examples
and appended claims are defined here. Other terms are defined as
they appear in the specification.
[0515] The singular forms "a", "an", and "the" include plural
references unless the context clearly dictates otherwise.
[0516] The term "agonist," as used herein, is meant to refer to an
agent that mimics or up-regulates (e.g., potentiates or
supplements) the bioactivity of a protein. An agonist can be a
wild-type protein or derivative thereof having at least one
bioactivity of the wild-type protein. An agonist can also be a
compound which increases at least one bioactivity of a protein. An
agonist can also be a compound which increases the interaction of a
polypeptide with another molecule, e.g., a target peptide or
nucleic acid.
[0517] "Antagonist" as used herein is meant to refer to an agent
that downregulates (e.g., suppresses or inhibits) at least one
bioactivity of a protein. An antagonist can be a compound which
inhibits or decreases the interaction between a protein and another
molecule, e.g., a target peptide or enzyme substrate. An antagonist
can also be a compound which reduces the amount of expressed
protein present.
[0518] The term "antibody" refers to a protein that includes at
least one immunoglobulin variable domain (variable region) or
immunoglobulin variable domain (variable region) sequence. For
example, an antibody can include a heavy (H) chain variable region
(abbreviated herein as VH or HV), and a light (L) chain variable
region (abbreviated herein as VL or LV). In another example, an
antibody includes two heavy (H) chain variable regions and two
light (L) chain variable regions. The term "antibody" encompasses
antigen-binding fragments of antibodies (e.g., single chain
antibodies, Fab and sFab fragments, F(ab').sub.2, Fd fragments, Fv
fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et
al., Eur J Immunol. 1996; 26(3):629-39)) as well as complete
antibodies. An antibody can have the structural features of IgA,
IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be
from any source, but primate (human and non-human primate) and
primatized are preferred.
[0519] The VH and VL regions can be further subdivided into regions
of hypervariability, termed "complementarity determining regions"
("CDRs"), interspersed with regions that are more conserved, termed
"framework regions" ("FRs"). The extent of the framework region and
CDRs have been defined (see, Kabat, E. A., et al. (1991) Sequences
of Proteins of Immunological Interest, Fifth Edition, U.S.
Department of Health and Human Services, NIH Publication No.
91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
Kabat definitions are used herein. Each VH and VL is typically
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0520] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence which can form the structure of an
immunoglobulin variable domain such that one or more CDR regions
are positioned in a conformation suitable for an antigen binding
site. For example, the sequence may include all or part of the
amino acid sequence of a naturally-occurring variable domain. For
example, the sequence may omit one, two or more N- or C-terminal
amino acids, internal amino acids, may include one or more
insertions or additional terminal amino acids, or may include other
alterations. In one embodiment, a polypeptide that includes
immunoglobulin variable domain sequence can associate with another
immunoglobulin variable domain sequence to form an antigen binding
site, e.g., a structure that preferentially interacts with plasma
kallikrein.
[0521] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region, to thereby form
a heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains, wherein the heavy and
light immunoglobulin chains are inter-connected by, e.g., disulfide
bonds. In IgGs, the heavy chain constant region includes three
immunoglobulin domains, CH1, CH2 and CH3. The light chain constant
region includes a CL domain. The variable region of the heavy and
light chains contains a binding domain that interacts with an
antigen. The constant regions of the antibodies typically mediate
the binding of the antibody to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system. The light
chains of the immunoglobulin may be of types kappa or lambda. In
one embodiment, the antibody is glycosylated. An antibody can be
functional for antibody-dependent cytotoxicity and/or
complement-mediated cytotoxicity.
[0522] One or more regions of an antibody can be human or
effectively human. For example, one or more of the variable regions
can be human or effectively human. For example, one or more of the
CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC
CDR2, and/or LC CDR3. Each of the light chain (LC) and/or heavy
chain (HC) CDRs can be human. HC CDR3 can be human. One or more of
the framework regions can be human, e.g., FR1, FR2, FR3, and/or FR4
of the HC and/or LC. For example, the Fc region can be human. In
one embodiment, all the framework regions are human, e.g., derived
from a human somatic cell, e.g., a hematopoietic cell that produces
immunoglobulins or a non-hematopoietic cell. In one embodiment, the
human sequences are germline sequences, e.g., encoded by a germline
nucleic acid. In one embodiment, the framework (FR) residues of a
selected Fab can be converted to the amino-acid type of the
corresponding residue in the most similar primate germline gene,
especially the human germline gene. One or more of the constant
regions can be human or effectively human. For example, at least
70, 75, 80, 85, 90, 92, 95, 98, or 100% of an immunoglobulin
variable domain, the constant region, the constant domains (CH1,
CH2, CH3, and/or CL1), or the entire antibody can be human or
effectively human.
[0523] All or part of an antibody can be encoded by an
immunoglobulin gene or a segment thereof. Exemplary human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the many immunoglobulin variable
region genes. Full-length immunoglobulin "light chains" (about 25
KDa or about 214 amino acids) are encoded by a variable region gene
at the NH2-terminus (about 110 amino acids) and a kappa or lambda
constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 KDa or about 446 amino
acids), are similarly encoded by a variable region gene (about 116
amino acids) and one of the other aforementioned constant region
genes, e.g., gamma (encoding about 330 amino acids). The length of
human HC varies considerably because HC CDR3 varies from about 3
amino-acid residues to over 35 amino-acid residues.
[0524] The term "antigen-binding fragment" of a full length
antibody refers to one or more fragments of a full-length antibody
that retain the ability to specifically bind to a target of
interest. Examples of binding fragments encompassed within the term
"antigen-binding fragment" of a full length antibody and that
retain functionality include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment including two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules known as single chain Fv (scFv). See e.g., U.S. Pat. Nos.
5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science
242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883.
[0525] Antibody fragments can be obtained using any appropriate
technique including conventional techniques known to those with
skill in the art. The term "monospecific antibody" refers to an
antibody that displays a single binding specificity and affinity
for a particular target, e.g., epitope. This term includes a
"monoclonal antibody" or "monoclonal antibody composition," which
as used herein refers to a preparation of antibodies or fragments
thereof of single molecular composition, irrespective of how the
antibody was generated.
[0526] Antibodies are "germlined" by reverting one or more
non-germline amino acids in framework regions to corresponding
germline amino acids of the antibody, so long as binding properties
are substantially retained.
[0527] The inhibition constant (Ki) provides a measure of inhibitor
potency; it is the concentration of inhibitor required to reduce
enzyme activity by half and is not dependent on enzyme or substrate
concentrations. The apparent Ki (K.sub.i,app) is obtained at
different substrate concentrations by measuring the inhibitory
effect of different concentrations of inhibitor (e.g., inhibitory
binding protein) on the extent of the reaction (e.g., enzyme
activity); fitting the change in pseudo-first order rate constant
as a function of inhibitor concentration to the Morrison equation
(Equation 1) yields an estimate of the apparent Ki value. The Ki is
obtained from the y-intercept extracted from a linear regression
analysis of a plot of Ki,app versus substrate concentration.
v = v o - v o ( ( K i , app + I + E ) - ( K i , app + I + E ) 2 - 4
I E 2 E ) Equation 1 ##EQU00001##
[0528] Where v=measured velocity; v.sub.0=velocity in the absence
of inhibitor; K.sub.i,app=apparent inhibition constant; I=total
inhibitor concentration; and E=total enzyme concentration.
[0529] As used herein, "binding affinity" refers to the apparent
association constant or K.sub.A. The K.sub.A is the reciprocal of
the dissociation constant (K.sub.D). A binding protein may, for
example, have a binding affinity of at least 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10 and 10.sup.11 M.sup.-1 for
a particular target molecule, e.g., plasma kallikrein. Higher
affinity binding of a binding protein to a first target relative to
a second target can be indicated by a higher K.sub.A (or a smaller
numerical value K.sub.D) for binding the first target than the
K.sub.A (or numerical value K.sub.D) for binding the second target.
In such cases, the binding protein has specificity for the first
target (e.g., a protein in a first conformation or mimic thereof)
relative to the second target (e.g., the same protein in a second
conformation or mimic thereof; or a second protein). Differences in
binding affinity (e.g., for specificity or other comparisons) can
be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100,
500, 1000, 10,000 or 10.sup.5 fold.
[0530] Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, surface plasmon resonance, or spectroscopy
(e.g., using a fluorescence assay). Exemplary conditions for
evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4,
150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be
used to measure the concentration of bound and free binding protein
as a function of binding protein (or target) concentration. The
concentration of bound binding protein ([Bound]) is related to the
concentration of free binding protein ([Free]) and the
concentration of binding sites for the binding protein on the
target where (N) is the number of binding sites per target molecule
by the following equation:
[Bound]=N[Free]/((1/K.sub.A)+[Free]).
[0531] It is not always necessary to make an exact determination of
K.sub.A, though, since sometimes it is sufficient to obtain a
quantitative measurement of affinity, e.g., determined using a
method such as ELISA or FACS analysis, is proportional to K.sub.A,
and thus can be used for comparisons, such as determining whether a
higher affinity is, e.g., 2-fold higher, to obtain a qualitative
measurement of affinity, or to obtain an inference of affinity,
e.g., by activity in a functional assay, e.g., an in vitro or in
vivo assay.
[0532] The term "binding protein" refers to a protein that can
interact with a target molecule. This term is used interchangeably
with "ligand." A "plasma kallikrein binding protein" refers to a
protein that can interact with (e.g., bind) plasma kallikrein, and
includes, in particular, proteins that preferentially or
specifically interact with and/or inhibit plasma kallikrein. A
protein inhibits plasma kallikrein if it causes a decrease in the
activity of plasma kallikrein as compared to the activity of plasma
kallikrein in the absence of the protein and under the same
conditions. In some embodiments, the plasma kallikrein binding
protein is an antibody.
[0533] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0534] It is possible for one or more framework and/or CDR amino
acid residues of a binding protein to include one or more mutations
(e.g., substitutions (e.g., conservative substitutions or
substitutions of non-essential amino acids), insertions, or
deletions) relative to a binding protein described herein. A plasma
kallikrein binding protein may have mutations (e.g., substitutions
(e.g., conservative substitutions or substitutions of non-essential
amino acids), insertions, or deletions) (e.g., at least one, two,
three, or four, and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3,
or 2 mutations) relative to a binding protein described herein,
e.g., mutations which do not have a substantial effect on protein
function. The mutations can be present in framework regions, CDRs,
and/or constant regions. In some embodiments, the mutations are
present in a framework region. In some embodiments, the mutations
are present in a CDR. In some embodiments, the mutations are
present in a constant region. Whether or not a particular
substitution will be tolerated, i.e., will not adversely affect
biological properties, such as binding activity, can be predicted,
e.g., by evaluating whether the mutation is conservative or by the
method of Bowie, et al. (1990) Science 247:1306-1310.
[0535] Motif sequences for biopolymers can include positions which
can be varied amino acids. For example, the symbol "X" in such a
context generally refers to any amino acid (e.g., any of the twenty
natural amino acids) unless otherwise specified, e.g., to refer to
any non-cysteine amino acid. Other allowed amino acids can also be
indicated for example, using parentheses and slashes. For example,
"(A/W/F/N/Q)" means that alanine, tryptophan, phenylalanine,
asparagine, and glutamine are allowed at that particular
position.
[0536] An "effectively human" immunoglobulin variable region is an
immunoglobulin variable region that includes a sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. An "effectively human" antibody is an antibody that includes
a sufficient number of human amino acid positions such that the
antibody does not elicit an immunogenic response in a normal
human.
[0537] An "epitope" refers to the site on a target compound that is
bound by a binding protein (e.g., an antibody such as a Fab or full
length antibody). In the case where the target compound is a
protein, the site can be entirely composed of amino acid
components, entirely composed of chemical modifications of amino
acids of the protein (e.g., glycosyl moieties), or composed of
combinations thereof. Overlapping epitopes include at least one
common amino acid residue, glycosyl group, phosphate group, sulfate
group, or other molecular feature.
[0538] A first binding protein (e.g., antibody) "binds to the same
epitope" as a second binding protein (e.g., antibody) if the first
binding protein binds to the same site on a target compound that
the second binding protein binds, or binds to a site that overlaps
(e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of
amino acid sequence or other molecular feature (e.g., glycosyl
group, phosphate group, or sulfate group)) with the site that the
second binding protein binds.
[0539] A first binding protein (e.g., antibody) "competes for
binding" with a second binding protein (e.g., antibody) if the
binding of the first binding protein to its epitope decreases
(e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or
more) the amount of the second binding protein that binds to its
epitope. The competition can be direct (e.g., the first binding
protein binds to an epitope that is the same as, or overlaps with,
the epitope bound by the second binding protein), or indirect
(e.g., the binding of the first binding protein to its epitope
causes a steric change in the target compound that decreases the
ability of the second binding protein to bind to its epitope).
[0540] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) are
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). The optimal alignment is determined as
the best score using the GAP program in the GCG software package
with a Blossum 62 scoring matrix with a gap penalty of 12, a gap
extend penalty of 4, and a frameshift gap penalty of 5. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences.
[0541] In a preferred embodiment, the length of a reference
sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, and even more preferably at least 70%,
80%, 90%, 92%, 95%, 97%, 98%, or 100% of the length of the
reference sequence. For example, the reference sequence may be the
length of the immunoglobulin variable domain sequence.
[0542] A "humanized" immunoglobulin variable region is an
immunoglobulin variable region that is modified to include a
sufficient number of human framework amino acid positions such that
the immunoglobulin variable region does not elicit an immunogenic
response in a normal human. Descriptions of "humanized"
immunoglobulins include, for example, U.S. Pat. No. 6,407,213 and
U.S. Pat. No. 5,693,762.
[0543] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described
in that reference and either can be used. Specific hybridization
conditions referred to herein are as follows: (1) low stringency
hybridization conditions in 6.times. sodium chloride/sodium citrate
(SSC) at about 45.degree. C., followed by two washes in
0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature
of the washes can be increased to 55.degree. C. for low stringency
conditions); (2) medium stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; (3) high stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C.; and (4) very high stringency hybridization
conditions are 0.5M sodium phosphate, 7% SDS at 65.degree. C.,
followed by one or more washes at 0.2.times.SSC, 1% SDS at
65.degree. C. Very high stringency conditions (4) are the preferred
conditions and the ones that should be used unless otherwise
specified. The disclosure includes nucleic acids that hybridize
with low, medium, high, or very high stringency to a nucleic acid
described herein or to a complement thereof, e.g., nucleic acids
encoding a binding protein described herein. The nucleic acids can
be the same length or within 30, 20, or 10% of the length of the
reference nucleic acid. The nucleic acid can correspond to a region
encoding an immunoglobulin variable domain sequence described
herein.
[0544] An "isolated composition" refers to a composition that is
removed from at least 90% of at least one component of a natural
sample from which the isolated composition can be obtained.
Compositions produced artificially or naturally can be
"compositions of at least" a certain degree of purity if the
species or population of species of interest is at least 5, 10, 25,
50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-weight
basis.
[0545] An "isolated" protein refers to a protein that is removed
from at least 90% of at least one component of a natural sample
from which the isolated protein can be obtained. Proteins can be
"of at least" a certain degree of purity if the species or
population of species of interest is at least 5, 10, 25, 50, 75,
80, 90, 92, 95, 98, or 99% pure on a weight-weight basis.
[0546] The term "modulator" refers to a polypeptide, nucleic acid,
macromolecule, complex, molecule, small molecule, compound, species
or the like (naturally-occurring or non-naturally-occurring), or an
extract made from biological materials such as bacteria, plants,
fungi, or animal cells or tissues, that may be capable of causing
modulation. Modulators may be evaluated for potential activity as
inhibitors or activators (directly or indirectly) of a functional
property, biological activity or process, or combination of them,
(e.g., agonist, partial antagonist, partial agonist, inverse
agonist, antagonist, anti-microbial agents, inhibitors of microbial
infection or proliferation, and the like) by inclusion in assays.
In such assays, many modulators may be screened at one time. The
activity of a modulator may be known, unknown or partially
known.
[0547] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of the binding agent, e.g.,
the antibody, without abolishing or more preferably, without
substantially altering a biological activity, whereas changing an
"essential" amino acid residue results in a substantial loss of
activity.
[0548] A "patient," "subject" or "host" (these terms are used
interchangeably) to be treated by the subject method may mean
either a human or non-human animal.
[0549] The terms "prekallikrein" and "preplasma kallikrein" are
used interchangeably herein and refer to the zymogen form of active
plasma kallikrein, which is also known as prekallikrein.
[0550] The term "preventing" or to "prevent" a disease in a subject
refers to subjecting the subject to a pharmaceutical treatment,
e.g., the administration of a drug, such that at least one symptom
of the disease is prevented, that is, administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) so that it protects the
host against developing the unwanted condition. "Preventing" a
disease may also be referred to as "prophylaxis" or "prophylactic
treatment."
[0551] As used herein, the term "substantially identical" (or
"substantially homologous") is used herein to refer to a first
amino acid or nucleic acid sequence that contains a sufficient
number of identical or equivalent (e.g., with a similar side chain,
e.g., conserved amino acid substitutions) amino acid residues or
nucleotides to a second amino acid or nucleic acid sequence such
that the first and second amino acid or nucleic acid sequences have
(or encode proteins having) similar activities, e.g., a binding
activity, a binding preference, or a biological activity. In the
case of antibodies, the second antibody has the same specificity
and has at least 50%, at least 25%, or at least 10% of the affinity
relative to the same antigen.
[0552] Sequences similar or homologous (e.g., at least about 85%
sequence identity) to the sequences disclosed herein are also part
of this application. In some embodiments, the sequence identity can
be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
higher. In some embodiments, a plasma kallikrein binding protein
can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or higher sequence identity to a binding protein described
herein. In some embodiments, a plasma kallikrein binding protein
can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or higher sequence identity in the HC and/or LC framework
regions (e.g., HC and/or LC FR 1, 2, 3, and/or 4) to a binding
protein described herein. In some embodiments, a plasma kallikrein
binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or higher sequence identity in the HC and/or LC
CDRs (e.g., HC and/or LC CDR1, 2, and/or 3) to a binding protein
described herein. In some embodiments, a plasma kallikrein binding
protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or higher sequence identity in the constant region (e.g.,
CH1, CH2, CH3, and/or CL1) to a binding protein described
herein.
[0553] In addition, substantial identity exists when the nucleic
acid segments hybridize under selective hybridization conditions
(e.g., highly stringent hybridization conditions), to the
complement of the strand. The nucleic acids may be present in whole
cells, in a cell lysate, or in a partially purified or
substantially pure form.
[0554] Statistical significance can be determined by any art known
method. Exemplary statistical tests include: the Students T-test,
Mann Whitney U non-parametric test, and Wilcoxon non-parametric
statistical test. Some statistically significant relationships have
a P value of less than 0.05 or 0.02. Particular binding proteins
may show a difference, e.g., in specificity or binding that are
statistically significant (e.g., P value <0.05 or 0.02). The
terms "induce", "inhibit", "potentiate", "elevate", "increase",
"decrease" or the like, e.g., which denote distinguishable
qualitative or quantitative differences between two states, may
refer to a difference, e.g., a statistically significant
difference, between the two states.
[0555] A "therapeutically effective dosage" preferably modulates a
measurable parameter, e.g., plasma kallikrein activity, by a
statistically significant degree or at least about 20%, more
preferably by at least about 40%, even more preferably by at least
about 60%, and still more preferably by at least about 80% relative
to untreated subjects. The ability of a compound to modulate a
measurable parameter, e.g., a disease-associated parameter, can be
evaluated in an animal model system predictive of efficacy in human
disorders and conditions, e.g., rheumatoid arthritis or oral
mucositis. Alternatively, this property of a composition can be
evaluated by examining the ability of the compound to modulate a
parameter in vitro.
[0556] "Treating" a disease (or condition) in a subject or
"treating" a subject having a disease refers to subjecting the
subject to a pharmaceutical treatment, e.g., the administration of
a drug, such that at least one symptom of the disease is cured,
alleviated or decreased.
[0557] The term "preventing" a disease in a subject refers to
subjecting the subject to a pharmaceutical treatment, e.g., the
administration of a drug, such that at least one symptom of the
disease is prevented, that is, administered prior to clinical
manifestation of the unwanted condition (e.g., disease or other
unwanted state of the host animal) so that it protects the host
against developing the unwanted condition. "Preventing" a disease
may also be referred to as "prophylaxis" or "prophylactic
treatment."
[0558] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, because a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0559] As used herein the term "DX-2922" as used inchangeably with
the term "X101-A01". Other variants of this antibody are described
below.
TABLE-US-00001 Antibody Identification Description X63-G06
Non-germlined Fab discovered using ROLIC, same HC but different LC
as M160-G12 X81-B01 Germlined IgG produced in HEK 293T cells
X101-A01 Germlined IgG produced in CHO cells, same HC and LC
sequence as X81-B01 DX-2922 Alternate nomenclature for X101-A01
Plasma Kallikrein Binding Proteins
[0560] Plasma kallikrein binding proteins can be full-length (e.g.,
an IgG (e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1,
IgA2), IgD, and IgE) or can include only an antigen-binding
fragment (e.g., a Fab, F(ab')2 or scFv fragment. The binding
protein can include two heavy chain immunoglobulins and two light
chain immunoglobulins, or can be a single chain antibody. Plasma
kallikrein binding proteins can be recombinant proteins such as
humanized, CDR grafted, chimeric, deimmunized, or in vitro
generated antibodies, and may optionally include constant regions
derived from human germline immunoglobulin sequences. In one
embodiment, the plasma kallikrein binding protein is a monoclonal
antibody.
[0561] In one aspect, the disclosure features a protein (e.g., an
isolated protein) that binds to plasma kallikrein (e.g., human
plasma kallikrein and/or murine kallikrein) and includes at least
one immunoglobulin variable region. For example, the protein
includes a heavy chain (HC) immunoglobulin variable domain sequence
and/or a light chain (LC) immunoglobulin variable domain sequence.
In one embodiment, the protein binds to and inhibits plasma
kallikrein, e.g., human plasma kallikrein and/or murine
kallikrein.
[0562] The protein can include one or more of the following
characteristics: (a) a human CDR or human framework region; (b) the
HC immunoglobulin variable domain sequence comprises one or more
(e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a HC
variable domain described herein; (c) the LC immunoglobulin
variable domain sequence comprises one or more (e.g., 1, 2, or 3)
CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% identical to a CDR of a LC variable domain
described herein; (d) the LC immunoglobulin variable domain
sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% identical to a LC variable domain described herein
(e.g., overall or in framework regions or CDRs); (e) the HC
immunoglobulin variable domain sequence is at least 85, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC
variable domain described herein (e.g., overall or in framework
regions or CDRs); (f) the protein binds an epitope bound by a
protein described herein, or competes for binding with a protein
described herein; (g) a primate CDR or primate framework region;
(h) the HC immunoglobulin variable domain sequence comprises a CDR1
that differs by at least one amino acid but by no more than 2 or 3
amino acids from the CDR1 of a HC variable domain described herein;
(i) the HC immunoglobulin variable domain sequence comprises a CDR2
that differs by at least one amino acid but by no more than 2, 3,
4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variable domain
described herein; (j) the HC immunoglobulin variable domain
sequence comprises a CDR3 that differs by at least one amino acid
but by no more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of a
HC variable domain described herein; (k) the LC immunoglobulin
variable domain sequence comprises a CDR1 that differs by at least
one amino acid but by no more than 2, 3, 4, or 5 amino acids from
the CDR1 of a LC variable domain described herein; (1) the LC
immunoglobulin variable domain sequence comprises a CDR2 that
differs by at least one amino acid but by no more than 2, 3, or 4
amino acids from the CDR2 of a LC variable domain described herein;
(m) the LC immunoglobulin variable domain sequence comprises a CDR3
that differs by at least one amino acid but by no more than 2, 3,
4, or 5 amino acids from the CDR3 of a LC variable domain described
herein; (n) the LC immunoglobulin variable domain sequence differs
by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7, 8,
9, or 10 amino acids from a LC variable domain described herein
(e.g., overall or in framework regions or CDRs); and (o) the HC
immunoglobulin variable domain sequence differs by at least one
amino acid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acids from a HC variable domain described herein (e.g., overall or
in framework regions or CDRs).
[0563] The plasma kallikrein binding protein may be an isolated
protein (e.g., at least 70, 80, 90, 95, or 99% free of other
proteins). In some embodiments, the plasma kallikrein binding
protein, or composition thereof, is isolated from antibody cleavage
fragments (e.g., cleaved DX-2922) that are inactive or partially
active (e.g., bind plasma kallikrein with a Ki, app of 5000 nM or
greater) compared to the plasma kallikrein binding protein. For
example, the plasma kallikrein binding protein is at least 70% free
of such antibody cleavage fragments; in other embodiments the
binding protein is at least 80%, at least 90%, at least 95%, at
least 99% or even 100% free from antibody cleavage fragments that
are inactive or partially active.
[0564] The plasma kallikrein binding protein may additionally
inhibit plasma kallikrein, e.g., human plasma kallikrein.
[0565] In some embodiments, the plasma kallikrein binding protein
does not bind prekallikrein (e.g., human prekallikrein and/or
murine prekallikrein), but binds to the active form of plasma
kallikrein (e.g., human plasma kallikrein and/or murine
kallikrein).
[0566] In certain embodiments, the protein binds at or near the
active site of the catalytic domain of plasma kallikrein, or a
fragment thereof, or binds an epitope that overlaps with the active
site of plasma kallikrein.
[0567] In some aspects, the protein binds the same epitope or
competes for binding with a protein described herein.
[0568] In some embodiments, the protein competes with or binds the
same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,
X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,
X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09
and M35-G04.
[0569] In some embodiments, the protein binds to (e.g., positions
on plasma kallikrein corresponding to) CLIPS peptide C1, C2, C3,
C4, C5, C6, or C7, or more than one of these peptides, e.g., the
protein binds to C5 and C6. CLIPS peptides C1-C7 are peptides in
plasma kallikrein identified by CLIPS epitope mapping (see FIGS. 9
and 10A-10C). C1 corresponds to positions 55-67 of the catalytic
domain, C2 to positions 81-94, C3 to positions 101-108, C4 to
positions 137-151, C5 to positions 162-178, C6 to positions
186-197, and C7 to positions 214-217 of plasma kallikrein.
[0570] In some embodiments, the protein binds to an epitope shown
in FIG. 9.
[0571] In some embodiments, the protein binds to one or more amino
acids that form the catalytic triad of plasma kallikrein: His434,
Asp483, and/or Ser578 (numbering based on the human sequence).
[0572] In some embodiments, the protein binds one or more amino
acids of: Arg551, Gln553, Tyr555, Thr558, and/or Arg560 (numbering
based on the human sequence). In some embodiments, the plasma
kallikrein binding protein binds one or more amino acids of: S478,
N481, S525, and K526 (numbering based on the human kallikrein
sequence).
[0573] In some embodiments, the protein binds to one or more amino
acids of Ser479, Tyr563, and/or Asp585 (numbering based on the
human sequence).
[0574] The active site cleft of plasma kallikrein contains three
amino acids that form the catalytic triad (His434, Asp483, and
Ser578) and result in enzymatic hydrolysis of bound substrate
(catalytic triad residues are underlined in FIG. 10). The peptides
selected for the CLIPS epitope mapping analysis were determined to
be surface accessible and either form or surround the vicinity of
the active site. Peptide C1 contains the active site histidine 434.
Peptide C3 contains the active site aspartate 483. Peptide C6
contains the active site serine 578. It is possible for an antibody
to bind multiple surface exposed amino acids that are discontinuous
in amino acid sequence. For example, by CLIPs analysis, X81-B01
appears to bind the C2, C3, C5 and the C6 peptides.
[0575] In some embodiments, the protein binds to an epitope that
includes one or more amino acids from CLIPS peptide C1, peptide C2,
peptide C3, peptide C4, peptide C5, peptide C6, or peptide C7.
[0576] In some embodiments, the protein binds to an epitope that
includes amino acids from at least 2 different CLIPS peptides,
e.g., from at least two of peptide C1, peptide C2, peptide C3,
peptide C4, peptide C5, peptide C6, or peptide C7.
[0577] The protein can bind to plasma kallikrein, e.g., human
plasma kallikrein, with a binding affinity of at least 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10 and 10.sup.11
M.sup.-1. In one embodiment, the protein binds to human plasma
kallikrein with a K.sub.off slower than 1.times.10.sup.-3,
5.times.10.sup.-4 s.sup.-1, or 1.times.10.sup.-4 s.sup.-1. In one
embodiment, the protein binds to human plasma kallikrein with a
K.sub.on faster than 1.times.10.sup.2, 1.times.10.sup.3, or
5.times.10.sup.3 M.sup.-1 s.sup.-1. In one embodiment, the protein
binds to plasma kallikrein, but does not bind to tissue kallikrein
and/or plasma prekallikrein (e.g., the protein binds to tissue
kallikrein and/or plasma prekallikrein less effectively (e.g., 5-,
10-, 50-, 100-, or 1000-fold less or not at all, e.g., as compared
to a negative control) than it binds to plasma kallikrein.
[0578] In one embodiment, the protein inhibits human plasma
kallikrein activity, e.g., with a Ki of less than 10.sup.-5,
10.sup.-6, 10.sup.-7, 10.sup.-8, 10.sup.-9, and 10.sup.-10 M. The
protein can have, for example, an IC.sub.50 of less than 100 nM, 10
nM, 1, 0.5, or 0.2 nM. For example, the protein may modulate plasma
kallikrein activity, as well as the production of Factor XIIa
(e.g., from Factor XII) and/or bradykinin (e.g., from
high-molecular-weight kininogen (HMWK)). The protein may inhibit
plasma kallikrein activity, and/or the production of Factor XIIa
(e.g., from Factor XII) and/or bradykinin (e.g., from
high-molecular-weight kininogen (HMWK)). The affinity of the
protein for human plasma kallikrein can be characterized by a
K.sub.D of less than 100 nm, less than 10 nM, less than 5 nM, less
than 1 nM, less than 0.5 nM. In one embodiment, the protein
inhibits plasma kallikrein, but does not inhibit tissue kallikrein
(e.g., the protein inhibits tissue kallikrein less effectively
(e.g., 5-, 10-, 50-, 100-, or 1000-fold less or not at all, e.g.,
as compared to a negative control) than it inhibits plasma
kallikrein.
[0579] In some embodiments, the protein has an apparent inhibition
constant (K.sub.i,app) of less than 1000, 500, 100, 5, 1, 0.5 or
0.2 nM.
[0580] Plasma kallikrein binding proteins may be antibodies. Plasma
kallikrein binding antibodies may have their HC and LC variable
domain sequences included in a single polypeptide (e.g., scFv), or
on different polypeptides (e.g., IgG or Fab).
[0581] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light and heavy chains of antibodies
selected from the group consisting of M162-A04, M160-G12, M142-H08
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, DX-2922, X115-B07,
X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,
X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0582] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the heavy chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08,
X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,
X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,
M29-D09, M145-D11, M06-D09 and M35-G04.
[0583] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having the light chain of an antibody selected
from the group consisting of: M162-A04, M160-G12, M142-H08 X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0584] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having light and heavy antibody variable regions
of an antibody selected from the group consisting of M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0585] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a heavy chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0586] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having a light chain antibody variable region of
an antibody selected from the group consisting of: M162-A04,
M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,
X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,
X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.
[0587] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs selected from the corresponding CDRs of the group of heavy
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0588] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) light chain
CDRs selected from the corresponding CDRs of the group of light
chains consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,
X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,
M145-D11, M06-D09 and M35-G04.
[0589] In a preferred embodiment, the protein is an antibody (e.g.,
a human antibody) having one or more (e.g., 1, 2, or 3) heavy chain
CDRs and one or more (e.g., 1, 2, or 3) light chain CDRs selected
from the corresponding CDRs of the group of light chains consisting
of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,
X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,
X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04.
[0590] In one embodiment, the HC and LC variable domain sequences
are components of the same polypeptide chain. In another, the HC
and LC variable domain sequences are components of different
polypeptide chains. For example, the protein is an IgG, e.g., IgG1,
IgG2, IgG3, or IgG4. The protein can be a soluble Fab. In other
implementations the protein includes a Fab2', scFv, minibody,
scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab
fusion, or other molecule that comprises the antigen combining site
of one of the binding proteins herein. The VH and VL regions of
these Fabs can be provided as IgG, Fab, Fab2, Fab2', scFv,
PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC,
HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other
appropriate construction.
[0591] In one embodiment, the protein is a human or humanized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more human antibody framework regions, e.g., all
human framework regions, or framework regions at least 85, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to human
framework regions. In one embodiment, the protein includes a human
Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99%
identical to a human Fc domain.
[0592] In one embodiment, the protein is a primate or primatized
antibody or is non-immunogenic in a human. For example, the protein
includes one or more primate antibody framework regions, e.g., all
primate framework regions, or framework regions at least 85, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to primate
framework regions. In one embodiment, the protein includes a
primate Fc domain, or an Fc domain that is at least 95, 96, 97, 98,
or 99% identical to a primate Fc domain. "Primate" includes humans
(Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus
(bonobos)), gorillas (Gorilla gorilla), gibons, monkeys, lemurs,
aye-ayes (Daubentonia madagascariensis), and tarsiers.
[0593] In some embodiments, the affinity of the primate antibody
for human plasma kallikrein is characterized by a K.sub.D of less
than 1000, 500, 100, 10, 5, 1, 0.5 nM, e.g., less than 10 nM, less
than 1 nM, or less than 0.5 nM.
[0594] In certain embodiments, the protein includes no sequences
from mice or rabbits (e.g., is not a murine or rabbit
antibody).
[0595] In some aspects, the disclosure provides the use of proteins
(e.g., binding proteins, e.g., antibodies) (e.g., the proteins
described herein) that bind to plasma kallikrein (e.g., human
plasma kallikrein) and include at least one immunoglobin variable
region in methods for treating (or preventing) a plasma kallikrein
associated disorder or condition. For example, the plasma
kallikrein binding protein includes a heavy chain (HC)
immunoglobulin variable domain sequence and a light chain (LC)
immunoglobulin variable domain sequence. A number of exemplary
plasma kallikrein binding proteins are described herein.
[0596] The plasma kallikrein binding protein may be an isolated
protein (e.g., at least 70, 80, 90, 95, or 99% free of other
proteins).
[0597] The plasma kallikrein binding protein may additionally
inhibit plasma kallikrein, e.g., human plasma kallikrein and/or
murine plasma kallikrein. In some embodiments, it may be preferred
to have a plasma kallikrein binding protein bind to both human and
murine plasma kallikrein, as these antibodies can be tested for
efficacy in a mouse model.
Plasma Kallikrein
[0598] Exemplary plasma kallikrein sequences against which plasma
kallikrein binding proteins may be developed can include human,
mouse, or rat plasma kallikrein amino acid sequences, a sequence
that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one
of these sequences, or a fragment thereof, e.g., of a sequence
provided below.
[0599] The sequence of human plasma kallikrein that was used in
selections and subsequent screening is shown below (accession
number NP_000883.2). The human plasma kallikrein (86 kDa) that was
used was purified from human plasma and activated with factor XIIa
by a commercial vendor. Factor XIIa activates prekallikrein by
cleaving the polypeptide sequence at a single site (between
Arg371-Ile372, cleavage site marked by "/" in the sequence below)
to generate active plasma kallikrein, which then consists of two
disulfide linked polypeptides; a heavy chain of approximately 52
kDa and a catalytic domain of approximately 34 kDa [Colman and
Schmaier, (1997) "Contact System: A Vascular Biology Modulator With
Anticoagulant, Profibrinolytic, Antiadhesive, and Proinflammatory
Attributes" Blood, 90, 3819-3843]
TABLE-US-00002 (SEQ ID NO: 1)
GCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSIN
DMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGV
DMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLK
YSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARV
LTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSS
TPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQ
ETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSG
YSLRLCNTGDNSVCTTKTSTR/IVGGTNSSWGEWPWQVSLQVKLTAQRHL
CGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKE
IIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCW
VTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKE
GGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYM
DWILEKTQSSDGKAQMQSPA
[0600] The human, mouse, and rat prekallikrein amino acid
sequences, and the mRNA sequences encoding the same, are
illustrated below. The sequences of prekallikrein are the same as
plasma kallikrein, except that active plasma kallikrein (pkal) has
the single polypeptide chain cleaved at a single position
(indicated by the "/") to generate two chains. The sequences
provided below are full sequences that include signal sequences. On
secretion from the expressing cell, it is expected that the signal
sequences are removed.
TABLE-US-00003 Human plasma kallikrein (ACCESSION: NP_000883.2)
>gi|78191798|ref|NP_000883.2|plasma kallikrein B1 precursor
[Homo sapiens] (SEQ ID NO: 2)
MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMR
CTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHS
LKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFF
SYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIG
CHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIE
SQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGV
DFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLR
LSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWG
EWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSG
ILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQK
PICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQK
RYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGE
GCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA Human plasma kallikrein mRNA
(ACCESSION: NM_000892) >gi|78191797|ref|NM_000892.3|Homo sapiens
kallikrein B, plasma (Fletcher factor) 1 (KLKB1), mRNA (SEQ ID NO:
3) AGAACAGCTTGAAGACCGTTCATTTTTAAGTGACAAGAGACTCACCTCCA
AGAAGCAATTGTGTTTTCAGAATGATTTTATTCAAGCAAGCAACTTATTT
CATTTCCTTGTTTGCTACAGTTTCCTGTGGATGTCTGACTCAACTCTATG
AAAACGCCTTCTTCAGAGGTGGGGATGTAGCTTCCATGTACACCCCAAAT
GCCCAATACTGCCAGATGAGGTGCACATTCCACCCAAGGTGTTTGCTATT
CAGTTTTCTTCCAGCAAGTTCAATCAATGACATGGAGAAAAGGTTTGGTT
GCTTCTTGAAAGATAGTGTTACAGGAACCCTGCCAAAAGTACATCGAACA
GGTGCAGTTTCTGGACATTCCTTGAAGCAATGTGGTCATCAAATAAGTGC
TTGCCATCGAGACATTTATAAAGGAGTTGATATGAGAGGAGTCAATTTTA
ATGTGTCTAAGGTTAGCAGTGTTGAAGAATGCCAAAAAAGGTGCACCAGT
AACATTCGCTGCCAGTTTTTTTCATATGCCACGCAAACATTTCACAAGGC
AGAGTACCGGAACAATTGCCTATTAAAGTACAGTCCCGGAGGAACACCTA
CCGCTATAAAGGTGCTGAGTAACGTGGAATCTGGATTCTCACTGAAGCCC
TGTGCCCTTTCAGAAATTGGTTGCCACATGAACATCTTCCAGCATCTTGC
GTTCTCAGATGTGGATGTTGCCAGGGTTCTCACTCCAGATGCTTTTGTGT
GTCGGACCATCTGCACCTATCACCCCAACTGCCTCTTCTTTACATTCTAT
ACAAATGTATGGAAAATCGAGTCACAAAGAAATGTTTGTCTTCTTAAAAC
ATCTGAAAGTGGCACACCAAGTTCCTCTACTCCTCAAGAAAACACCATAT
CTGGATATAGCCTTTTAACCTGCAAAAGAACTTTACCTGAACCCTGCCAT
TCTAAAATTTACCCGGGAGTTGACTTTGGAGGAGAAGAATTGAATGTGAC
TTTTGTTAAAGGAGTGAATGTTTGCCAAGAGACTTGCACAAAGATGATTC
GCTGTCAGTTTTTCACTTATTCTTTACTCCCAGAAGACTGTAAGGAAGAG
AAGTGTAAGTGTTTCTTAAGATTATCTATGGATGGTTCTCCAACTAGGAT
TGCGTATGGGACACAAGGGAGCTCTGGTTACTCTTTGAGATTGTGTAACA
CTGGGGACAACTCTGTCTGCACAACAAAAACAAGCACACGCATTGTTGGA
GGAACAAACTCTTCTTGGGGAGAGTGGCCCTGGCAGGTGAGCCTGCAGGT
GAAGCTGACAGCTCAGAGGCACCTGTGTGGAGGGTCACTCATAGGACACC
AGTGGGTCCTCACTGCTGCCCACTGCTTTGATGGGCTTCCCCTGCAGGAT
GTTTGGCGCATCTATAGTGGCATTTTAAATCTGTCAGACATTACAAAAGA
TACACCTTTCTCACAAATAAAAGAGATTATTATTCACCAAAACTATAAAG
TCTCAGAAGGGAATCATGATATCGCCTTGATAAAACTCCAGGCTCCTTTG
AATTACACTGAATTCCAAAAACCAATATGCCTACCTTCCAAAGGTGACAC
AAGCACAATTTATACCAACTGTTGGGTAACCGGATGGGGCTTCTCGAAGG
AGAAAGGTGAAATCCAAAATATTCTACAAAAGGTAAATATTCCTTTGGTA
ACAAATGAAGAATGCCAGAAAAGATATCAAGATTATAAAATAACCCAACG
GATGGTCTGTGCTGGCTATAAAGAAGGGGGAAAAGATGCTTGTAAGGGAG
ATTCAGGTGGTCCCTTAGTTTGCAAACACAATGGAATGTGGCGTTTGGTG
GGCATCACCAGCTGGGGTGAAGGCTGTGCCCGCAGGGAGCAACCTGGTGT
CTACACCAAAGTCGCTGAGTACATGGACTGGATTTTAGAGAAAACACAGA
GCAGTGATGGAAAAGCTCAGATGCAGTCACCAGCATGAGAAGCAGTCCAG
AGTCTAGGCAATTTTTACAACCTGAGTTCAAGTCAAATTCTGAGCCTGGG
GGGTCCTCATCTGCAAAGCATGGAGAGTGGCATCTTCTTTGCATCCTAAG
GACGAAAAACACAGTGCACTCAGAGCTGCTGAGGACAATGTCTGGCTGAA
GCCCGCTTTCAGCACGCCGTAACCAGGGGCTGACAATGCGAGGTCGCAAC
TGAGATCTCCATGACTGTGTGTTGTGAAATAAAATGGTGAAAGATCAAAA AA Mouse plasma
kallikrein (ACCESSION: NP_032481.1)
>gi|6680584|ref|NP_032481.1|kallikrein B, plasma 1 [Mus
musculus] (SEQ ID NO: 4)
MILFNRVGYFVSLFATVSCGCMTQLYKNTFFRGGDLAAIYTPDAQYCQKM
CTFHPRCLLFSFLAVTPPKETNKRFGCFMKESITGTLPRIHRTGAISGHS
LKQCGHQISACHRDIYKGLDMRGSNFNISKTDNIEECQKLCTNNFHCQFF
TYATSAFYRPEYRKKCLLKHSASGTPTSIKSADNLVSGFSLKSCALSEIG
CPMDIFQHSAFADLNVSQVITPDAFVCRTICTFHPNCLFFTFYTNEWETE
SQRNVCFLKTSKSGRPSPPIPQENAISGYSLLTCRKTRPEPCHSKIYSGV
DFEGEELNVTFVQGADVCQETCTKTIRCQFFIYSLLPQDCKEEGCKCSLR
LSTDGSPTRITYGMQGSSGYSLRLCKLVDSPDCTTKINARIVGGTNASLG
EWPWQVSLQVKLVSQTHLCGGSIIGRQWVLTAAHCFDGIPYPDVWRIYGG
ILSLSEITKETPSSRIKELIIHQEYKVSEGNYDIALIKLQTPLNYTEFQK
PICLPSKADTNTIYTNCWVTGWGYTKEQGETQNILQKATIPLVPNEECQK
KYRDYVINKQMICAGYKEGGTDACKGDSGGPLVCKHSGRWQLVGITSWGE
GCGRKDQPGVYTKVSEYMDWILEKTQSSDVRALETSSA Mouse plasma kallikrein mRNA
(ACCESSION: NM_008455.2) >gi|236465804|ref|NM_008455.2|Mus
musculus kallikrein B, plasma 1 (Klkb1), mRNA (SEQ ID NO: 5)
AGACCGCCCTCGGTGCCATATTCAGAGGGCTTGAAGACCATCTTCATGTG
AAGACTCCCTCTCCTCCAGAACCACAACGTGACCATCCTTCCAGGATGAT
TTTATTCAACCGAGTGGGTTATTTTGTTTCCTTGTTTGCTACCGTCTCCT
GTGGGTGTATGACTCAACTGTATAAAAATACCTTCTTCAGAGGTGGGGAT
CTAGCTGCCATCTACACCCCAGATGCCCAGTACTGTCAGAAGATGTGCAC
TTTTCACCCCAGGTGCCTGCTGTTCAGCTTTCTCGCCGTGACTCCACCCA
AAGAGACAAATAAACGGTTTGGTTGCTTCATGAAAGAGAGCATTACAGGG
ACTTTGCCAAGAATACACCGGACAGGGGCCATTTCTGGTCATTCTTTAAA
GCAGTGTGGCCATCAAATAAGTGCTTGCCACCGAGACATATACAAAGGAC
TTGATATGAGAGGGTCCAACTTTAATATCTCTAAGACCGACAATATTGAA
GAATGCCAGAAACTGTGCACAAATAATTTTCACTGCCAATTTTTCACATA
TGCTACAAGTGCATTTTACAGACCAGAGTACCGGAAGAAGTGCCTGCTGA
AGCACAGTGCAAGCGGAACACCCACCAGCATAAAGTCAGCGGACAACCTG
GTGTCTGGATTCTCACTGAAGTCCTGTGCGCTTTCGGAGATAGGTTGCCC
CATGGATATTTTCCAGCACTCTGCCTTTGCAGACCTGAATGTAAGCCAGG
TCATCACCCCCGATGCCTTTGTGTGTCGCACCATCTGCACCTTCCATCCC
AACTGCCTTTTCTTCACGTTCTACACGAATGAATGGGAGACAGAATCACA
GAGAAATGTTTGTTTTCTTAAGACGTCTAAAAGTGGAAGACCAAGTCCCC
CTATTCCTCAAGAAAACGCTATATCTGGATATAGTCTCCTCACCTGCAGA
AAAACTCGCCCTGAACCCTGCCATTCCAAAATTTACTCTGGAGTTGACTT
TGAAGGGGAAGAACTGAATGTGACCTTCGTGCAAGGAGCAGATGTCTGCC
AAGAGACTTGTACAAAGACAATCCGCTGCCAGTTTTTTATTTACTCCTTA
CTCCCCCAAGACTGCAAGGAGGAGGGGTGTAAATGTTCCTTAAGGTTATC
CACAGATGGCTCCCCAACTAGGATCACCTATGGCATGCAGGGGAGCTCCG
GTTATTCTCTGAGATTGTGTAAACTTGTGGACAGCCCTGACTGTACAACA
AAAATAAATGCACGTATTGTGGGAGGAACAAACGCTTCTTTAGGGGAGTG
GCCATGGCAGGTCAGCCTGCAAGTGAAGCTGGTATCTCAGACCCATTTGT
GTGGAGGGTCCATCATTGGTCGCCAATGGGTACTGACAGCTGCCCATTGC
TTTGATGGAATTCCCTATCCAGATGTGTGGCGTATATATGGCGGAATTCT
TAGTCTGTCCGAGATTACGAAAGAAACGCCTTCCTCGAGAATAAAGGAGC
TTATTATTCATCAGGAATACAAAGTCTCAGAAGGCAATTATGATATTGCC
TTAATAAAGCTTCAGACGCCCCTGAATTATACTGAATTCCAAAAACCAAT
ATGCCTGCCTTCCAAAGCTGACACAAATACAATTTATACCAACTGTTGGG
TGACTGGATGGGGCTACACGAAGGAACAAGGTGAAACGCAAAATATTCTA
CAAAAGGCTACTATTCCTTTGGTACCAAATGAAGAATGCCAGAAAAAATA
CAGAGATTATGTTATAAACAAGCAGATGATCTGTGCTGGCTACAAAGAAG
GCGGAACAGACGCTTGTAAGGGAGATTCCGGTGGCCCCTTAGTCTGTAAA
CACAGTGGACGGTGGCAGTTGGTGGGTATCACCAGCTGGGGTGAAGGCTG
CGCCCGCAAGGACCAACCAGGAGTCTACACCAAAGTTTCTGAGTACATGG
ACTGGATATTGGAGAAGACACAGAGCAGTGATGTAAGAGCTCTGGAGACA
TCTTCAGCCTGAGGAGGCTGGGTACCAAGGAGGAAGAACCCAGCTGGCTT
TACCACCTGCCCTCAAGGCAAACTAGAGCTCCAGGATTCTCGGCTGTAAA
ATGTTGATAATGGTGTCTACCTCACATCCGTATCATTGGATTGAAAATTC
AAGTGTAGATATAGTTGCTGAAGACAGCGTTTTGCTCAAGTGTGTTTCCT
GCCTTGAGTCACAGGAGCTCCAATGGGAGCATTACAAAGATCACCAAGCT
TGTTAGGAAAGAGAATGATCAAAGGGTTTTATTAGGTAATGAAATGTCTA
GATGTGATGCAATTGAAAAAAAGACCCCAGATTCTAGCACAGTCCTTGGG
ACCATTCTCATGTAACTGTTGACTCTGGACCTCAGCAGATCTCAGAGTTA
CCTGTCCACTTCTGACATTTGTTTATTAGAGCCTGATGCTATTCTTTCAA
GTGGAGCAAAAAAAAAAAAAAA Rat plasma kallikrein (ACCESSION:
NP_036857.2) >gi|162138905|ref|NP_036857.2|kallikrein B, plasma
1 [Rattus norvegicus] (SEQ ID NO: 6)
MILFKQVGYFVSLFATVSCGCLSQLYANTFFRGGDLAAIYTPDAQHCQKM
CTFHPRCLLFSFLAVSPTKETDKRFGCFMKESITGTLPRIHRTGAISGHS
LKQCGHQLSACHQDIYEGLDMRGSNFNISKTDSIEECQKLCTNNIHCQFF
TYATKAFHRPEYRKSCLLKRSSSGTPTSIKPVDNLVSGFSLKSCALSEIG
CPMDIFQHFAFADLNVSHVVTPDAFVCRTVCTFHPNCLFFTFYTNEWETE
SQRNVCFLKTSKSGRPSPPIIQENAVSGYSLFTCRKARPEPCHFKIYSGV
AFEGEELNATFVQGADACQETCTKTIRCQFFTYSLLPQDCKAEGCKCSLR
LSTDGSPTRITYEAQGSSGYSLRLCKVVESSDCTTKINARIVGGTNSSLG
EWPWQVSLQVKLVSQNHMCGGSIIGRQWILTAAHCFDGIPYPDVWRIYGG
ILNLSEITNKTPFSSIKELIIHQKYKMSEGSYDIALIKLQTPLNYTEFQK
PICLPSKADTNTIYTNCWVTGWGYTKERGETQNILQKATIPLVPNEECQK
KYRDYVITKQMICAGYKEGGIDACKGDSGGPLVCKHSGRWQLVGITSWGE
GCARKEQPGVYTKVAEYIDWILEKIQSSKERALETSPA Rat plasma kallikrein mRNA
(ACCESSION: NM_012725) >gi|162138904|ref|NM_012725.2|Rattus
norvegicus kallikrein B, plasma 1 (Klkb1), mRNA (SEQ ID NO: 7)
TGAAGACTAGCTTCATGTGAAGACTCCTTCTCCTCCAGCAGCACAAAGCA
ACCATCCTTCCAGGATGATTTTATTCAAACAAGTGGGTTATTTTGTTTCC
TTGTTCGCTACAGTTTCCTGTGGGTGTCTGTCACAACTGTATGCAAATAC
CTTCTTCAGAGGTGGGGATCTGGCTGCCATCTACACCCCGGATGCCCAGC
ACTGTCAGAAGATGTGCACGTTTCACCCCAGGTGCCTGCTCTTCAGCTTC
CTTGCCGTGAGTCCAACCAAGGAGACAGATAAAAGGTTTGGGTGCTTCAT
GAAAGAGAGCATTACAGGGACTTTGCCAAGAATACACCGGACAGGGGCCA
TTTCTGGTCATTCTTTAAAACAGTGTGGCCATCAATTAAGTGCTTGCCAC
CAAGACATATACGAAGGACTGGATATGAGAGGGTCCAACTTTAATATATC
TAAGACCGACAGTATTGAAGAATGCCAGAAACTGTGCACAAATAATATTC
ACTGCCAATTTTTCACATATGCTACAAAAGCATTTCACAGACCAGAGTAC
AGGAAGAGTTGCCTGCTGAAGCGCAGTTCAAGTGGAACGCCCACCAGTAT
AAAGCCAGTGGACAACCTGGTGTCTGGATTCTCACTGAAGTCCTGTGCTC
TCTCAGAGATCGGTTGCCCCATGGATATTTTCCAGCACTTTGCCTTTGCA
GACCTGAATGTAAGCCATGTCGTCACCCCCGATGCCTTCGTGTGTCGCAC
CGTTTGCACCTTCCATCCCAACTGCCTCTTCTTCACATTCTACACGAATG
AGTGGGAGACGGAATCACAGAGGAATGTTTGTTTTCTTAAGACATCTAAA
AGTGGAAGACCAAGTCCCCCTATTATTCAAGAAAATGCTGTATCTGGATA
CAGTCTCTTCACCTGCAGAAAAGCTCGCCCTGAACCCTGCCATTTCAAGA
TTTACTCTGGAGTTGCCTTCGAAGGGGAAGAACTGAACGCGACCTTCGTG
CAGGGAGCAGATGCGTGCCAAGAGACTTGTACAAAGACCATCCGCTGTCA
GTTTTTTACTTACTCATTGCTTCCCCAAGACTGCAAGGCAGAGGGGTGTA
AATGTTCCTTAAGGTTATCCACGGATGGCTCTCCAACTAGGATCACCTAT
GAGGCACAGGGGAGCTCTGGTTATTCTCTGAGACTGTGTAAAGTTGTGGA
GAGCTCTGACTGTACGACAAAAATAAATGCACGTATTGTGGGAGGAACAA
ACTCTTCTTTAGGAGAGTGGCCATGGCAGGTCAGCCTGCAAGTAAAGTTG
GTTTCTCAGAATCATATGTGTGGAGGGTCCATCATTGGACGCCAATGGAT
ACTGACGGCTGCCCATTGCTTTGATGGGATTCCCTATCCAGACGTGTGGC
GTATATATGGCGGGATTCTTAATCTGTCAGAGATTACAAACAAAACGCCT
TTCTCAAGTATAAAGGAGCTTATTATTCATCAGAAATACAAAATGTCAGA
AGGCAGTTACGATATTGCCTTAATAAAGCTTCAGACACCGTTGAATTATA
CTGAATTCCAAAAACCAATATGCCTGCCTTCCAAAGCTGACACAAATACA
ATTTATACCAACTGCTGGGTGACTGGATGGGGCTACACAAAGGAACGAGG
TGAGACCCAAAATATTCTACAAAAGGCAACTATTCCCTTGGTACCAAATG
AAGAATGCCAGAAAAAATATAGAGATTATGTTATAACCAAGCAGATGATC
TGTGCTGGCTACAAAGAAGGTGGAATAGATGCTTGTAAGGGAGATTCCGG
TGGCCCCTTAGTTTGCAAACATAGTGGAAGGTGGCAGTTGGTGGGTATCA
CCAGCTGGGGCGAAGGCTGTGCCCGCAAGGAGCAACCAGGAGTCTACACC
AAAGTTGCTGAGTACATTGACTGGATATTGGAGAAGATACAGAGCAGCAA
GGAAAGAGCTCTGGAGACATCTCCAGCATGAGGAGGCTGGGTACTGATGG
GGAAGAGCCCAGCTGGCACCAGCTTTACCACCTGCCCTCAAGTCCTACTA
GAGCTCCAGAGTTCTCTTCTGCAAAATGTCGATAGTGGTGTCTACCTCGC
ATCCTTACCATAGGATTAAAAGTCCAAATGTAGACACAGTTGCTAAAGAC
AGCGCCATGCTCAAGCGTGCTTCCTGCCTTGAGCAACAGGAACGCCAATG
AGAACTATCCAAAGATTACCAAGCCTGTTTGGAAATAAAATGGTCAAAGG
ATTTTTATTAGGTAGTGAAATTAGGTAGTTGTCCTTGGAACCATTCTCAT
GTAACTGTTGACTCTGGACCTCAGCAGATCACAGTTACCTTCTGTCCACT
TCTGACATTTGTGTACTGGAACCTGATGCTGTTCTTCCACTTGGAGCAAA
GAACTGAGAAACCTGGTTCTATCCATTGGGAAAAAGAGATCTTTGTAACA
TTTCCTTTACAATAAAAAGATGTTCTACTTGGACTTGAAAAAAAAAAAAA
AAAAAAAAAAAAA
Display Libraries
[0601] A display library is a collection of entities; each entity
includes an accessible polypeptide component and a recoverable
component that encodes or identifies the polypeptide component. The
polypeptide component is varied so that different amino acid
sequences are represented. The polypeptide component can be of any
length, e.g. from three amino acids to over 300 amino acids. A
display library entity can include more than one polypeptide
component, for example, the two polypeptide chains of a sFab. In
one exemplary implementation, a display library can be used to
identify proteins that bind to plasma kallikrein. In a selection,
the polypeptide component of each member of the library is probed
with plasma kallikrein (or fragment thereof) and if the polypeptide
component binds to the plasma kallikrein, the display library
member is identified, typically by retention on a support.
[0602] Retained display library members are recovered from the
support and analyzed. The analysis can include amplification and a
subsequent selection under similar or dissimilar conditions. For
example, positive and negative selections can be alternated. The
analysis can also include determining the amino acid sequence of
the polypeptide component and purification of the polypeptide
component for detailed characterization.
[0603] A variety of formats can be used for display libraries.
Examples include the following.
[0604] Phage Display:
[0605] The protein component is typically covalently linked to a
bacteriophage coat protein. The linkage results from translation of
a nucleic acid encoding the protein component fused to the coat
protein. The linkage can include a flexible peptide linker, a
protease site, or an amino acid incorporated as a result of
suppression of a stop codon. Phage display is described, for
example, in U.S. Pat. No. 5,223,409; Smith (1985) Science
228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;
WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al.
(1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998)
Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today
2:371-8 and Hoet et al. (2005) Nat Biotechnol. 23(3)344-8.
Bacteriophage displaying the protein component can be grown and
harvested using standard phage preparatory methods, e.g. PEG
precipitation from growth media. After selection of individual
display phages, the nucleic acid encoding the selected protein
components can be isolated from cells infected with the selected
phages or from the phage themselves, after amplification.
Individual colonies or plaques can be picked, the nucleic acid
isolated and sequenced.
[0606] Other Display Formats.
[0607] Other display formats include cell based display (see, e.g.,
WO 03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat.
No. 6,207,446), ribosome display (See, e.g., Mattheakis et al.
(1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000)
Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol.
328:404-30; and Schaffitzel et al. (1999) J Immunol Methods.
231(1-2):119-35), and E. coli periplasmic display (J Immunol
Methods. 2005 Nov. 22; PMID: 16337958).
[0608] Scaffolds.
[0609] Scaffolds useful for display include: antibodies (e.g., Fab
fragments, single chain Fv molecules (scFv), single domain
antibodies, camelid antibodies, and camelized antibodies); T-cell
receptors; MHC proteins; extracellular domains (e.g., fibronectin
Type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz
domains, ecotin, BPTI, and so forth); TPR repeats; trifoil
structures; zinc finger domains; DNA-binding proteins; particularly
monomeric DNA binding proteins; RNA binding proteins; enzymes,
e.g., proteases (particularly inactivated proteases), RNase;
chaperones, e.g., thioredoxin and heat shock proteins;
intracellular signaling domains (such as SH2 and SH3 domains);
linear and constrained peptides; and linear peptide substrates.
Display libraries can include synthetic and/or natural diversity.
See, e.g., U.S. 2004-0005709.
[0610] Display technology can also be used to obtain binding
proteins (e.g., antibodies) that bind particular epitopes of a
target. This can be done, for example, by using competing
non-target molecules that lack the particular epitope or are
mutated within the epitope, e.g., with alanine. Such non-target
molecules can be used in a negative selection procedure as
described below, as competing molecules when binding a display
library to the target, or as a pre-elution agent, e.g., to capture
in a wash solution dissociating display library members that are
not specific to the target.
[0611] Iterative Selection.
[0612] In one preferred embodiment, display library technology is
used in an iterative mode. A first display library is used to
identify one or more binding proteins for a target. These
identified binding proteins are then varied using a mutagenesis
method to form a second display library. Higher affinity binding
proteins are then selected from the second library, e.g., by using
higher stringency or more competitive binding and washing
conditions.
[0613] In some implementations, the mutagenesis is targeted to
regions at the binding interface. If, for example, the identified
binding proteins are antibodies, then mutagenesis can be directed
to the CDR regions of the heavy or light chains as described
herein. Further, mutagenesis can be directed to framework regions
near or adjacent to the CDRs. In the case of antibodies,
mutagenesis can also be limited to one or a few of the CDRs, e.g.,
to make precise step-wise improvements. Exemplary mutagenesis
techniques include: error-prone PCR, recombination, DNA shuffling,
site-directed mutagenesis and cassette mutagenesis.
[0614] In one example of iterative selection, the methods described
herein are used to first identify a protein from a display library
that binds plasma kallikrein, with at least a minimal binding
specificity for a target or a minimal activity, e.g., an
equilibrium dissociation constant for binding of less than 0.5 nM,
1 nM, 10 nM, or 100 nM. The nucleic acid sequences encoding the
initial identified proteins are used as a template nucleic acid for
the introduction of variations, e.g., to identify a second protein
that has enhanced properties (e.g., binding affinity, kinetics, or
stability) relative to the initial protein.
[0615] Off-Rate Selection.
[0616] Since a slow dissociation rate can be predictive of high
affinity, particularly with respect to interactions between
polypeptides and their targets, the methods described herein can be
used to isolate binding proteins with a desired (e.g., reduced)
kinetic dissociation rate for a binding interaction to a
target.
[0617] To select for slow dissociating binding proteins from a
display library, the library is contacted to an immobilized target.
The immobilized target is then washed with a first solution that
removes non-specifically or weakly bound biomolecules. Then the
bound binding proteins are eluted with a second solution that
includes a saturating amount of free target or a target specific
high-affinity competing monoclonal antibody, i.e., replicates of
the target that are not attached to the particle. The free target
binds to biomolecules that dissociate from the target. Rebinding is
effectively prevented by the saturating amount of free target
relative to the much lower concentration of immobilized target.
[0618] The second solution can have solution conditions that are
substantially physiological or that are stringent. Typically, the
solution conditions of the second solution are identical to the
solution conditions of the first solution. Fractions of the second
solution are collected in temporal order to distinguish early from
late fractions. Later fractions include biomolecules that
dissociate at a slower rate from the target than biomolecules in
the early fractions.
[0619] Further, it is also possible to recover display library
members that remain bound to the target even after extended
incubation. These can either be dissociated using chaotropic
conditions or can be amplified while attached to the target. For
example, phage bound to the target can be contacted to bacterial
cells.
[0620] Selecting or Screening for Specificity.
[0621] The display library screening methods described herein can
include a selection or screening process that discards display
library members that bind to a non-target molecule. Examples of
non-target molecules include streptavidin on magnetic beads,
blocking agents such as bovine serum albumin, non-fat bovine milk,
soy protein, any capturing or target immobilizing monoclonal
antibody, or non-transfected cells which do not express the
target.
[0622] In one implementation, a so-called "negative selection" step
is used to discriminate between the target and related non-target
molecule and a related, but distinct non-target molecule. The
display library or a pool thereof is contacted to the non-target
molecule. Members of the sample that do not bind the non-target are
collected and used in subsequent selections for binding to the
target molecule or even for subsequent negative selections. The
negative selection step can be prior to or after selecting library
members that bind to the target molecule.
[0623] In another implementation, a screening step is used. After
display library members are isolated for binding to the target
molecule, each isolated library member is tested for its ability to
bind to a non-target molecule (e.g., a non-target listed above).
For example, a high-throughput ELISA screen can be used to obtain
this data. The ELISA screen can also be used to obtain quantitative
data for binding of each library member to the target as well as
for cross species reactivity to related targets or subunits of the
target (e.g., plasma kallikrein) and also under different condition
such as pH 6 or pH 7.5. The non-target and target binding data are
compared (e.g., using a computer and software) to identify library
members that specifically bind to the target.
Other Exemplary Expression Libraries
[0624] Other types of collections of proteins (e.g., expression
libraries) can be used to identify proteins with a particular
property (e.g., ability to bind plasma kallikrein), including,
e.g., protein arrays of antibodies (see, e.g., De Wildt et al.
(2000) Nat. Biotechnol. 18:989-994), lambda gt11 libraries,
two-hybrid libraries and so forth.
Exemplary Libraries
[0625] It is possible to immunize a non-human primate and recover
primate antibody genes that can be displayed on phage (see below).
From such a library, one can select antibodies that bind the
antigen used in immunization. See, for example, Vaccine. (2003)
22(2):257-67 or Immunogenetics. (2005) 57(10):730-8. Thus one could
obtain primate antibodies that bind and inhibit plasma kallikrein
by immunizing a chimpanzee or macaque and using a variety of means
to select or screen for primate antibodies that bind and inhibit
plasma kallikrein. One can also make chimeras of primatized Fabs
with human constant regions, see Curr Opin Mol Ther. (2004)
6(6):675-83. "PRIMATIZED antibodies, genetically engineered from
cynomolgus macaque monkey and human components, are structurally
indistinguishable from human antibodies. They may, therefore, be
less likely to cause adverse reactions in humans, making them
potentially suited for long-term, chronic treatment" Curr Opin
Investig Drugs. (2001) 2(5):635-8.
[0626] One exemplary type of library presents a diverse pool of
polypeptides, each of which includes an immunoglobulin domain,
e.g., an immunoglobulin variable domain. Of interest are display
libraries where the members of the library include primate or
"primatized" (e.g., such as human, non-human primate or
"humanized") immunoglobin domains (e.g., immunoglobin variable
domains) or chimeric primatized Fabs with human constant regions.
Human or humanized immunoglobin domain libraries may be used to
identify human or "humanized" antibodies that, for example,
recognize human antigens. Because the constant and framework
regions of the antibody are human, these antibodies may avoid
themselves being recognized and targeted as antigens when
administered to humans. The constant regions may also be optimized
to recruit effector functions of the human immune system. The in
vitro display selection process surmounts the inability of a normal
human immune system to generate antibodies against
self-antigens.
[0627] A typical antibody display library displays a polypeptide
that includes a VH domain and a VL domain. An "immunoglobulin
domain" refers to a domain from the variable or constant domain of
immunoglobulin molecules. Immunoglobulin domains typically contain
two .beta.-sheets formed of about seven .beta.-strands, and a
conserved disulphide bond (see, e.g., A. F. Williams and A. N.
Barclay, 1988, Ann. Rev. Immunol. 6:381-405). The display library
can display the antibody as a Fab fragment (e.g., using two
polypeptide chains) or a single chain Fv (e.g., using a single
polypeptide chain). Other formats can also be used.
[0628] As in the case of the Fab and other formats, the displayed
antibody can include one or more constant regions as part of a
light and/or heavy chain. In one embodiment, each chain includes
one constant region, e.g., as in the case of a Fab. In other
embodiments, additional constant regions are displayed.
[0629] Antibody libraries can be constructed by a number of
processes (see, e.g., de Haard et al., 1999, J. Biol. Chem.
274:18218-30; Hoogenboom et al., 1998, Immunotechnology 4:1-20;
Hoogenboom et al., 2000, Immunol. Today 21:371-378, and Hoet et al.
(2005) Nat Biotechnol. 23(3):344-8. Further, elements of each
process can be combined with those of other processes. The
processes can be used such that variation is introduced into a
single immunoglobulin domain (e.g., VH or VL) or into multiple
immunoglobulin domains (e.g., VH and VL). The variation can be
introduced into an immunoglobulin variable domain, e.g., in the
region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and/or
FR4, referring to such regions of either and both of heavy and
light chain variable domains. For example, the variation(s) may be
introduced into all three CDRs of a given variable domain, or into
CDR1 and CDR2, e.g., of a heavy chain variable domain. Any
combination is feasible. In one process, antibody libraries are
constructed by inserting diverse oligonucleotides that encode CDRs
into the corresponding regions of the nucleic acid. The
oligonucleotides can be synthesized using monomeric nucleotides or
trinucleotides. For example, Knappik et al., 2000, J. Mol. Biol.
296:57-86 describe a method for constructing CDR encoding
oligonucleotides using trinucleotide synthesis and a template with
engineered restriction sites for accepting the
oligonucleotides.
[0630] In another process, an animal (e.g., a rodent) is immunized
with plasma kallikrein. The animal is optionally boosted with the
antigen to further stimulate the response. Then spleen cells are
isolated from the animal, and nucleic acid encoding VH and/or VL
domains is amplified and cloned for expression in the display
library.
[0631] In yet another process, antibody libraries are constructed
from nucleic acid amplified from naive germline immunoglobulin
genes. The amplified nucleic acid includes nucleic acid encoding
the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic
acids are described below. Amplification can include PCR, e.g.,
with primers that anneal to the conserved constant region, or
another amplification method.
[0632] Nucleic acid encoding immunoglobulin domains can be obtained
from the immune cells of, e.g., a primate (e.g., a human), mouse,
rabbit, camel, or rodent. In one example, the cells are selected
for a particular property. B cells at various stages of maturity
can be selected. In another example, the B cells are naive.
[0633] In one embodiment, fluorescent-activated cell sorting (FACS)
is used to sort B cells that express surface-bound IgM, IgD, or IgG
molecules. Further, B cells expressing different isotypes of IgG
can be isolated. In another preferred embodiment, the B or T cells
are cultured in vitro. The cells can be stimulated in vitro, e.g.,
by culturing with feeder cells or by adding mitogens or other
modulatory reagents, such as antibodies to CD40, CD40 ligand or
CD20, phorbol myristate acetate, bacterial lipopolysaccharide,
concanavalin A, phytohaemagglutinin, or pokeweed mitogen.
[0634] In another embodiment, the cells are isolated from a subject
that has a disease of condition described herein, e.g., a plasma
kallikrein associated disease or condition.
[0635] In one preferred embodiment, the cells have activated a
program of somatic hypermutation. Cells can be stimulated to
undergo somatic mutagenesis of immunoglobulin genes, for example,
by treatment with anti-immunoglobulin, anti-CD40, and anti-CD38
antibodies (see, e.g., Bergthorsdottir et al., 2001, J. Immunol.
166:2228). In another embodiment, the cells are naive.
[0636] The nucleic acid encoding an immunoglobulin variable domain
can be isolated from a natural repertoire by the following
exemplary method. First, RNA is isolated from the immune cell. Full
length (i.e., capped) mRNAs are separated (e.g. by degrading
uncapped RNAs with calf intestinal phosphatase). The cap is then
removed with tobacco acid pyrophosphatase and reverse transcription
is used to produce the cDNAs.
[0637] The reverse transcription of the first (antisense) strand
can be done in any manner with any suitable primer. See, e.g., de
Haard et al., 1999, J. Biol. Chem. 274:18218-30. The primer binding
region can be constant among different immunoglobulins, e.g., in
order to reverse transcribe different isotypes of immunoglobulin.
The primer binding region can also be specific to a particular
isotype of immunoglobulin. Typically, the primer is specific for a
region that is 3' to a sequence encoding at least one CDR. In
another embodiment, poly-dT primers may be used (and may be
preferred for the heavy-chain genes).
[0638] A synthetic sequence can be ligated to the 3' end of the
reverse transcribed strand. The synthetic sequence can be used as a
primer binding site for binding of the forward primer during PCR
amplification after reverse transcription. The use of the synthetic
sequence can obviate the need to use a pool of different forward
primers to fully capture the available diversity.
[0639] The variable domain-encoding gene is then amplified, e.g.,
using one or more rounds. If multiple rounds are used, nested
primers can be used for increased fidelity. The amplified nucleic
acid is then cloned into a display library vector.
Secondary Screening Methods
[0640] After selecting candidate library members that bind to a
target, each candidate library member can be further analyzed,
e.g., to further characterize its binding properties for the
target, e.g., plasma kallikrein. Each candidate library member can
be subjected to one or more secondary screening assays. The assay
can be for a binding property, a catalytic property, an inhibitory
property, a physiological property (e.g., cytotoxicity, renal
clearance, immunogenicity), a structural property (e.g., stability,
conformation, oligomerization state) or another functional
property. The same assay can be used repeatedly, but with varying
conditions, e.g., to determine pH, ionic, or thermal
sensitivities.
[0641] As appropriate, the assays can use a display library member
directly, a recombinant polypeptide produced from the nucleic acid
encoding the selected polypeptide, or a synthetic peptide
synthesized based on the sequence of the selected polypeptide. In
the case of selected Fabs, the Fabs can be evaluated or can be
modified and produced as intact IgG proteins. Exemplary assays for
binding properties include the following.
[0642] ELISA.
[0643] Binding proteins can be evaluated using an ELISA assay. For
example, each protein is contacted to a microtitre plate whose
bottom surface has been coated with the target, e.g., a limiting
amount of the target. The plate is washed with buffer to remove
non-specifically bound polypeptides. Then the amount of the binding
protein bound to the target on the plate is determined by probing
the plate with an antibody that can recognize the binding protein,
e.g., a tag or constant portion of the binding protein. The
antibody is linked to a detection system (e.g., an enzyme such as
alkaline phosphatase or horse radish peroxidase (HRP) which
produces a colorimetric product when appropriate substrates are
provided).
[0644] Homogeneous Binding Assays.
[0645] The ability of a binding protein described herein to bind a
target can be analyzed using a homogenous assay, i.e., after all
components of the assay are added, additional fluid manipulations
are not required. For example, fluorescence resonance energy
transfer (FRET) can be used as a homogenous assay (see, for
example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos,
et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first
molecule (e.g., the molecule identified in the fraction) is
selected such that its emitted fluorescent energy can be absorbed
by a fluorescent label on a second molecule (e.g., the target) if
the second molecule is in proximity to the first molecule. The
fluorescent label on the second molecule fluoresces when it absorbs
to the transferred energy. Since the efficiency of energy transfer
between the labels is related to the distance separating the
molecules, the spatial relationship between the molecules can be
assessed. In a situation in which binding occurs between the
molecules, the fluorescent emission of the `acceptor` molecule
label in the assay should be maximal. A binding event that is
configured for monitoring by FRET can be conveniently measured
through standard fluorometric detection means, e.g., using a
fluorimeter. By titrating the amount of the first or second binding
molecule, a binding curve can be generated to estimate the
equilibrium binding constant.
[0646] Another example of a homogenous assay is ALPHASCREEN.TM.
(Packard Bioscience, Meriden Conn.). ALPHASCREEN.TM. uses two
labeled beads. One bead generates singlet oxygen when excited by a
laser. The other bead generates a light signal when singlet oxygen
diffuses from the first bead and collides with it. The signal is
only generated when the two beads are in proximity. One bead can be
attached to the display library member, the other to the target.
Signals are measured to determine the extent of binding.
[0647] Surface Plasmon Resonance (SPR).
[0648] The interaction of binding protein and a target can be
analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA)
detects biospecific interactions in real time, without labeling any
of the interactants. Changes in the mass at the binding surface
(indicative of a binding event) of the BIA chip result in
alterations of the refractive index of light near the surface (the
optical phenomenon of surface plasmon resonance (SPR)). The changes
in the refractivity generate a detectable signal, which are
measured as an indication of real-time reactions between biological
molecules. Methods for using SPR are described, for example, in
U.S. Pat. No. 5,641,640; Raether, 1988, Surface Plasmons Springer
Verlag; Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345;
Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705 and on-line
resources provide by BIAcore International AB (Uppsala,
Sweden).
[0649] Information from SPR can be used to provide an accurate and
quantitative measure of the equilibrium dissociation constant
(K.sub.D), and kinetic parameters, including K.sub.on and
K.sub.off, for the binding of a binding protein to a target. Such
data can be used to compare different biomolecules. For example,
selected proteins from an expression library can be compared to
identify proteins that have high affinity for the target or that
have a slow K.sub.off. This information can also be used to develop
structure-activity relationships (SAR). For example, the kinetic
and equilibrium binding parameters of matured versions of a parent
protein can be compared to the parameters of the parent protein.
Variant amino acids at given positions can be identified that
correlate with particular binding parameters, e.g., high affinity
and slow K.sub.off. This information can be combined with
structural modeling (e.g., using homology modeling, energy
minimization, or structure determination by x-ray crystallography
or NMR). As a result, an understanding of the physical interaction
between the protein and its target can be formulated and used to
guide other design processes.
[0650] Cellular Assays.
[0651] Binding proteins can be screened for ability to bind to
cells which transiently or stably express and display the target of
interest on the cell surface. For example, plasma kallikrein
binding proteins can be fluorescently labeled and binding to plasma
kallikrein in the presence of absence of antagonistic antibody can
be detected by a change in fluorescence intensity using flow
cytometry e.g., a FACS machine.
Other Exemplary Methods for Obtaining Plasma Kallikrein Binding
Proteins
[0652] In addition to the use of display libraries, other methods
can be used to obtain a plasma kallikrein binding protein (e.g.,
antibody). For example, plasma kallikrein protein or a fragment
thereof can be used as an antigen in a non-human animal, e.g., a
rodent.
[0653] In one embodiment, the non-human animal includes at least a
part of a human immunoglobulin gene. For example, it is possible to
engineer mouse strains deficient in mouse antibody production with
large fragments of the human Ig loci. Using the hybridoma
technology, antigen-specific monoclonal antibodies (Mabs) derived
from the genes with the desired specificity may be produced and
selected. See, e.g., XENOMOUSE.TM., Green et al., 1994, Nat. Gen.
7:13-21; U.S. 2003-0070185, WO 96/34096, published Oct. 31, 1996,
and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
[0654] In another embodiment, a monoclonal antibody is obtained
from the non-human animal, and then modified, e.g., humanized or
deimmunized. Winter describes a CDR-grafting method that may be
used to prepare the humanized antibodies (UK Patent Application GB
2188638A, filed on Mar. 26, 1987; U.S. Pat. No. 5,225,539. All of
the CDRs of a particular human antibody may be replaced with at
least a portion of a non-human CDR or only some of the CDRs may be
replaced with non-human CDRs. It is only necessary to replace the
number of CDRs required for binding of the humanized antibody to a
predetermined antigen.
[0655] Humanized antibodies can be generated by replacing sequences
of the Fv variable region that are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986,
BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089,
5,693,761 and 5,693,762. Those methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of immunoglobulin Fv variable regions from at least one
of a heavy or light chain. Numerous sources of such nucleic acid
are available. For example, nucleic acids may be obtained from a
hybridoma producing an antibody against a predetermined target, as
described above. The recombinant DNA encoding the humanized
antibody, or fragment thereof, can then be cloned into an
appropriate expression vector.
[0656] Reducing Immunogenicity of Plasma Kallikrein Binding
Proteins
[0657] Immunoglobin plasma kallikrein binding proteins (e.g., IgG
or Fab plasma kallikrein binding proteins) may be modified to
reduce immunogenicity. Reduced immunogenicity is desirable in
plasma kallikrein binding proteins intended for use as
therapeutics, as it reduces the chance that the subject will
develop an immune response against the therapeutic molecule.
Techniques useful for reducing immunogenicity of plasma kallikrein
binding proteins include deletion/modification of potential human T
cell epitopes and "germlining" of sequences outside of the CDRs
(e.g., framework and Fc).
[0658] A plasma kallikrein-binding antibody may be modified by
specific deletion of human T cell epitopes or "deimmunization,"
e.g., by the methods disclosed in WO 98/52976 and WO 00/34317.
Briefly, the heavy and light chain variable regions of an antibody
are analyzed for peptides that bind to MHC Class II; these peptides
represent potential T-cell epitopes (as defined in WO 98/52976 and
WO 00/34317). For detection of potential T-cell epitopes, a
computer modeling approach termed "peptide threading" can be
applied, and in addition a database of human MHC class II binding
peptides can be searched for motifs present in the VH and VL
sequences, as described in WO 98/52976 and WO 00/34317. These
motifs bind to any of the 18 major MHC class II DR allotypes, and
thus constitute potential T cell epitopes. Potential T-cell
epitopes detected can be eliminated by substituting small numbers
of amino acid residues in the variable regions, or preferably, by
single amino acid substitutions. As far as possible conservative
substitutions are made, often but not exclusively, an amino acid
common at this position in human germline antibody sequences may be
used. Human germline sequences are disclosed in Tomlinson, I. A. et
al., 1992, J. Mol. Biol. 227:776-798; Cook, G. P. et al., 1995,
Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al., 1992, J.
Mol. Bio. 227:799-817. The V BASE directory provides a
comprehensive directory of human immunoglobulin variable region
sequences (compiled by Tomlinson, I. A. et al. MRC Centre for
Protein Engineering, Cambridge, UK). After the deimmunizing changes
are identified, nucleic acids encoding V.sub.H and V.sub.L can be
constructed by mutagenesis or other synthetic methods (e.g., de
novo synthesis, cassette replacement, and so forth). Mutagenized
variable sequence can, optionally, be fused to a human constant
region, e.g., human IgG1 or .kappa. constant regions.
[0659] In some cases a potential T cell epitope will include
residues which are known or predicted to be important for antibody
function. For example, potential T cell epitopes are usually biased
towards the CDRs. In addition, potential T cell epitopes can occur
in framework residues important for antibody structure and binding.
Changes to eliminate these potential epitopes will in some cases
require more scrutiny, e.g., by making and testing chains with and
without the change. Where possible, potential T cell epitopes that
overlap the CDRs were eliminated by substitutions outside the CDRs.
In some cases, an alteration within a CDR is the only option, and
thus variants with and without this substitution should be tested.
In other cases, the substitution required to remove a potential T
cell epitope is at a residue position within the framework that
might be critical for antibody binding. In these cases, variants
with and without this substitution should be tested. Thus, in some
cases several variant deimmunized heavy and light chain variable
regions were designed and various heavy/light chain combinations
tested in order to identify the optimal deimmunized antibody. The
choice of the final deimmunized antibody can then be made by
considering the binding affinity of the different variants in
conjunction with the extent of deimmunization, i.e., the number of
potential T cell epitopes remaining in the variable region.
Deimmunization can be used to modify any antibody, e.g., an
antibody that includes a non-human sequence, e.g., a synthetic
antibody, a murine antibody other non-human monoclonal antibody, or
an antibody isolated from a display library.
[0660] Plasma kallikrein binding antibodies are "germlined" by
reverting one or more non-germline amino acids in framework regions
to corresponding germline amino acids of the antibody, so long as
binding properties are substantially retained. Similar methods can
also be used in the constant region, e.g., in constant
immunoglobulin domains.
[0661] Antibodies that bind to plasma kallikrein, e.g., an antibody
described herein, may be modified in order to make the variable
regions of the antibody more similar to one or more germline
sequences. For example, an antibody can include one, two, three, or
more amino acid substitutions, e.g., in a framework, CDR, or
constant region, to make it more similar to a reference germline
sequence. One exemplary germlining method can include identifying
one or more germline sequences that are similar (e.g., most similar
in a particular database) to the sequence of the isolated antibody.
Mutations (at the amino acid level) are then made in the isolated
antibody, either incrementally or in combination with other
mutations. For example, a nucleic acid library that includes
sequences encoding some or all possible germline mutations is made.
The mutated antibodies are then evaluated, e.g., to identify an
antibody that has one or more additional germline residues relative
to the isolated antibody and that is still useful (e.g., has a
functional activity). In one embodiment, as many germline residues
are introduced into an isolated antibody as possible.
[0662] In one embodiment, mutagenesis is used to substitute or
insert one or more germline residues into a framework and/or
constant region. For example, a germline framework and/or constant
region residue can be from a germline sequence that is similar
(e.g., most similar) to the non-variable region being modified.
After mutagenesis, activity (e.g., binding or other functional
activity) of the antibody can be evaluated to determine if the
germline residue or residues are tolerated (i.e., do not abrogate
activity). Similar mutagenesis can be performed in the framework
regions.
[0663] Selecting a germline sequence can be performed in different
ways. For example, a germline sequence can be selected if it meets
a predetermined criteria for selectivity or similarity, e.g., at
least a certain percentage identity, e.g., at least 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The
selection can be performed using at least 2, 3, 5, or 10 germline
sequences. In the case of CDR1 and CDR2, identifying a similar
germline sequence can include selecting one such sequence. In the
case of CDR3, identifying a similar germline sequence can include
selecting one such sequence, but may include using two germline
sequences that separately contribute to the amino-terminal portion
and the carboxy-terminal portion of the sequence. In other
implementations more than one or two germline sequences are used,
e.g., to form a consensus sequence.
[0664] In one embodiment, with respect to a particular reference
variable domain sequence, e.g., a sequence described herein, a
related variable domain sequence has at least 30, 40, 50, 60, 70,
80, 90, 95 or 100% of the CDR amino acid positions that are not
identical to residues in the reference CDR sequences, residues that
are identical to residues at corresponding positions in a human
germline sequence (i.e., an amino acid sequence encoded by a human
germline nucleic acid).
[0665] In one embodiment, with respect to a particular reference
variable domain sequence, e.g., a sequence described herein, a
related variable domain sequence has at least 30, 50, 60, 70, 80,
90 or 100% of the FR regions identical to FR sequence from a human
germline sequence, e.g., a germline sequence related to the
reference variable domain sequence.
[0666] Accordingly, it is possible to isolate an antibody which has
similar activity to a given antibody of interest, but is more
similar to one or more germline sequences, particularly one or more
human germline sequences. For example, an antibody can be at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to a
germline sequence in a region outside the CDRs (e.g., framework
regions). Further, an antibody can include at least 1, 2, 3, 4, or
5 germline residues in a CDR region, the germline residue being
from a germline sequence of similar (e.g., most similar) to the
variable region being modified. Germline sequences of primary
interest are human germline sequences. The activity of the antibody
(e.g., the binding activity as measured by K.sub.A) can be within a
factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original
antibody.
[0667] Germline sequences of human immunoglobin genes have been
determined and are available from a number of sources, including
the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM.RTM. (IMGT),
and the V BASE directory (compiled by Tomlinson, I. A. et al. MRC
Centre for Protein Engineering, Cambridge, UK).
[0668] Exemplary germline reference sequences for V.sub.kappa
include: O12/O2, O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19,
L8, L23, L9, L24, L11, L12, O11/O1, A17, A1, A18, A2, A19/A3, A23,
A27, A11, L2/L16, L6, L20, L25, B3, B2, A26/A10, and A14. See,
e.g., Tomlinson et al., 1995, EMBO J. 14(18):4628-3.
[0669] A germline reference sequence for the HC variable domain can
be based on a sequence that has particular canonical structures,
e.g., 1-3 structures in the H1 and H2 hypervariable loops. The
canonical structures of hypervariable loops of an immunoglobulin
variable domain can be inferred from its sequence, as described in
Chothia et al., 1992, J. Mol. Biol. 227:799-817; Tomlinson et al.,
1992, J. Mol. Biol. 227:776-798); and Tomlinson et al., 1995, EMBO
J. 14(18):4628-38. Exemplary sequences with a 1-3 structure
include: DP-1, DP-8, DP-12, DP-2, DP-25, DP-15, DP-7, DP-4, DP-31,
DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3005f3, DP-46, DP-47,
DP-58, DP-49, DP-50, DP-51, DP-53, and DP-54.
Protein Production
[0670] Standard recombinant nucleic acid methods can be used to
express a protein that binds to plasma kallikrein. Generally, a
nucleic acid sequence encoding the protein is cloned into a nucleic
acid expression vector. Of course, if the protein includes multiple
polypeptide chains, each chain can be cloned into an expression
vector, e.g., the same or different vectors, that are expressed in
the same or different cells.
[0671] Antibody Production.
[0672] Some antibodies, e.g., Fabs, can be produced in bacterial
cells, e.g., E. coli cells (see e.g., Nadkarni, A. et al., 2007
Protein Expr Purif 52(1):219-29). For example, if the Fab is
encoded by sequences in a phage display vector that includes a
suppressible stop codon between the display entity and a
bacteriophage protein (or fragment thereof), the vector nucleic
acid can be transferred into a bacterial cell that cannot suppress
a stop codon. In this case, the Fab is not fused to the gene III
protein and is secreted into the periplasm and/or media.
[0673] Antibodies can also be produced in eukaryotic cells. In one
embodiment, the antibodies (e.g., scFv's) are expressed in a yeast
cell such as Pichia (see, e.g., Powers et al., 2001, J. Immunol.
Methods. 251:123-35; Schoonooghe S. et al., 2009 BMC Biotechnol.
9:70; Abdel-Salam, H A. et al., 2001 Appl Microbiol Biotechnol
56(1-2):157-64; Takahashi K. et al., 2000 Biosci Biotechnol Biochem
64(10):2138-44; Edqvist, J. et al., 1991 J Biotechnol
20(3):291-300), Hanseula, or Saccharomyces. One of skill in the art
can optimize antibody production in yeast by optimizing, for
example, oxygen conditions (see e.g., Baumann K., et al. 2010 BMC
Syst. Biol. 4:141), osmolarity (see e.g., Dragosits, M. et al.,
2010 BMC Genomics 11:207), temperature (see e.g., Dragosits, M. et
al., 2009 J Proteome Res. 8(3):1380-92), fermentation conditions
(see e.g., Ning, D. et al. 2005 J. Biochem. and Mol. Biol. 38(3):
294-299), strain of yeast (see e.g., Kozyr, A V et al. 2004 Mol
Biol (Mosk) 38(6):1067-75; Horwitz, A H. et al., 1988 Proc Natl
Acad Sci USA 85(22):8678-82; Bowdish, K. et al. 1991 J Biol Chem
266(18):11901-8), overexpression of proteins to enhance antibody
production (see e.g., Gasser, B. et al., 2006 Biotechol. Bioeng.
94(2):353-61), level of acidity of the culture (see e.g., Kobayashi
H., et al., 1997 FEMS Microbiol Lett 152(2):235-42), concentrations
of substrates and/or ions (see e.g., Ko J H. et al., 2996 Appl
Biochem Biotechnol 60(1):41-8). In addition, yeast systems can be
used to produce antibodies with an extended half-life (see e.g.,
Smith, B J. et al. 2001 Bioconjug Chem 12(5):750-756),
[0674] In one preferred embodiment, antibodies are produced in
mammalian cells. Preferred mammalian host cells for expressing the
clone antibodies or antigen-binding fragments thereof include
Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells,
described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA
77:4216-4220, used with a DHFR selectable marker, e.g., as
described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621),
lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS
cells, HEK293T cells (J. Immunol. Methods (2004) 289(1-2):65-80),
and a cell from a transgenic animal, e.g., a transgenic mammal. For
example, the cell is a mammary epithelial cell.
[0675] In some embodiments, plasma kallikrein binding proteins are
produced in a plant or cell-free based system (see e.g., Galeffi,
P., et al., 2006 J Transl Med 4:39).
[0676] In addition to the nucleic acid sequence encoding the
diversified immunoglobulin domain, the recombinant expression
vectors may carry additional sequences, such as sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0677] In an exemplary system for recombinant expression of an
antibody, or antigen-binding portion thereof, a recombinant
expression vector encoding both the antibody heavy chain and the
antibody light chain is introduced into dhfr.sup.- CHO cells by
calcium phosphate-mediated transfection. Within the recombinant
expression vector, the antibody heavy and light chain genes are
each operatively linked to enhancer/promoter regulatory elements
(e.g., derived from SV40, CMV, adenovirus and the like, such as a
CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G coupled
matrix.
[0678] For antibodies that include an Fc domain, the antibody
production system may produce antibodies in which the Fc region is
glycosylated. For example, the Fc domain of IgG molecules is
glycosylated at asparagine 297 in the CH2 domain. This asparagine
is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation
is required for effector functions mediated by Fcg receptors and
complement C1q (Burton and Woof, 1992, Adv. Immunol. 51:1-84;
Jefferis et al., 1998, Immunol. Rev. 163:59-76). In one embodiment,
the Fc domain is produced in a mammalian expression system that
appropriately glycosylates the residue corresponding to asparagine
297. The Fc domain can also include other eukaryotic
post-translational modifications.
[0679] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method of expressing
an antibody in the mammary gland of a transgenic mammal. A
transgene is constructed that includes a milk-specific promoter and
nucleic acids encoding the antibody of interest and a signal
sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the antibody of
interest. The antibody can be purified from the milk, or for some
applications, used directly.
Characterization of Plasma Kallikrein Binding Proteins
[0680] IC.sub.50 (Inhibitory Concentration 50%) and EC.sub.50
(Effective Concentration 50%).
[0681] Within a series or group of binding proteins, those having
lower IC.sub.50 or EC.sub.50 values are considered more potent
inhibitors of plasma kallikrein than those binding proteins having
higher IC.sub.50 or EC.sub.50 values. Exemplary binding proteins
have an IC.sub.50 value of less than 800 nM, 400 nM, 100 nM, 25 nM,
5 nM, or 1 nM, e.g., as measured in an in vitro assay for
inhibition of plasma kallikrein activity when the plasma kallikrein
is at 2 pM.
[0682] Plasma kallikrein binding proteins may also be characterized
with reference to the activity of Factor XII and HMWK
(high-molecular-weight kininogen) signaling events, e.g., the
production of Factor XIIa and/or bradykinin.
[0683] The binding proteins can also be evaluated for selectivity
toward plasma kallikrein. For example, a plasma kallikrein binding
protein can be assayed for its potency toward plasma kallikrein and
a panel of kallikreins and an IC.sub.50 value or EC.sub.50 value
can be determined for each kallikrein. In one embodiment, a
compound that demonstrates a low IC.sub.50 value or EC.sub.50 value
for the plasma kallikrein, and a higher IC.sub.50 value or
EC.sub.50 value, e.g., at least 2-, 5-, or 10-fold higher, for
another kallikrein within the test panel is considered to be
selective toward plasma kallikrein.
[0684] A pharmacokinetics study in rat, mice, or monkey can be
performed with plasma kallikrein binding proteins for determining
plasma kallikrein half-life in the serum. Likewise, the effect of
the binding protein can be assessed in vivo, e.g., in an animal
model for a disease (e.g., carrageenin-induced edema in rat hind
paw (Winter et al. Proc Soc Exp Biol Med. 1962; 111:544-7)), for
use as a therapeutic, for example, to treat a disease or condition
described herein, e.g., a plasma kallikrein associated
disorder.
Pharmaceutical Compositions
[0685] Proteins (e.g., binding proteins) that bind to plasma
kallikrein (e.g., human plasma kallikrein and/or murine plasma
kallikrein) and, e.g., include at least one immunoglobin variable
region can be used in methods for treating (or preventing) a plasma
kallikrein associated disease or condition. The binding proteins
can be present in a composition, e.g., a pharmaceutically
acceptable composition or pharmaceutical composition, which
includes a plasma kallikrein-binding protein, e.g., an antibody
molecule or other polypeptide or peptide identified as binding to
plasma kallikrein, as described herein. The plasma kallikrein
binding protein can be formulated together with a pharmaceutically
acceptable carrier. Pharmaceutical compositions include therapeutic
compositions and diagnostic compositions, e.g., compositions that
include labeled plasma kallikrein binding proteins for in vivo
imaging, and compositions that include labeled plasma kallikrein
binding proteins for treating (or preventing) a plasma kallikrein
associated disease.
[0686] A pharmaceutically acceptable carrier includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Preferably, the carrier is suitable
for intravenous, intramuscular, subcutaneous, parenteral, spinal,
or epidermal administration (e.g., by injection or infusion),
although carriers suitable for inhalation and intranasal
administration are also contemplated. Depending on the route of
administration, the plasma kallikrein binding protein may be coated
in a material to protect the compound from the action of acids and
other natural conditions that may inactivate the compound.
[0687] A pharmaceutically acceptable salt is a salt that retains
the desired biological activity of the compound and does not impart
any undesired toxicological effects (see e.g., Berge, S. M., et
al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include
acid addition salts and base addition salts. Acid addition salts
include those derived from nontoxic inorganic acids, such as
hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous, and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids, and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium, and the like, as
well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine, and the
like.
[0688] The compositions may be in a variety of forms. These
include, for example, liquid, semi-solid and solid dosage forms,
such as liquid solutions (e.g., injectable and infusible
solutions), dispersions or suspensions, tablets, pills, powders,
liposomes and suppositories. The form can depend on the intended
mode of administration and therapeutic application. Many
compositions are in the form of injectable or infusible solutions,
such as compositions similar to those used for administration of
humans with antibodies. An exemplary mode of administration is
parenteral (e.g., intravenous, subcutaneous, intraperitoneal,
intramuscular). In one embodiment, the plasma kallikrein binding
protein is administered by intravenous infusion or injection. In
another preferred embodiment, the plasma kallikrein binding protein
is administered by intramuscular or subcutaneous injection.
[0689] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0690] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable to high drug concentration. Sterile injectable solutions
can be prepared by incorporating the binding protein in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying that yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0691] A plasma kallikrein binding protein can be administered by a
variety of methods, although for many applications, the preferred
route/mode of administration is intravenous injection or infusion.
For example, for therapeutic applications, the plasma kallikrein
binding protein can be administered by intravenous infusion at a
rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of
about 1 to 100 mg/m.sup.2 or 7 to 25 mg/m.sup.2. The route and/or
mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are available. See, e.g., Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson, ed., 1978, Marcel
Dekker, Inc., New York.
[0692] Pharmaceutical compositions can be administered with medical
devices. For example, in one embodiment, a pharmaceutical
composition disclosed herein can be administered with a device,
e.g., a needleless hypodermic injection device, a pump, or
implant.
[0693] In certain embodiments, a plasma kallikrein binding protein
can be formulated to ensure proper distribution in vivo. For
example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic compounds
disclosed herein cross the BBB (if desired), they can be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties that are
selectively transported into specific cells or organs, thus enhance
targeted drug delivery (see, e.g., V.V. Ranade, 1989, J. Clin.
Pharmacol. 29:685).
[0694] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms can be dictated by and
directly dependent on (a) the unique characteristics of the active
compound and the particular therapeutic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0695] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a binding protein (e.g., an
antibody) disclosed herein is 0.1-20 mg/kg, more preferably 1-10
mg/kg. An anti-plasma kallikrein antibody can be administered,
e.g., by intravenous infusion, e.g., at a rate of less than 30, 20,
10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m.sup.2 or
about 5 to 30 mg/m.sup.2. For binding proteins smaller in molecular
weight than an antibody, appropriate amounts can be proportionally
less. Dosage values may vary with the type and severity of the
condition to be alleviated. For a particular subject, specific
dosage regimens can be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions.
[0696] The pharmaceutical compositions disclosed herein may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a plasma kallikrein binding protein disclosed
herein. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the composition may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the protein to elicit a desired response in the
individual. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the composition are outweighed
by the therapeutically beneficial effects.
[0697] A "therapeutically effective dosage" preferably modulates a
measurable parameter, e.g., levels of circulating IgG antibodies by
a statistically significant degree or at least about 20%, more
preferably by at least about 40%, even more preferably by at least
about 60%, and still more preferably by at least about 80% relative
to untreated subjects. The ability of a compound to modulate a
measurable parameter, e.g., a disease-associated parameter, can be
evaluated in an animal model system predictive of efficacy in human
disorders and conditions, e.g., a plasma kallikrein associated
disease. Alternatively, this property of a composition can be
evaluated by examining the ability of the compound to modulate a
parameter in vitro.
[0698] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, because a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
Stabilization and Retention
[0699] In one embodiment, a plasma kallikrein binding protein is
physically associated with a moiety that improves its stabilization
and/or retention in circulation, e.g., in blood, serum, lymph, or
other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For
example, a plasma kallikrein binding protein can be associated with
a polymer, e.g., a substantially non-antigenic polymer, such as
polyalkylene oxides or polyethylene oxides. Suitable polymers will
vary substantially by weight. Polymers having molecular number
average weights ranging from about 200 to about 35,000 (or about
1,000 to about 15,000, and 2,000 to about 12,500) can be used. For
example, a plasma kallikrein binding protein can be conjugated to a
water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.,
polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained.
[0700] A plasma kallikrein binding protein can also be associated
with a carrier protein, e.g., a serum albumin, such as a human
serum albumin (see e.g., Smith, B J. et al., 2001 Bioconjug Chem
12(5): 750-756). For example, a translational fusion can be used to
associate the carrier protein with the plasma kallikrein binding
protein.
[0701] A plasma kallikrein binding protein can also be modified as
a HESylation derivative. Processes for HESylation of a plasma
kallikrein binding protein utilize hydroxyethyl starch to modify
the protein. HESylation of a protein can extend the circulating
half-life of the protein and also reduce renal clearance.
Kits
[0702] A plasma kallikrein binding protein described herein can be
provided in a kit, e.g., as a component of a kit. For example, the
kit includes (a) a plasma kallikrein binding protein, e.g., a
composition (e.g., a pharmaceutical composition) that includes a
plasma kallikrein binding protein, and, optionally (b)
informational material. The informational material can be
descriptive, instructional, marketing or other material that
relates to a method described herein and/or the use of a plasma
kallikrein binding protein, e.g., for a method described
herein.
[0703] The informational material of the kit is not limited in its
form. In one embodiment, the informational material can include
information about production of the compound, molecular weight of
the compound, concentration, date of expiration, batch or
production site information, and so forth. In one embodiment, the
informational material relates to using the binding protein to
treat, prevent, or diagnosis of disorders and conditions, e.g., a
plasma kallikrein associated disease or condition.
[0704] In one embodiment, the informational material can include
instructions to administer a plasma kallikrein binding protein in a
suitable manner to perform the methods described herein, e.g., in a
suitable dose, dosage form, or mode of administration (e.g., a
dose, dosage form, or mode of administration described herein). In
another embodiment, the informational material can include
instructions to administer a plasma kallikrein binding protein to a
suitable subject, e.g., a human, e.g., a human having, or at risk
for, a disorder or condition described herein, e.g., a plasma
kallikrein associated disease or condition. For example, the
material can include instructions to administer a plasma kallikrein
binding protein to a patient with a disorder or condition described
herein, e.g., a plasma kallikrein associated disease. The
informational material of the kits is not limited in its form. In
many cases, the informational material, e.g., instructions, is
provided in print but may also be in other formats, such as
computer readable material.
[0705] A plasma kallikrein binding protein can be provided in any
form, e.g., liquid, dried or lyophilized form. It is preferred that
a plasma kallikrein binding protein be substantially pure and/or
sterile. When a plasma kallikrein binding protein is provided in a
liquid solution, the liquid solution preferably is an aqueous
solution, with a sterile aqueous solution being preferred. When a
plasma kallikrein binding protein is provided as a dried form,
reconstitution generally is by the addition of a suitable solvent.
The solvent, e.g., sterile water or buffer, can optionally be
provided in the kit.
[0706] The kit can include one or more containers for the
composition containing a plasma kallikrein binding protein. In some
embodiments, the kit contains separate containers, dividers or
compartments for the composition and informational material. For
example, the composition can be contained in a bottle, vial, or
syringe, and the informational material can be contained in
association with the container. In other embodiments, the separate
elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle,
vial or syringe that has attached thereto the informational
material in the form of a label. In some embodiments, the kit
includes a plurality (e.g., a pack) of individual containers, each
containing one or more unit dosage forms (e.g., a dosage form
described herein) of a plasma kallikrein binding protein. For
example, the kit includes a plurality of syringes, ampules, foil
packets, or blister packs, each containing a single unit dose of a
plasma kallikrein binding protein. The containers of the kits can
be air tight, waterproof (e.g., impermeable to changes in moisture
or evaporation), and/or light-tight.
[0707] The kit optionally includes a device suitable for
administration of the composition, e.g., a syringe, inhalant,
dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden
swab), or any such delivery device. In one embodiment, the device
is an implantable device that dispenses metered doses of the
binding protein. The disclosure also features a method of providing
a kit, e.g., by combining components described herein.
Treatments
[0708] Proteins that bind to plasma kallikrein, e.g., as described
herein, have therapeutic and prophylactic utilities, particularly
in human subjects. These binding proteins are administered to a
subject to treat, prevent, and/or diagnose a variety of disorders
and conditions, including e.g., a plasma kallikrein associated
disease, or even to cells in culture, e.g., in vitro or ex vivo.
For example, these binding proteins can be used to modify the
effects of plasma kallikrein released from cells in culture (Lilla
et al., J Biol Chem. 284(20):13792-13803 (2009)). Treating includes
administering an amount effective to alleviate, relieve, alter,
remedy, ameliorate, improve or affect the disorder, the symptoms of
the disorder or the predisposition toward the disorder. The
treatment may also delay onset, e.g., prevent onset, or prevent
deterioration of a disease or condition.
[0709] As used herein, an amount of a target-binding agent
effective to prevent a disorder, or a prophylactically effective
amount of the binding agent refers to an amount of a target binding
agent, e.g., an plasma kallikrein binding protein, e.g., an
anti-plasma kallikrein antibody described herein, which is
effective, upon single- or multiple-dose administration to the
subject, for preventing or delaying the occurrence of the onset or
recurrence of a disorder, e.g., a disorder described herein, e.g.,
a plasma kallikrein associated disease.
[0710] Methods of administering plasma kallikrein binding proteins
and other agents are also described in "Pharmaceutical
Compositions." Suitable dosages of the molecules used can depend on
the age and weight of the subject and the particular drug used. The
binding proteins can be used as competitive agents to inhibit,
reduce an undesirable interaction, e.g., between plasma kallikrein
and its substrate (e.g., Factor XII or HMWK). The dose of the
plasma kallikrein binding protein can be the amount sufficient to
block 90%, 95%, 99%, or 99.9% of the activity of plasma kallikrein
in the patient, especially at the site of disease. Depending on the
disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg/Kg. For an
IgG having a molecular mass of 150,000 g/mole (two binding sites),
these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08
.mu.M, and 1.8 .mu.M of binding sites for a 5 L blood volume.
[0711] In one embodiment, the plasma kallikrein binding proteins
are used to inhibit an activity (e.g., inhibit at least one
activity of plasma kallikrein, e.g., reduce Factor XIIa and/or
bradykinin production) of plasma kallikrein, e.g., in vivo. The
binding proteins can be used by themselves or conjugated to an
agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope.
This method includes: administering the binding protein alone or
attached to an agent (e.g., a cytotoxic drug), to a subject
requiring such treatment. For example, plasma kallikrein binding
proteins that do not substantially inhibit plasma kallikrein may be
used to deliver nanoparticles containing agents, such as toxins, to
plasma kallikrein associated cells or tissues, e.g., to treat a
plasma kallikrein-associate disorder.
[0712] Because the plasma kallikrein binding proteins recognize
plasma kallikrein expressing cells and can bind to cells that are
associated with (e.g., in proximity of or intermingled with) a
plasma kallikrein associated disorder or condition, plasma
kallikrein binding proteins can be used to inhibit an activity
(e.g., inhibit at least one activity of plasma kallikrein, e.g.,
reduce Factor XIIa and/or bradykinin production) any such cells and
inhibit the plasma kallikrein associated disease. Reducing plasma
kallikrein activity can indirectly inhibit cells which may be
dependent on the plasma kallikrein activity for the development
and/or progression of a plasma kallikrein-associated disorder.
[0713] The binding proteins may be used to deliver an agent (e.g.,
any of a variety of cytotoxic and therapeutic drugs) to cells and
tissues where plasma kallikrein is present. Exemplary agents
include a compound emitting radiation, molecules of plants, fungal,
or bacterial origin, biological proteins, and mixtures thereof. The
cytotoxic drugs can be intracellularly acting cytotoxic drugs, such
as toxins short range radiation emitters, e.g., short range, high
energy .alpha.-emitters.
[0714] To target plasma kallikrein expressing cells, a prodrug
system can be used. For example, a first binding protein is
conjugated with a prodrug which is activated only when in close
proximity with a prodrug activator. The prodrug activator is
conjugated with a second binding protein, preferably one which
binds to a non competing site on the target molecule. Whether two
binding proteins bind to competing or non competing binding sites
can be determined by conventional competitive binding assays.
Exemplary drug prodrug pairs are described in Blakely et al.,
(1996) Cancer Research, 56:3287 3292.
[0715] The plasma kallikrein binding proteins can be used directly
in vivo to eliminate antigen-expressing cells via natural
complement-dependent cytotoxicity (CDC) or antibody dependent
cellular cytotoxicity (ADCC). The binding proteins described herein
can include complement binding effector domain, such as the Fc
portions from IgG1, -2, or -3 or corresponding portions of IgM
which bind complement. In one embodiment, a population of target
cells is ex vivo treated with a binding agent described herein and
appropriate effector cells. The treatment can be supplemented by
the addition of complement or serum containing complement. Further,
phagocytosis of target cells coated with a binding protein
described herein can be improved by binding of complement proteins.
In another embodiment target, cells coated with the binding protein
which includes a complement binding effector domain are lysed by
complement.
[0716] Methods of administering plasma kallikrein binding proteins
are described in "Pharmaceutical Compositions." Suitable dosages of
the molecules used will depend on the age and weight of the subject
and the particular drug used. The binding proteins can be used as
competitive agents to inhibit or reduce an undesirable interaction,
e.g., between a natural or pathological agent and the plasma
kallikrein.
[0717] The plasma kallikrein binding protein can be used to deliver
macro and micromolecules, e.g., a gene into the cell for gene
therapy purposes into the endothelium or epithelium and target only
those tissues expressing the plasma kallikrein. The binding
proteins may be used to deliver a variety of cytotoxic drugs
including therapeutic drugs, a compound emitting radiation,
molecules of plants, fungal, or bacterial origin, biological
proteins, and mixtures thereof. The cytotoxic drugs can be
intracellularly acting cytotoxic drugs, such as short range
radiation emitters, including, for example, short range, high
energy a emitters, as described herein.
[0718] In the case of polypeptide toxins, recombinant nucleic acid
techniques can be used to construct a nucleic acid that encodes the
binding protein (e.g., antibody or antigen-binding fragment
thereof) and the cytotoxin (or a polypeptide component thereof) as
translational fusions. The recombinant nucleic acid is then
expressed, e.g., in cells and the encoded fusion polypeptide
isolated.
[0719] Alternatively, the plasma kallikrein binding protein can be
coupled to high energy radiation emitters, for example, a
radioisotope, such as .sup.131I, a .gamma.-emitter, which, when
localized at a site, results in a killing of several cell
diameters. See, e.g., S. E. Order, "Analysis, Results, and Future
Prospective of the Therapeutic Use of Radiolabeled Antibody in
Cancer Therapy", Monoclonal Antibodies for Cancer Detection and
Therapy, R. W. Baldwin et al. (eds.), pp 303 316 (Academic Press
1985). Other suitable radioisotopes include a emitters, such as
.sup.212Bi, .sup.213Bi, and .sup.211At, and b emitters, such as
.sup.186Re and .sup.90Y. Moreover, .sup.177Lu may also be used as
both an imaging and cytotoxic agent.
[0720] Radioimmunotherapy (RIT) using antibodies labeled with 131I,
.sup.90Y and .sup.177Lu is under intense clinical investigation.
There are significant differences in the physical characteristics
of these three nuclides and as a result, the choice of radionuclide
is very critical in order to deliver maximum radiation dose to a
tissue of interest. The higher beta energy particles of .sup.90Y
may be good for bulky tumors. The relatively low energy beta
particles of .sup.131I are ideal, but in vivo dehalogenation of
radioiodinated molecules is a major disadvantage for internalizing
antibody. In contrast, .sup.177Lu has low energy beta particle with
only 0.2-0.3 mm range and delivers much lower radiation dose to
bone marrow compared to .sup.90Y. In addition, due to longer
physical half-life (compared to .sup.90Y), the residence times are
higher. As a result, higher activities (more mCi amounts) of
.sup.177Lu labeled agents can be administered with comparatively
less radiation dose to marrow. There have been several clinical
studies investigating the use of .sup.177Lu labeled antibodies in
the treatment of various cancers. (Mulligan T et al., 1995, Clin.
Canc. Res. 1: 1447-1454; Meredith R F, et al., 1996, J. Nucl. Med.
37:1491-1496; Alvarez R D, et al., 1997, Gynecol. Oncol. 65:
94-101).
Exemplary Diseases and Conditions
[0721] A plasma kallikrein binding protein described herein is
useful to treat (or prevent) a disease or condition in which plasma
kallikrein activity is implicated, e.g., a disease or condition
described herein, or to treat (or prevent) one or more symptoms
associated therewith. In some embodiments, the plasma kallikrein
binding protein (e.g., plasma kallikrein binding IgG or Fab)
inhibits plasma kallikrein activity.
[0722] Examples of such diseases and conditions which can be
treated (or prevented) by a plasma kallikrein binding protein
described herein include: rheumatoid arthritis, gout, intestinal
bowel disease, oral mucositis, neuropathic pain, inflammatory pain,
spinal stenosis-degenerative spine disease, arterial or venous
thrombosis, post operative ileus, aortic aneurysm, osteoarthritis,
vasculitis, edema, hereditary angioedema, cerebral edema, pulmonary
embolism, stroke, clotting induced by ventricular assistance
devices or stents, head trauma or peri-tumor brain edema, sepsis,
acute middle cerebral artery (MCA) ischemic event (stroke),
restenosis (e.g., after angioplasty), systemic lupus erythematosis
nephritis, and burn injury. A plasma kallikrein binding protein
described herein can also be used to promote wound healing. A
plasma kallikrein binding protein described herein can also be used
as an oncology treatment by mechanisms that include, but are not
limited to, blocking production of pro-angiogenic bradykinin.
[0723] A therapeutically effective amount of a plasma kallikrein
binding protein can be administered to a subject having or
suspected of having a disorder in which plasma kallikrein activity
is implicated, thereby treating (e.g., ameliorating or improving a
symptom or feature of a disorder, slowing, stabilizing and/or
halting disease progression) the disorder.
[0724] The plasma kallikrein binding protein can be administered in
a therapeutically effective amount. A therapeutically effective
amount of a plasma kallikrein binding protein is the amount which
is effective, upon single or multiple dose administration to a
subject, in treating a subject, e.g., curing, alleviating,
relieving or improving at least one symptom of a disorder in a
subject to a degree beyond that expected in the absence of such
treatment. A therapeutically effective amount of the composition
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the compound to
elicit a desired response in the individual. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the composition are outweighed by the therapeutically
beneficial effects. A therapeutically effective dosage preferably
modulates a measurable parameter, favorably, relative to untreated
subjects. The ability of a compound to affect (e.g., inhibit) a
measurable parameter can be evaluated in an animal model system
predictive of efficacy in a human disorder.
[0725] Dosage regimens can be adjusted to provide the optimum
desired response (e.g., a therapeutic response). For example, a
single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Rheumatoid Arthritis
[0726] Rheumatoid arthritis (RA) is an autoimmune, chronic
inflammatory disease that causes joint swelling and pain and
normally results in joint destruction. RA generally follows a
relapsing/remitting course, with "flares" of disease activity
interspersed with remissions of disease symptoms. RA is associated
with a number of additional inflammatory disorders, including
Sjogren's syndrome (dry eyes and mouth caused by inflammation of
tear and saliva glands), pleuritis (inflammation of the pleura that
causes pain upon deep breath and coughing), rheumatoid nodules
(nodular sites of inflammation that develop within the lungs),
pericarditis (inflammation of the pericardium that causes pain when
lying down or leaning forward), Felty syndrome (splenomegaly and
leucopenia observed in conjunction with RA, making the subject
prone to infection), and vasculitis (an inflammation of the blood
vessels which can block blood flow). Plasma kallikrein has been
implicated in rheumatoid arthritis.
[0727] Symptoms of active RA include fatigue, lack of appetite, low
grade fever, muscle and joint aches, and stiffness. Muscle and
joint stiffness are usually most notable in the morning and after
periods of inactivity. During flares, joints frequently become red,
swollen, painful, and tender, generally as a consequence of
synovitis.
[0728] Treatment for rheumatoid arthritis involves a combination of
medications, rest, joint strengthening exercises, and joint
protection. Two classes of medications are used in treating
rheumatoid arthritis: anti-inflammatory "first-line drugs," and
"Disease-Modifying Antirheumatic Drugs" (DMARDs). The first-line
drugs include NSAIDS (e.g., aspirin, naproxen, ibuprofen, and
etodolac) and cortisone (corticosteroids). DMARDs, such as gold
(e.g., gold salts, gold thioglucose, gold thiomalate, oral gold),
methotrexate, sulfasalazine, D-penicillamine, azathioprine,
cyclophosphamide, chlorambucil, and cyclosporine, leflunomide,
etanercept, infliximab, anakinra, and adalimumab, and
hydroxychloroquine, promote disease remission and prevent
progressive joint destruction, but they are not anti-inflammatory
agents.
[0729] The disclosure provides methods of treating (e.g.,
ameliorating, stabilizing, or eliminating one or more symptoms or
ameliorating or stabilizing the subject's score on a RA scale)
rheumatoid arthritis by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having RA. Additionally provided are methods of treating RA by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., with at least
one anti-inflammatory "first line drug" (e.g., an NSAID and/or
cortisone) and/or a DMARD. The disclosure also provides methods of
preventing rheumatoid arthritis or a symptom thereof by
administering a plasma kallikrein binding protein (e.g., a
prophylactically effective amount of a plasma kallikrein binding
protein) to a subject at risk of developing RA (e.g., a subject
having a family member with RA or a genetic predisposition
thereto).
[0730] Further provided are methods of treating (e.g.,
ameliorating, stabilizing, or eliminating one or more symptoms)
rheumatoid arthritis associated disorders (Sjogren's syndrome,
pleuritis, pulmonary rheumatoid nodules, pericarditis, Felty
syndrome, and vasculitis) by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) to a subject having or suspected
of having RA.
[0731] Scales useful for assessing RA and symptoms of RA include,
e.g., the Rheumatoid Arthritis Severity Scale (RASS; Bardwell et
al., (2002) Rheumatology 41(1):38-45), SF-36 Arthritis Specific
Health Index (ASHI; Ware et al., (1999) Med. Care. 37(5
Suppl):MS40-50), Arthritis Impact Measurement Scales or Arthritis
Impact Measurement Scales 2 (AIMS or AIMS2; Meenan et al. (1992)
Arthritis Rheum. 35(1):1-10); the Stanford Health Assessment
Questionnaire (HAQ), HAQII, or modified HAQ (see, e.g., Pincus et
al. (1983) Arthritis Rheum. 26(11):1346-53).
[0732] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of rheumatoid arthritis,
such as collagen-induced arthritis (CIA), which is induced,
typically in rodents, by immunization with autologous or
heterologous type II collagen in adjuvant (Williams et al. Methods
Mol Med. 98:207-16 (2004)).
Gout
[0733] Gout is a condition that results from crystals of uric acid
depositing in tissues of the body. Gout is characterized by an
overload of uric acid in the body and recurring attacks of joint
inflammation (arthritis). Chronic gout can lead to deposits of hard
lumps of uric acid in and around the joints, decreased kidney
function, and kidney stones. Gout is often related to an inherited
abnormality in the body's ability to process uric acid. Uric acid
is a breakdown product of purines, which are part of many foods. An
abnormality in handling uric acid can cause attacks of painful
arthritis (gout attack), kidney stones, and blockage of the kidney
filtering tubules with uric acid crystals, leading to kidney
failure. Some patients may only develop elevated blood uric acid
levels (hyperuricemia) without having arthritis or kidney
problems.
[0734] Symptoms of gout include, e.g., excruciating and unexpected
pain, swelling, redness, warmth and stiffness in the affected foot
or other parts of the body, and low-grade fever.
[0735] Treatments for gout include, e.g., nonsteroidal
anti-inflammatory drugs (NSAIDs), colchicine and oral
glucocorticoids, intra-articular glucocorticoids administered via a
joint injection, xanthine oxidase inhibitors (e.g., allopurinol,
febuxostat), uricosurics (e.g., probenecid, EDTA), urate oxidases
(e.g., pegloticase), sodium bicarbonate, and low purine diet.
[0736] The disclosure provides methods of treating (e.g.,
ameliorating, stabilizing, or eliminating one or more symptoms or
the worsening of) gout by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having gout. Additionally provided are methods of treating gout by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a kallikrein binding protein)
in combination with a second therapy, e.g., an NSAID, a colchicine,
an oral glucocorticoid, an intra-articular glucocorticoid
administered via a joint injection, a xanthine oxidase inhibitor
(e.g., allopurinol, febuxostat), a uricosuric (e.g., probenecid,
EDTA), a urate oxidase (e.g., pegloticase), sodium bicarbonate,
and/or low purine diet. The disclosure also provides methods of
preventing gout or a symptom thereof by administering a plasma
kallikrein binding protein (e.g., a prophylactically effective
amount of a plasma kallikrein binding protein) to a subject at risk
of developing gout (e.g., a subject having a family member with
gout or a genetic predisposition thereto).
[0737] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of gout, see, e.g.,
Reginato and Olsen, Curr Opin Rheumatol. 19(2):134-45 (2007) and
references cited therein.
Intestinal Bowel Disease (IBD)
[0738] Inflammatory bowel disease (IBD) is a group of inflammatory
conditions of the large intestine and, in some cases, the small
intestine. The main forms of IBD are Crohn's disease and ulcerative
colitis (UC). Accounting for far fewer cases are other forms of
IBD: collagenous colitis, lymphocytic colitis, ischaemic colitis,
diversion colitis, Behcet's syndrome, infective colitis, and
indeterminate colitis. The main difference between Crohn's disease
and UC is the location and nature of the inflammatory changes.
Crohn's can affect any part of the gastrointestinal tract, from
mouth to anus (skip lesions), although a majority of the cases
start in the terminal ileum. Ulcerative colitis, in contrast, is
restricted to the colon and the rectum. Microscopically, ulcerative
colitis is restricted to the mucosa (epithelial lining of the gut),
while Crohn's disease affects the whole bowel wall. Finally,
Crohn's disease and ulcerative colitis present with
extra-intestinal manifestations (such as liver problems, arthritis,
skin manifestations and eye problems) in different proportions.
[0739] Symptoms of IBD include abdominal pain, vomiting, diarrhea,
hematochezia, weight loss, weight gain and various associated
complaints or diseases (arthritis, pyoderma gangrenosum, primary
sclerosing cholangitis). Diagnosis is generally by colonoscopy with
biopsy of pathological lesions. Rarely, a definitive diagnosis of
neither Crohn's disease nor ulcerative colitis can be made because
of idiosyncrases in the presentation. In this case, a diagnosis of
indeterminate colitis may be made.
[0740] Treatment for IBD, depending on the level of severity, may
require immunosuppression to control the symptoms.
Immunosuppressives such as azathioprine, methotrexate, or
6-mercaptopurine can be used. More commonly, treatment of IBD
requires a form of mesalamine. Often, steroids are used to control
disease flares and were once acceptable as a maintenance drug.
Biologicals, such as infliximab, have been used to treat patients
with Crohn's disease or Ulcerative Colitis. Severe cases may
require surgery, such as bowel resection, strictureplasty or a
temporary or permanent colostomy or ileostomy. Alternative medicine
treatments for IBD exist in various forms however such methods
concentrate on controlling underlying pathology in order to avoid
prolonged steroidal exposure or surgical excision. Usually the
treatment is started by administering drugs, such as prednisone,
with high anti-inflammatory affects. Once the inflammation is
successfully controlled, the patient is usually switched to a
lighter drug, such as asacol--a mesalamine--to keep the disease in
remission. If unsuccessful, a combination of the aforementioned
immunosuppressant drugs with a mesalamine (which may also have an
anti-inflammatory effect) may or may not be administered, depending
on the patient.
[0741] The disclosure provides methods of treating (e.g.,
ameliorating, stabilizing, or eliminating one or more symptoms of)
IBD by administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) to a subject having or suspected of having IBD.
Additionally provided are methods of treating IBD by administering
a plasma kallikrein binding protein (e.g., a therapeutically
effective amount of a kallikrein binding protein) in combination
with a second therapy, e.g., an immunosuppressive (e.g.,
azathioprine, methotrexate, 6-mercaptopurine), a mesalamine, a
steroid, and/or infliximab. The disclosure also provides methods of
preventing IBD or a symptom thereof by administering a plasma
kallikrein binding protein (e.g., a prophylactically effective
amount of a plasma kallikrein binding protein) to a subject at risk
of developing IBD (e.g., a subject having a family member with IBD
or a genetic predisposition thereto).
[0742] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of IBD, see, e.g., those
described in U.S. Pat. No. 6,114,382, WO 2004/071186, and
references cited therein.
Oral Mucositis
[0743] Oral mucositis is the painful inflammation and ulceration of
the mucous membranes in the mouth, usually as an adverse effect of
chemotherapy and radiotherapy treatment for cancer.
[0744] Symptoms of oral mucositis include, e.g., ulcers, peripheral
erythema, burning sensation accompanied by reddening, trouble
speaking, eating, or even opening the mouth, and dyseusia
(alteration in taste perception).
[0745] Treatment for oral mucositis includes oral hygiene (salt
mouthwash, GELCLAIR.RTM., CAPHOSOL.RTM., MUGARD.RTM.), palifermin
(a human keratinocyte growth factor), cytokines and other modifiers
of inflammation (e.g., IL-1, IL-11, TGF-beta3), amino acid
supplementation (e.g., glutamine), vitamins, colony-stimulating
factors, cryotherapy, and laser therapy.
[0746] The disclosure provides methods of treating (e.g.,
ameliorating, reducing, or eliminating one or more symptoms, or
stabilizing the subject's score on a mucositis scale) oral
mucositis by administering a plasma kallikrein binding protein
(e.g., a therapeutically effective amount of a plasma kallikrein
binding protein) to a subject having or suspected of having oral
mucositis. Additionally provided are methods of treating oral
mucositis by administering a plasma kallikrein binding protein
(e.g., a therapeutically effective amount of a plasma kallikrein
binding protein) in combination with a second therapy, e.g., oral
hygiene (salt mouthwash, GELCLAIR.RTM., CAPHOSOL.RTM.,
MUGARD.RTM.), palifermin (a human keratinocyte growth factor), a
cytokine and/or a modifier of inflammation (e.g., IL-1, IL-11,
TGF-beta3), an amino acid supplementation (e.g., glutamine), a
vitamin, a colony-stimulating factor, cryotherapy, and/or laser
therapy. The disclosure also provides methods of preventing oral
mucositis or a symptom thereof by administering a plasma kallikrein
binding protein (e.g., a prophylactically effective amount of a
plasma kallikrein binding protein) to a subject at risk of
developing oral mucositis (e.g., a subject that has undergone or is
undergoing chemotherapy or radiotherapy).
[0747] Scales useful for assessing oral mucositis include the World
Health Organization (WHO) Oral Toxicity score (Handbook for
reporting results of cancer treatment. Geneva, Switzerland: World
Health Organization; 1979:15-22), National Cancer Institute Common
Toxicity Criteria (NCI-CTC) for Oral Mucositis (National Cancer
Institute Common Toxicity Criteria. Version 2.0, Jun. 1, 1999,
Sonis et al., Cancer. 85:2103-2113 (1999)), and Oral Mucositis
Assessment Scale (OMAS).
[0748] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of oral mucositis, such
as an animal model of oral mucositis induced by conditioning
regimen of haematopoietic stem cell transplantation (Chen et al.,
Zhonghua Kou Qiang Yi Xue Za Zhi. 42(11):672-6 (2007)).
Neuropathic Pain
[0749] Neuropathic pain is a complex, chronic pain state that
usually is accompanied by tissue injury. With neuropathic pain, the
nerve fibers themselves may be damaged, dysfunctional or injured.
These damaged nerve fibers send incorrect signals to other pain
centers. The impact of nerve fiber injury includes a change in
nerve function both at the site of injury and areas around the
injury.
[0750] Symptoms of neuropathic pain include, e.g., shooting and
burning pain and tingling and numbness.
[0751] Treatments for neuropathic pain include, e.g., medications
(e.g., non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.,
ALEVE.RTM., MOTRIN.RTM., or morphine), anticonvulsant, and
antidepressant drugs), and invasive or implantable devices (e.g.,
electrical stimulation).
[0752] The disclosure provides methods of treating (e.g.,
ameliorating, reducing, or eliminating one or more symptoms of or
stabilizing the subject's score on a pain scale) neuropathic pain
by administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) to a subject having or suspected of having neuropathic
pain. Additionally provided are methods of treating neuropathic
pain by administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., a nonsurgical
treatment ((e.g., a non-steroidal anti-inflammatory drug (NSAID)
(e.g., ALEVE.RTM., MOTRIN.RTM., or morphine), an anticonvulsant,
and/or an antidepressant drug), and/or an invasive or implantable
device (e.g., electrical stimulation). The disclosure also provides
methods of preventing neuropathic pain or a symptom thereof by
administering a plasma kallikrein binding protein (e.g., a
prophylactically effective amount of a plasma kallikrein binding
protein) to a subject at risk of developing neuropathic pain (e.g.,
a subject that has experienced tissue injury).
[0753] Scales useful for the assessment of neuropathic pain
include, e.g., Wong-Baker FACES Pain Rating Scale (Wong-Baker FACES
Pain Rating Scale Foundation), Visual analog scale (VAS)
(Huskisson, J. Rheumatol. 9 (5): 768-9 (1982)), McGill Pain
Questionnaire (MPQ) (Melzack, Pain 1 (3): 277-99 (1975)),
Descriptor differential scale (DDS) (Gracely and Kwilosz, Pain 35
(3): 279-88 (1988)), Faces Pain Scale--Revised (FPS-R) (Hicks et
al., Pain 93 (2): 173-83 (2001)), Numerical 11 point box (BS-11)
(Jensen et al., Clin J Pain 5 (2): 153-9 (1989)), Numeric Rating
Scale (NRS-11) (Hartrick et al., Pain Pract 3 (4): 310-6 (2003)),
Dolorimeter Pain Index (DPI) (Hardy et al., (1952). Pain Sensations
and Reactions. Baltimore: The Williams & Wilkins Co.), and
Brief Pain Inventory (BPI) (Cleeland and Ryan Ann. Acad. Med.
Singap. 23 (2): 129-38 (1994)).
[0754] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of neuropathic pain,
see, e.g., those described in Martin et al., Methods Mol Med.
84:233-42 (2003) and references cited therein.
Inflammatory Pain
[0755] Inflammatory pain is caused by an insult such as penetration
wounds, burns, extreme cold, fractures, arthritis, autoimmune
conditions, excessive stretching, infections and vasoconstriction
to the integrity of tissues at a cellular level. During
inflammation a complex neuro-immune interaction results in primary
hyperalgesia, in which a large range of inflammatory molecules
including prostaglandins and bradykinin induce and maintain the
altered nociceptor sensitivity.
[0756] Treatments for inflammatory pain include, e.g.,
non-steroidal anti-inflammatory drugs (NSAIDs) and
corticosteroids.
[0757] The disclosure provides methods of treating (e.g.,
ameliorating, reducing, or eliminating one or more symptoms of)
inflammatory pain by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having inflammatory pain. Additionally provided are methods of
treating inflammatory pain by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) in combination with a second
therapy, e.g., a non-steroidal anti-inflammatory drug (NSAID)
and/or a corticosteroid. The disclosure also provides methods of
preventing inflammatory pain or a symptom thereof by administering
a plasma kallikrein binding protein (e.g., a prophylactically
effective amount of a plasma kallikrein binding protein) to a
subject at risk of developing inflammatory pain (e.g., a subject
that has experienced an insult, e.g., such as a penetration wound,
a burn, extreme cold, a fracture, arthritis, an autoimmune
condition, excessive stretching, or infection).
[0758] Scales useful for the assessment of inflammatory pain
include, e.g., Wong-Baker FACES Pain Rating Scale (Wong-Baker FACES
Pain Rating Scale Foundation), Visual analog scale (VAS)
(Huskisson, J. Rheumatol. 9 (5): 768-9 (1982)), McGill Pain
Questionnaire (MPQ) (Melzack, Pain 1 (3): 277-99 (1975)),
Descriptor differential scale (DDS) (Gracely and Kwilosz, Pain 35
(3): 279-88 (1988)), Faces Pain Scale--Revised (FPS-R) (Hicks et
al., Pain 93 (2): 173-83 (2001)), Numerical 11 point box (BS-11)
(Jensen et al., Clin J Pain 5 (2): 153-9 (1989)), Numeric Rating
Scale (NRS-11) (Hartrick et al., Pain Pract 3 (4): 310-6 (2003)),
Dolorimeter Pain Index (DPI) (Hardy et al., (1952). Pain Sensations
and Reactions. Baltimore: The Williams & Wilkins Co.), and
Brief Pain Inventory (BPI) (Cleeland and Ryan Ann. Acad. Med.
Singap. 23 (2): 129-38 (1994)).
[0759] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of inflammatory pain
such as an animal model of chronic inflammatory pain (Wilson et
al., Eur J Pain. 10(6):537-49 (2006)) and an inflammatory model of
pain and hyperalgesia (Ren and Dubner, ILAR J. 40(3):111-118
(1999)).
Spinal Stenosis
[0760] Spinal stenosis is a medical condition in which the spinal
canal narrows and compresses the spinal cord and nerves. This is
usually due to the common occurrence of spinal degeneration that
occurs with aging. It can also sometimes be caused by spinal disc
herniation, osteoporosis or a tumor. Spinal stenosis may affect the
cervical, thoracic or lumbar spine. In some cases, it may be
present in all three places in the same patient.
[0761] Symptoms of spinal stenosis include, e.g., pain or cramping
in the legs, radiating back and hip pain, pain in the neck and
shoulders, loss of balance, and loss of bowel or bladder function
(cauda equina syndrome).
[0762] Treatments for spinal stenosis include, e.g., nonsurgical
treatments (e.g., physical therapy, non-steroidal anti-inflammatory
drugs (NSAIDs) (e.g., aspirin, ibuprofen and indomethacin),
analgesics (e.g., acetaminophen), chondroitin sulfate, glucosamine,
rest or restricted activity, back brace or corset, epidural steroid
injections (e.g., corticosteroid)), and surgery (e.g.,
decompressive laminectomy, laminotomy and fusion).
[0763] The disclosure provides methods of treating (e.g.,
ameliorating, reducing, or eliminating one or more symptoms of)
spinal stenosis by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having spinal stenosis. Additionally provided are methods of
treating spinal stenosis by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) in combination with a second
therapy, e.g., a nonsurgical treatment (e.g., physical therapy
and/or a nonsteroidal anti-inflammatory drug (NSAID) (e.g.,
aspirin, ibuprofen or indomethacin), an analgesic (e.g.,
acetaminophen), chondroitin sulfate, glucosamine, rest or
restricted activity, a back brace or corset, an epidural steroid
injection (e.g., corticosteroid), and/or surgery (e.g.,
decompressive laminectomy, laminotomy and/or fusion). The
disclosure also provides methods of preventing spinal stenosis or a
symptom thereof by administering a plasma kallikrein binding
protein (e.g., a prophylactically effective amount of a plasma
kallikrein binding protein) to a subject at risk of developing
spinal stenosis (e.g., a subject that has spinal degeneration).
[0764] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of spinal stenosis, such
as a model of lumbar spinal stenosis (Sekiguchi et al., Spine 29,
1105-1111 (2004)).
Arterial and Venous Thrombosis
[0765] Arterial thrombosis is the formation of a thrombus within an
artery. In most cases, arterial thrombosis follows rupture of
atheroma, and is therefore referred to as atherothrombosis.
[0766] Arterial thrombosis is associated with a number of
disorders, including stroke and myocardial infarction. In
thrombotic stroke, a thrombus (blood clot) usually forms around
atherosclerotic plaques. Since blockage of the artery is gradual,
onset of symptomatic thrombotic strokes is slower. Thrombotic
stroke can be divided into two categories--large vessel disease and
small vessel disease. The former affects vessels such as the
internal carotids, vertebral and the circle of Willis. The latter
can affect smaller vessels such as the branches of the circle of
Willis. Myocardial infarction (MI) is caused by an infarct (death
of tissue due to ischemia), often due to the obstruction of the
coronary artery by a thrombus. MI can quickly become fatal if
emergency medical treatment is not received promptly.
[0767] Venous thrombosis is a blood clot that forms within a vein.
If a piece of a blood clot formed in a vein breaks off, it can be
transported to the right side of the heart, and from there into the
lungs. A piece of thrombus that is transported in this way is an
embolism and the process of forming a thrombus that becomes embolic
is called a thromboembolism. An embolism that lodges in the lungs
is a pulmonary embolism (PE). A pulmonary embolus is a very serious
condition that can be fatal if not recognized and treated
promptly.
[0768] Superficial venous thromboses can cause discomfort but
generally do not cause serious consequences, unlike the deep venous
thromboses (DVTs) that form in the deep veins of the legs or in the
pelvic veins. Systemic embolisms of venous origin can occur in
patients with an atrial or ventricular septal defect, through which
an embolus may pass into the arterial system. Such an event is
termed a paradoxical embolism.
[0769] Prevention of arterial and/or venous thrombosis includes
medications (e.g., anticoagulants (e.g., heparin), aspirin, and
vitamin E) and mechanical methods (e.g., mechanical leg pumps
(pneumatic compression stockings)).
[0770] The disclosure provides methods of treating (e.g.,
ameliorating, reducing, or eliminating one or more symptoms of)
arterial and/or venous thrombosis by administering a plasma
kallikrein binding protein (e.g., a therapeutically effective
amount of a plasma kallikrein binding protein) to a subject having
or suspected of having arterial and/or venous thrombosis.
Additionally provided are methods of treating arterial and/or
venous thrombosis by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) in combination with a second therapy,
e.g., an anticoagulant (e.g., heparin), aspirin, and/or vitamin E
and/or a mechanical method (e.g., a mechanical leg pump (pneumatic
compression stockings). The disclosure also provides methods of
preventing arterial and/or venous thrombosis or a symptom thereof
by administering a plasma kallikrein binding protein (e.g., a
prophylactically effective amount of a plasma kallikrein binding
protein) to a subject at risk of developing arterial and/or venous
thrombosis (e.g., a subject that has experienced a stroke or
myocardial infarction).
[0771] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of arterial or venous
thrombosis, such as a double-tuck model of arterial thrombosis
(Gomez-Jorge et al., J. Vasc. Inter. Rad. 9(4): 633-638 (1998), a
model of venous thrombosis in rat with low flow conditions in the
venous blood stream (Fredrich et al., Blood Coagul Fibrinolysis.
5(2):243-8 (1994)), and a canine model for venous thrombosis and
spontaneous pulmonary embolism (Frisbiel, Spinal Cord 43, 635-639
(2005)).
Postoperative Ileus
[0772] Postoperative ileus is a temporary paralysis of a portion of
the intestines typically after an abdominal surgery. Postoperative
ileus commonly occurs for 24 to 72 hours after abdominal
surgery.
[0773] Symptoms of postoperative ileus include, e.g., moderate and
diffuse abdominal discomfort, constipation, abdominal distension,
nausea or vomiting, lack of bowel movement and/or flatulence, and
excessive belching.
[0774] Treatments for postoperative ileus include, e.g., nil per os
(NPO or "Nothing by Mouth") until peristaltic sound is heard from
auscultation of the area where this portion lies, nasogastric
suction, parenteral feeds, and medications (e.g., lactulose and
erythromycin).
[0775] The disclosure provides methods of treating (e.g.,
ameliorating, reducing, or eliminating one or more symptoms of)
postoperative ileus by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having postoperative ileus. Additionally provided are methods of
treating postoperative ileus by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) in combination with a second
therapy, e.g., nil per os, nasogastric suction, parenteral feeds,
and/or a medication (e.g., lactulose and/or erythromycin). The
disclosure also provides methods of preventing postoperative ileus
or a symptom thereof by administering a plasma kallikrein binding
protein (e.g., a prophylactically effective amount of a plasma
kallikrein binding protein) to a subject at risk of developing
postoperative ileus (e.g., a subject that has had abdominal
surgery).
[0776] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of postoperative ileus,
such as a model to investigate postoperative ileus with strain
gauge transducers in awake rats (Huge et al. J Surg Res.
74(2):112-8 (1998)).
Aortic Aneurysm
[0777] An aortic aneurysm is a general term for any swelling
(dilatation or aneurysm) of the aorta, usually representing an
underlying weakness in the wall of the aorta at that location.
Types of aortic aneurysms include aortic root aneurysm, thoracic
aortic aneurysm, abdominal aortic aneurysm, and thoracoabdominal
aortic aneurysm.
[0778] Most intact aortic aneurysms do not produce symptoms. As
they enlarge, symptoms of aortic aneurysm include, e.g., anxiety or
feeling of stress, nausea or vomiting, clammy skin, rapid heart
rate, abdominal pain, back pain may develop, leg pain or numbness,
erythema nodosum (leg lesions typically found near the ankle
region), and a hoarse voice as the left recurrent laryngeal nerve
winding around the arch of the aorta is stretched. Once an aneurysm
is ruptured, it can cause severe pain and massive internal
hemorrhage, and is fatal in the absence of prompt treatment.
[0779] Treatments for aortic aneurysm include, e.g., medications,
surgical treatment and endovascular treatment. Smaller aneurysms
that are not at high risk for rupturing can be treated with drugs
to treat high blood pressure, such as beta-blockers; or doxycycline
for matrix metalloproteinase-9 inhibition. Surgical treatment
typically involves opening up of the dilated portion of the aorta
and insertion of a synthetic (Dacron or Gore-tex) patch tube.
Endovascular treatment, as a minimally invasive alternative to open
surgery repair, involves the placement of an endovascular stent via
a percutaneous technique (usually through the femoral arteries)
into the diseased portion of the aorta.
[0780] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms of)
aortic aneurysm by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having aortic aneurysm. Additionally provided are methods of
treating aortic aneurysm by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) in combination with a second
therapy, e.g., a medication (e.g., a drug to treat high blood
pressure (e.g., a beta-blocker) or doxycycline), surgery, and/or an
endovascular treatment. The disclosure also provides methods of
preventing aortic aneurysm or a symptom thereof by administering a
plasma kallikrein binding protein (e.g., a prophylactically
effective amount of a plasma kallikrein binding protein) to a
subject at risk of developing aortic aneurysm (e.g., a subject that
has high blood pressure).
[0781] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from an animal model of aortic aneurysm,
e.g., a rat model of abdominal aortic aneurysm using a combination
of intraluminal elastase infusion and extraluminal calcium chloride
exposure (Tanaka et al. J Vasc Surg. 50(6):1423-32 (2009)).
Osteoarthritis
[0782] Osteoarthritis, also known as degenerative arthritis, is
characterized by the breakdown and eventual loss of the cartilage
of one or more joints. Osteoarthritis occurs when the cartilage
that cushions the ends of bones in the joints deteriorates over
time. The smooth surface of the cartilage becomes rough, causing
irritation. If the cartilage wears down completely, the ends of the
bones will be damaged. Osteoarthritis commonly affects the hands,
feet, spine, and large weight-bearing joints, such as the hips and
knees.
[0783] Symptoms of osteoarthritis include, e.g., pain, tenderness,
stiffness, loss of flexibility, grating sensation, and bone
spurs.
[0784] Treatments for osteoarthritis include, e.g., conservative
measures (e.g., rest, weight reduction, physical and occupational
therapy) and medications (e.g., acetaminophen, pain-relieving
creams applied to the skin over the joints (e.g., capsaicin,
salycin, methyl salicylate, and menthol), non-steroidal
anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen,
nabumetone and naproxen), and Cox-2 inhibitors.
[0785] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on an osteoarthritis scale)
osteoarthritis by administering a plasma kallikrein binding protein
(e.g., a therapeutically effective amount of a plasma kallikrein
binding protein) to a subject having or suspected of having
osteoarthritis. Additionally provided are methods of treating
osteoarthritis by administering a plasma kallikrein binding protein
(e.g., a therapeutically effective amount of a plasma kallikrein
binding protein) in combination with a second therapy, e.g., a
conservative measure (e.g., rest, weight reduction, physical and/or
occupational therapy) and/or a medication (e.g., acetaminophen, a
topical pain-relieving cream, an NSAID (e.g., aspirin, ibuprofen,
nabumetone, or naproxen), and/or a Cox-2 inhibitor. The disclosure
also provides methods of preventing osteoarthritis or a symptom
thereof by administering a plasma kallikrein binding protein (e.g.,
a prophylactically effective amount of a plasma kallikrein binding
protein) to a subject at risk of developing osteoarthritis (e.g., a
subject that has had a joint injury).
[0786] Scales useful for the assessment of osteoarthritis include,
e.g., the Knee Injury and Osteoarthritis Outcome Score (KOOS; Roos
et al. (1998) J. Orthop. Sports Phys. Ther. 28(2):88-96), Western
Ontario and McMaster Universities Osteoarthrtis Index (WOMAC; Roos
et al. (2003) Health Qual. Life Outcomes 1(1):17), and the 36-item
Short Form General Health Scale (SF-36 GHS), as well as other
assessment tools known in the art.
[0787] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from an animal model of osteoarthritis,
e.g., injection of mono-iodoacetate (MIA) into the femorotibial
joint of rodents which promotes loss of articular cartilage similar
to that noted in human osteoarthritis (Guzman et al. Toxicol
Pathol. 31(6):619-24 (2003)), and transection of the anterior
cruciate ligament (ACL) in canines to induce osteoarthritis (Fife
and Brandt J Clin Invest. 84(5): 1432-1439 (1989)).
Vasculitis
[0788] Vasculitis refers to a heterogeneous group of disorders that
are characterized by inflammatory destruction of blood vessels.
Both arteries and veins can be affected. Lymphangitis is sometimes
considered a type of vasculitis. Vasculitis is primarily due to
leukocyte migration and resultant damage. Vasculitis can be
classified by the underlying cause, the location of the affected
vessels, or the type or size of the blood vessels. Vasculitis is
associated with a number of additional disorders and conditions,
e.g., Kawasaki disease, Behcet's disease, Polyarteritis nodosa,
Wegener's granulomatosis, Cryoglobulinemia, Takayasu's arteritis,
Churg-Strauss syndrome, Giant cell arteritis (temporal arteritis),
Henoch-Schonlein purpura, Rheumatic diseases (e.g., rheumatoid
arthritis and systemic lupus erythematosus), cancer (e.g.,
lymphomas), infections (e.g., hepatitis C), exposure to chemicals
and drugs (e.g., amphetamines, cocaine, and anthrax vaccines which
contain the Anthrax Protective Antigen as the primary
ingredient).
[0789] Symptoms of vasculitis include, e.g., fever, weight loss,
palpable purpura, livedo reticularis, myalgia or myositis,
arthralgia or arthritis, mononeuritis multiplex, headache, stroke,
tinnitus, reduced visual acuity, acute visual loss, myocardial
infarction, hypertension, gangrene, nose bleeds, bloody cough, lung
infiltrates, abdominal pain, bloody stool, perforations, and
glomerulonephritis.
[0790] Treatments for vasculitis include, e.g., cortisone-related
medications (e.g., prednisone) and immune suppression drugs (e.g.,
cyclophosphamide).
[0791] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a vasculitis scale) vasculitis
by administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) to a subject having or suspected of having vasculitis.
Additionally provided are methods of treating vasculitis by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy (e.g., a
cortisone-related medication (e.g., prednisone) and/or an immune
suppression drug (e.g., cyclophosphamide)). The disclosure also
provides methods of preventing vasculitis or a symptom thereof by
administering a plasma kallikrein binding protein (e.g., a
prophylactically effective amount of a plasma kallikrein binding
protein) to a subject at risk of developing vasculitis (e.g., a
subject that has had Kawasaki disease, Behcet's disease,
Polyarteritis nodosa, Wegener's granulomatosis, Cryoglobulinemia,
or Takayasu's arteritis, and so forth).
[0792] The disclosure also provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a vasculitis scale) vasculitis
associated with systemic lupus erythematosis by administering a
plasma kallikrein binding protein (e.g., a therapeutically
effective amount of a plasma kallikrein binding protein) to a
subject having or suspected of having vasculitis associated with
systemic lupus erythematosis. Additionally provided are methods of
treating vasculitis associated with systemic lupus erythematosis by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., a
cortisone-related medication (e.g., prednisone) and/or an immune
suppression drug (e.g., cyclophosphamide).
[0793] Further provided are methods of treating (e.g.,
ameliorating, stabilizing, or eliminating one or more symptoms) a
vasculitis associated disorder (Kawasaki disease, Behcet's disease,
Polyarteritis nodosa, Wegener's granulomatosis, Cryoglobulinemia,
Takayasu's arteritis, Churg-Strauss syndrome, Giant cell arteritis
(temporal arteritis), Henoch-Schonlein purpura, Rheumatic diseases
(e.g., rheumatoid arthritis and systemic lupus erythematosus),
cancer (e.g., lymphomas), infections (e.g., hepatitis C), exposure
to chemicals and drugs (e.g., amphetamines, cocaine, and anthrax
vaccines which contain the Anthrax Protective Antigen as the
primary ingredient)) by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) to a subject having or suspected of
having a vasculitis associated disorder. The disclosure also
provides methods of preventing a vasculitis associated disorder or
a symptom thereof by administering a plasma kallikrein binding
protein (e.g., a prophylactically effective amount of a plasma
kallikrein binding protein) to a subject at risk of developing a
vasculitis associated disorder.
[0794] Scales useful for the assessment of osteoarthritis include,
e.g., Birmingham Vasculitis Activity score (BVAS) version 3
(Mukhtyar et al. Ann Rheum Dis. 68(12):1827-32 (2009)), as well as
other assessment tools known in the art.
[0795] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from an animal model of vasculitis, see
e.g., those described in Katz et al., Clin Rev Allergy Immunol.
35(1-2):11-8 (2008) and references cited therein.
Head Trauma
[0796] Head trauma refers to trauma to the head, which may or may
not include injury to the brain. Types of head trauma include
concussion, epidural hematoma, subdural hematoma, cerebral
contusion, and diffuse axonal injury.
[0797] Symptoms of head trauma include, e.g., coma, confusion,
drowsiness, personality change, seizures, nausea and vomiting,
headache and a lucid interval, during which a patient appears
conscious only to deteriorate later, leaking cerebrospinal fluid,
visible deformity or depression in the head or face, an eye that
cannot move or is deviated to one side can indicate that a broken
facial bone is pinching a nerve that innervates eye muscles, wounds
or bruises on the scalp or face, basilar skull fractures, a
subcutaneous bleed over the mastoid, hemotympanum, cerebrospinal
fluid rhinorrhea, and otorrhea.
[0798] Treatments for head trauma include, e.g., controlling
elevated intracranial pressure (e.g., sedation, paralytics,
cerebrospinal fluid diversion), decompressive craniectomy,
barbiturate coma, hypertonic saline, and hypothermia.
[0799] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a head trauma scale) head trauma
by administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) to a subject having or suspected of having head trauma.
Additionally provided are methods of treating head trauma by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., controlling
elevated intracranial pressure (e.g., sedation, a paralytic, and/or
cerebrospinal fluid diversion), decompressive craniectomy,
barbiturate coma, hypertonic saline, and/or hypothermia. The
disclosure also provides methods of preventing head trauma or a
symptom thereof by administering a plasma kallikrein binding
protein (e.g., a prophylactically effective amount of a plasma
kallikrein binding protein) to a subject at risk of developing head
trauma (e.g., a subject that will be participating in a dangerous
activity or contact sport).
[0800] Scales useful for assessing head trauma and symptoms of head
trauma include, e.g., the Glasgow Coma Scale (Teasdale and Jennett,
Lancet 13; 2(7872):81-4 (1974)), as well as other assessment tools
known in the art.
[0801] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of head trauma, see,
e.g., those described in Cernak, NeuroRx. 2(3): 410-422 (2005) and
references cited therein.
Brain Edema
[0802] Brain edema (cerebral edema) is an excess accumulation of
water in the intracellular and/or extracellular spaces of the
brain. Types of brain edema include, e.g., vasogenic cerebral
edema, cytotoxic cerebral edema, osmotic cerebral edema, and
interstitial cerebral edema.
[0803] Vasogenic cerebral edema is due to a breakdown of tight
endothelial junctions which make up the blood-brain barrier (BBB).
This allows normally excluded intravascular proteins and fluid to
penetrate into cerebral parenchymal extracellular space. Once
plasma constituents cross the BBB, the edema spreads; this may be
quite fast and widespread. As water enters white matter it moves
extracellularly along fiber tracts and can also affect the gray
matter. This type of edema is seen in response to trauma, tumors,
focal inflammation, late stages of cerebral ischemia and
hypertensive encephalopathy. Some of the mechanisms contributing to
BBB dysfunction are: physical disruption by arterial hypertension
or trauma, tumor-facilitated release of vasoactive and endothelial
destructive compounds (e.g., arachidonic acid, excitatory
neurotransmitters, eicosanoids, bradykinin, histamine and free
radicals). Some of the special subcategories of vasogenic edema
include: hydrostatic cerebral edema, cerebral edema from brain
cancer, high altitude cerebral edema.
[0804] Cytotoxic cerebral edema is due to the derangement in
cellular metabolism resulting in inadequate functioning of the
sodium and potassium pump in the glial cell membrane. As a result
there is cellular retention of sodium and water. Cytoxotic edema is
seen with various intoxications (dinitrophenol, triethyltin,
hexachlorophene, isoniazid), in Reye's syndrome, severe
hypothermia, early ischemia, encephalopathy, early stroke or
hypoxia, cardiac arrest, pseudotumor cerebri, and cerebral
toxins.
[0805] Osmotic cerebral edema occurs when plasma is diluted by
excessive water intake (or hyponatremia), syndrome of inappropriate
antidiuretic hormone secretion (SIADH), hemodialysis, or rapid
reduction of blood glucose in hyperosmolar hyperglycemic state
(HHS), formerly hyperosmolar non-ketotic acidosis (HONK) and brain
osmolality exceeds the serum osmolality creating an abnormal
pressure.
[0806] Interstitial cerebral edema occurs in obstructive
hydrocephalus. This form of edema is due to rupture of
cerebral-spinal fluid (CSF)-brain barrier resulting in
trans-ependymal flow of CSF, which permits CSF to penetrate brain
and spread in the extracellular space of white matter.
[0807] Symptoms of brain edema (e.g., peritumoral brain edema)
include, e.g., headache, loss of coordination (ataxia), weakness,
and decreasing levels of consciousness including disorientation,
loss of memory, hallucinations, psychotic behavior, and coma.
[0808] Treatments for brain edema (e.g., peritumoral brain edema)
include, e.g., medications (e.g. dexamethasone, mannitol,
diuretics) and surgical decompression.
[0809] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms of)
brain edema (e.g., peritumoral brain edema) by administering a
plasma kallikrein binding protein (e.g., a therapeutically
effective amount of a plasma kallikrein binding protein) to a
subject having or suspected of having brain edema (e.g.,
peritumoral brain edema). Additionally provided are methods of
treating brain edema (e.g., peritumoral brain edema) by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., a medication
(e.g. dexamethasone, mannitol, and/or diuretics) and/or surgical
decompression. The disclosure also provides methods of preventing
brain edema or a symptom thereof by administering a plasma
kallikrein binding protein (e.g., a prophylactically effective
amount of a plasma kallikrein binding protein) to a subject at risk
of developing brain edema (e.g., a subject that has been diagnosed
with a brain tumor).
[0810] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of brain edema, e.g., a
rat model of cerebral embolism in which recirculation can be
introduced in the ischemic area (Koizumi et al., Jpn J Stroke 8:
1-8 (1986)).
Sepsis
[0811] Sepsis is a serious medical condition that is characterized
by a whole-body inflammatory state and the presence of a known or
suspected infection. This immunological response may be caused by
microbes in the blood, urine, lungs, skin, or other tissues and can
lead to widespread activation of acute-phase proteins, affecting
the complement system and the coagulation pathways, which then
cause damage to the vasculature as well as to the organs. Different
levels of sepsis include systemic inflammatory response syndrome
(SIRS), sepsis (SIRS in response to a confirmed infectious
process), severe sepsis (sepsis with organ dysfunction,
hypoperfusion, or hypotension), and septic shock (sepsis with
refractory arterial hypotension or hypoperfusion abnormalities in
spite of adequate fluid resuscitation).
[0812] Symptoms of sepsis include, e.g., general symptoms related
to the infection, acute inflammation present throughout the entire
body, hypothermia or fever, tachycardia, tachypnea or hypocapnia
due to hyperventilation, leukopenia, leukocytosis, bandemia, and
organ (e.g., lung, brain, liver, kidney, and/or heart)
dysfunction.
[0813] Treatments for sepsis include, e.g., antibiotics,
vasopressor drugs, insulin, corticosteroids, drotrecogin alfa,
surgical drainage of infected fluid collections, fluid replacement,
and appropriate support for organ dysfunction (e.g., hemodialysis
in kidney failure, mechanical ventilation in pulmonary dysfunction,
transfusion of blood products, and drug and fluid therapy for
circulatory failure). Early Goal Directed Therapy (EGDT), a
systematic approach to resuscitation, can be used to treat severe
sepsis and septic shock.
[0814] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a sepsis scale) sepsis by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) to a subject having or suspected of having sepsis.
Additionally provided are methods of treating sepsis by
administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., an antibiotic,
a vasopressor drug, insulin, a corticosteroid, drotrecogin alfa,
surgical drainage of infected fluid collections, fluid replacement,
an appropriate support for organ dysfunction (e.g., hemodialysis in
kidney failure, mechanical ventilation in pulmonary dysfunction,
transfusion of blood products, and/or drug and fluid therapy for
circulatory failure), and/or an Early Goal Directed Therapy (EGDT).
The disclosure also provides methods of preventing sepsis or a
symptom thereof by administering a plasma kallikrein binding
protein (e.g., a prophylactically effective amount of a plasma
kallikrein binding protein) to a subject at risk of developing
sepsis (e.g., a subject that has been diagnosed as having an
infection).
[0815] Scales useful for assessing sepsis and symptoms of sepsis
include, e.g., the Baltimore Sepsis Scale (Meek et al. J Burn Care
Rehabil. 12(6):564-8 (1991)) as well as other assessment tools
known in the art.
[0816] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of sepsis, see, e.g.,
those described in U.S. Pat. No. 6,964,856, and Buras et al. Nat
Rev Drug Discov. 4(10):854-65 (2005) and references cited
therein.
Acute Middle Cerebral Artery (MCA) Ischemic Event (Stroke)
[0817] An acute middle cerebral artery (MCA) ischemic event
(stroke) is the rapidly developing loss of brain function(s) due to
disturbance in the blood supply to the brain due to ischemia (lack
of glucose and oxygen supply) caused by thrombosis (e.g., venous
thrombosis), embolism, or systemic hypoperfusion. As a result, the
affected area of the brain is unable to function, leading to
inability to move one or more limbs on one side of the body,
inability to understand or formulate speech, or inability to see
one side of the visual field. A stroke is a medical emergency and
can cause permanent neurological damage, complications, and/or
death.
[0818] Symptoms of acute middle cerebral artery (MCA) ischemic
event (stroke) include, e.g., hemiplegia, decreased sensation and
muscle weakness of the face, numbness, reduction in sensory or
vibratory sensation, altered smell, taste, hearing or vision (total
or partial), drooping of eyelid (ptosis) and weakness of ocular
muscles, decreased reflexes, balance problems and nystagmus,
altered breathing and heart rate, weakness in sternocleidomastoid
muscle with inability to turn head to one side, weakness in tongue
(inability to protrude and/or move from side to side), aphasia,
apraxia, visual field defect, memory deficits, hemineglect,
disorganized thinking, confusion, hypersexual gestures,
anosognosia, trouble walking, altered movement coordination, and
vertigo and/or disequilibrium.
[0819] Treatment for acute middle cerebral artery (MCA) ischemic
event (stroke) includes, e.g., thrombolysis (e.g., tissue
plasminogen activator (tPA)), thrombectomy, angioplasty and
stenting, therapeutic hypothermia, and medications (e.g., aspirin,
clopidogrel and dipyridamole).
[0820] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a stroke scale) acute middle
cerebral artery (MCA) ischemic event (stroke) by administering a
plasma kallikrein binding protein (e.g., a therapeutically
effective amount of a plasma kallikrein binding protein) to a
subject having or suspected of having acute middle cerebral artery
(MCA) ischemic event (stroke). Additionally provided are methods of
treating acute middle cerebral artery (MCA) ischemic event (stroke)
by administering a plasma kallikrein binding protein (e.g., a
therapeutically effective amount of a plasma kallikrein binding
protein) in combination with a second therapy, e.g., thrombolysis
(e.g., tissue plasminogen activator (tPA)), thrombectomy,
angioplasty and stenting, therapeutic hypothermia, and/or a
medication (e.g., aspirin, clopidogrel and dipyridamole). The
disclosure also provides methods of preventing acute middle
cerebral artery (MCA) ischemic event (stroke) or a symptom thereof
by administering a plasma kallikrein binding protein (e.g., a
prophylactically effective amount of a plasma kallikrein binding
protein) to a subject at risk of developing acute middle cerebral
artery (MCA) ischemic event (stroke) (e.g., a subject that has
experienced systemic hypoperfusion).
[0821] Scales useful for assessing acute middle cerebral artery
(MCA) ischemic event (stroke) and symptoms of acute middle cerebral
artery (MCA) ischemic event (stroke) include, e.g., Oxford
Community Stroke Project classification (OCSP, also known as the
Bamford or Oxford classification) (Bamford et al., Lancet 337
(8756): 1521-6 (1991)), and TOAST (Trial of Org 10172 in Acute
Stroke Treatment) (Adams et al., Stroke 24 (1): 35-41 (1993)).
[0822] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of acute middle cerebral
artery (MCA) ischemic event (stroke), see, e.g., those described in
Beech et al., Brain Res 895: 18-24 (2001), Buchan et al., Stroke 23
(2): 273-9 (1992), Carmichael, NeuroRx 2: 396-409 (2005), Chen et
al., Stroke 17 (4): 738-43 (1986), Dittmar et al., Stroke 34:
2252-7 (2003), Dittmar et al., J Neurosci Methods 156: 50 (2006),
Gerriets et al., J Neurosci Methods 122: 201-11 (2003), Gerriets et
al., Stroke 35: 2372-2377 (2004), Graham et al., Comp Med 54:
486-496 (2004), Koizumi et al., Jpn J Stroke 8: 1-8 (2004), Longa
et al., Stroke 20 (1): 84-91 (1989), Mayzel-Oreg, Magn Reson Med
51: 1232-8 (2004), Schmid-Elsaesser et al., Stroke 29 (10): 2162-70
(1989), Tamura et al., J Cereb Blood Flow Metab 1: 53-60 (1981),
Watson et al., Ann Neurol 17: 497-504 (1985), and Zhang et al., J
Cereb Blood Flow Metab 17: 123-35 (1997).
Restenosis
[0823] Restenosis is the reoccurrence of stenosis, a narrowing of a
blood vessel, leading to restricted blood flow. Restenosis usually
pertains to an artery or other large blood vessel that has become
narrowed, received treatment to clear the blockage such as
angioplasty, and subsequently become renarrowed. It can be defined
as a reduction in the circumference of the lumen of 50% or more,
and had a high incidence rate (25-50%) in patients who had
undergone balloon angioplasty, with the majority of patients
needing further angioplasty within 6 months.
[0824] Treatments for restenosis include, e.g., additional
angioplasty if restenosis occurs without a stent or at either end
of a stent, repeated angioplasty and insertion of another stent
inside the original if restenosis occurs within a stent,
drug-eluted stents, brachytherapy, and intracoronary radiation.
[0825] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms of)
restenosis (e.g., after angioplasty) by administering a plasma
kallikrein binding protein (e.g., a therapeutically effective
amount of a plasma kallikrein binding protein) to a subject having
or suspected of having restenosis (e.g., after angioplasty).
Additionally provided are methods of treating restenosis (e.g.,
after angioplasty) by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) in combination with a second therapy,
e.g., angioplasty if restenosis occurs without a stent or at either
end of a stent, repeated angioplasty and insertion of another stent
inside the original if restenosis occurs within a stent, a
drug-eluted stent, brachytherapy, and/or intracoronary radiation.
The disclosure also provides methods of preventing restenosis or a
symptom thereof by administering a plasma kallikrein binding
protein (e.g., a prophylactically effective amount of a plasma
kallikrein binding protein) to a subject at risk of developing
restenosis (e.g., a subject that has had stenosis).
[0826] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of restenosis, see,
e.g., those described in U.S. Pat. Nos. 5,304,122 and 6,034,053,
and Kantor et al., Cardiovasc Radiat Med. 1(1):48-54 (1999), and
references cited therein.
Systemic Lupus Erythematosus Nephritis
[0827] Systemic lupus erythematosus nephritis is an inflammation of
the kidney caused by systemic lupus erythematosus (SLE), a chronic
autoimmune connective tissue disease. SLE can be associated with
vasculitis which are disorders characterized by inflammatory
destruction of blood vessels.
[0828] Symptoms of systemic lupus erythematosus nephritis include,
e.g., general symptoms of kidney disease, weight gain, high blood
pressure, darker foamy urine, and swelling around the eyes, legs,
ankles or fingers.
[0829] Treatments for systemic lupus erythematosus nephritis
include, e.g., steroid therapy (e.g., corticosteroids),
chemotherapy (e.g., cyclophosphamide, azathioprine, mycophenolate
mofetil, or cyclosporine), and immunosuppressant agents (e.g.,
mycophenolate mofetil and intravenous cyclophosphamide).
[0830] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a lupus scale) systemic lupus
erythematosus nephritis by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) to a subject having or suspected
of having systemic lupus erythematosus nephritis. Additionally
provided are methods of treating systemic lupus erythematosus
nephritis by administering a plasma kallikrein binding protein
(e.g., a therapeutically effective amount of a plasma kallikrein
binding protein) in combination with a second therapy, e.g.,
steroid therapy (e.g., a corticosteroid), chemotherapy (e.g.,
cyclophosphamide, azathioprine, mycophenolate mofetil, and/or
cyclosporine), and/or an immunosuppressant agent (e.g.,
mycophenolate mofetil and/or intravenous cyclophosphamide). The
disclosure also provides methods of preventing systemic lupus
erythematosus nephritis or a symptom thereof by administering a
plasma kallikrein binding protein (e.g., a prophylactically
effective amount of a plasma kallikrein binding protein) to a
subject at risk of developing systemic lupus erythematosus
nephritis (e.g., a subject that has been diagnosed with lupus or a
subject having a family member with lupus or a genetic
predisposition thereto).
[0831] Scales useful for assessing systemic lupus erythematosus
nephritis and symptoms of systemic lupus erythematosus nephritis
include, e.g., World Health Organization (WHO) classification based
on the biopsy (Weening et al., J. Am. Soc. Nephrol. 15 (2): 241-50
(2004)) as well as other assessment tools known in the art.
[0832] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of systemic lupus
erythematosus nephritis, see, e.g., those described in U.S. Pat.
No. 7,265,261, Peng, Methods Mol Med. 102:227-72 (2004), and
references cited therein.
Burn Injury and Wound Healing
[0833] A burn injury is a type of injury that may be caused by
heat, electricity, chemicals, light, radiation, or friction.
Muscle, bone, blood vessel, dermal and epidermal tissue can all be
damaged with subsequent pain due to profound injury to nerves.
Depending on the location affected and the degree of severity, a
burn victim may experience a wide number of potentially fatal
complications including shock, infection, electrolyte imbalance and
respiratory distress. In burn injuries, the damage to epidermis and
dermal elements is the result of several key insults which can be
divided into initial (e.g., heat injury, inflammatory mediator
injury, ischemia induced injury) and delayed insults. Excess heat
causes rapid protein denaturation and cell damage. Much of the
tissue damage, e.g., in the perfused subsurface burn, can be caused
by toxic mediators of inflammation (e.g., oxidants and/or
proteases) which are activated with the burn. Consumption of wound
oxygen by neutrophils can lead to tissue hypoxia. Instant surface
vascular thrombosis occurs along with cell death from the heat
insult and causes ischemia and further tissue damage. Delayed
injury after the initial heat and mediator damage includes, e.g.,
inflammation caused by neurotic tissue, bacteria on surface,
caustic topical agents, and surface exudate; and continued damage
to viable cells and new tissue growth by excess wound proteolytic
activity and oxidant release.
[0834] Treatments of burn injury include, e.g., intravenous fluids,
dressings, pain management (e.g., analgesics (e.g., ibuprofen and
acetaminophen), narcotics, and local anesthetics), inflammatory
mediator inhibitors, and antibiotics.
[0835] The disclosure provides methods of treating (e.g.,
stabilizing, reducing, or eliminating one or more symptoms or
stabilizing the subject's score on a burn scale) a burn injury
and/or promoting wound healing by administering a plasma kallikrein
binding protein (e.g., a therapeutically effective amount of a
plasma kallikrein binding protein) to a subject having or suspected
of having a burn injury. Additionally provided are methods of
treating a burn injury by administering a plasma kallikrein binding
protein (e.g., a therapeutically effective amount of a plasma
kallikrein binding protein) in combination with a second therapy,
e.g., intravenous fluid, a dressing, pain management (e.g., an
analgesic (e.g., ibuprofen and acetaminophen), a narcotic, and a
local anesthetic), an inflammatory mediator inhibitor, and an
antibiotic. The disclosure also provides methods of preventing burn
injuries or a symptom thereof by administering a plasma kallikrein
binding protein (e.g., a prophylactically effective amount of a
plasma kallikrein binding protein) to a subject at risk of
developing burn injuries (e.g., a subject whose occupation creates
a risk of a burn injury, e.g., firefighter or cook).
[0836] Scales useful for assessing burns and symptoms of burns
include, e.g., burn scales by degrees, by thickness, and by total
body surface area (TBSA) (Meek et al. J Burn Care Rehabil.
12(6):564-8 (1991)) as well as other assessment tools known in the
art.
[0837] Guidance for the determination of the dosage that delivers a
therapeutically effective amount of a plasma kallikrein binding
protein may be obtained from animal models of burn, such as a
porcine burn model (Singer and McClain, Methods Mol Med. 78:107-19
(2003), a sheep model of thermal injury (Jonkam et al., Shock,
28:704-709 (2007)), a rabbit model of thermal injury (Nwariaku et
al., Burns, 22:324-327 (1996)), and a mouse model of burn wounding
(Stevenson et al., Methods Mol Med. 78:95-105 (2003)).
Combination Therapies
[0838] A plasma kallikrein binding protein described herein, e.g.,
an anti-plasma kallikrein antibody, e.g., an anti-plasma kallikrein
Fab or IgG, can be administered in combination with one or more of
the other therapies for treating a disease or condition associated
with plasma kallikrein activity, e.g., a disease or condition
described herein. For example, a plasma kallikrein binding protein
can be used therapeutically or prophylactically with surgery,
another anti-plasma kallikrein Fab or IgG (e.g., another Fab or IgG
described herein), another plasma kallikrein inhibitor, a peptide
inhibitor, or small molecule inhibitor. Examples of plasma
kallikrein inhibitors that can be used in combination therapy with
a plasma kallikrein binding protein described herein include plasma
kallikrein inhibitors described in, e.g., WO 95/21601 or WO
2003/103475.
[0839] One or more plasma kallikrein inhibitors can be used in
combination with one or more plasma kallikrein binding proteins
described herein. For example, the combination can result in a
lower dose of the inhibitor being needed, such that side effects
are reduced.
[0840] A plasma kallikrein binding protein described herein can be
administered in combination with one or more current therapies for
treating a plasma kallikrein associated disease or condition,
including, but not limited to the current therapies for treating
the disorder, e.g., a current therapy for rheumatoid arthritis,
gout, intestinal bowel disease, oral mucositis, neuropathic pain,
inflammatory pain, spinal stenosis-degenerative spine disease,
arterial or venous thrombosis, post operative ileus, aortic
aneurysm, osteoarthritis, vasculitis, head trauma or peri-tumor
brain edema, sepsis, acute middle cerebral artery (MCA) ischemic
event (stroke), restenosis (e.g., after angioplasty), systemic
lupus erythematosis nephritis, burn injury, or wound healing. For
example, pKal inhibition is a novel mechanism of treating disease
and therefore could provide effects that are synergistic or
additive with other therapeutics. For example, a protein described
herein that inhibits plasma kallikrein or that inhibits a
downstream event of plasma kallikrein activity can also be used in
combination with another treatment for a plasma kallikrein
associated disease, such as surgery or administration of a second
agent, e.g., as described herein. For example, the second agent can
include ecallantide, a C1 esterase inhibitor (e.g., CINRYZE.TM.),
aprotinin (TRASYLOL.RTM.), a bradykinin B2 receptor inhibitor
(e.g., icatibant (FIRAZYR.RTM.)).
[0841] The term "combination" refers to the use of the two or more
agents or therapies to treat the same patient, wherein the use or
action of the agents or therapies overlap in time. The agents or
therapies can be administered at the same time (e.g., as a single
formulation that is administered to a patient or as two separate
formulations administered concurrently) or sequentially in any
order. Sequential administrations are administrations that are
given at different times. The time between administration of the
one agent and another agent can be minutes, hours, days, or weeks.
The use of a plasma kallikrein binding protein described herein can
also be used to reduce the dosage of another therapy, e.g., to
reduce the side effects associated with another agent that is being
administered. Accordingly, a combination can include administering
a second agent at a dosage at least 10, 20, 30, or 50% lower than
would be used in the absence of the plasma kallikrein binding
protein.
[0842] The second agent or therapy can also be another agent for a
plasma kallikrein associated therapy. Non-limiting examples of
another treatment for a plasma kallikrein associated disease or
condition include, e.g., ecallantide, a C1 esterase inhibitor
(e.g., CINRYZE.TM.), aprotinin (TRASYLOL.RTM.), a bradykinin B2
receptor inhibitor (e.g., icatibant (FIRAZYR.RTM.)) or a second
binding protein described herein.
[0843] A combination therapy can include administering an agent
that reduces the side effects of other therapies. The agent can be
an agent that reduces the side effects of a plasma kallikrein
associated disease treatment. For example, for inflammatory
diseases, a pKal inhibitor could be steroid sparring. Also, there
could be synergism with a TNF-alpha inhibitor for treating
inflammation or a VEGF blocker for treating cancer and/or
angiogenesis.
Diagnostic Uses
[0844] A protein that binds to plasma kallikrein described herein
can have in vitro and in vivo diagnostic utilities. A plasma
kallikrein binding protein described herein (e.g., a protein that
binds or binds and inhibits plasma kallikrein) can be used, e.g.,
for in vivo imaging, e.g., during a course of treatment for a
disease or condition in which plasma kallikrein is active, e.g., a
disease or condition described herein, or in diagnosing a disease
or condition described herein.
[0845] In one aspect, the disclosure provides a diagnostic method
for detecting the presence of plasma kallikrein, in vitro or in
vivo (e.g., in vivo imaging in a subject). The method can include
localizing plasma kallikrein within a subject or within a sample
from a subject. With respect to sample evaluation, the method can
include, for example: (i) contacting a sample with plasma
kallikrein binding protein; and (ii) detecting the location of the
plasma kallikrein binding protein in the sample.
[0846] A plasma kallikrein binding protein can also be used to
determine the qualitative or quantitative level of expression of
plasma kallikrein in a sample. The method can also include
contacting a reference sample (e.g., a control sample, e.g., a
negative control) with the binding protein, and determining a
corresponding assessment of the reference sample. A difference
(e.g., increase), e.g., a statistically significant difference, in
the formation of the complex in the sample or subject relative to
the control sample or subject can be indicative of the presence of
plasma kallikrein in the sample. In one embodiment, the plasma
kallikrein binding protein does not cross react with another
kallikrein protein, such as tissue kallikrein and/or with plasma
prekallikrein. E.g., the binding protein binds to another
kallikrein protein or to prekallikrein 5- to 10-fold less well (or
even less well) than it binds to plasma kallikrein. For example,
the binding protein can bind to plasma kallikrein with a KD of
.about.10-50 pM, whereas it binds to tissue kallikrein and/or
prekallikrein at .about.10 nM.
[0847] The plasma kallikrein binding protein can be directly or
indirectly labeled with a detectable substance to facilitate
detection of the bound or unbound antibody. Suitable detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials and radioactive materials.
[0848] Complex formation between the plasma kallikrein binding
protein and plasma kallikrein can be detected by evaluating the
binding protein bound to the plasma kallikrein or unbound binding
protein. Conventional detection assays can be used, e.g., an
enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay
(RIA) or tissue immunohistochemistry. Further to labeling the
plasma kallikrein binding protein, the presence of plasma
kallikrein can be assayed in a sample by a competition immunoassay
utilizing standards labeled with a detectable substance and an
unlabeled plasma kallikrein binding protein. In one example of this
assay, the biological sample, the labeled standards, and the plasma
kallikrein binding protein are combined and the amount of labeled
standard bound to the unlabeled binding protein is determined. The
amount of plasma kallikrein in the sample is inversely proportional
to the amount of labeled standard bound to the plasma kallikrein
binding protein.
[0849] Fluorophore and chromophore labeled proteins can be
prepared. Because antibodies and other proteins absorb light having
wavelengths up to about 310 nm, the fluorescent moieties should be
selected to have substantial absorption at wavelengths above 310 nm
and preferably above 400 nm. A variety of suitable fluorescers and
chromophores are described by Stryer, 1968, Science 162:526 and
Brand, L. et al., 1972, Annu. Rev. Biochem. 41:843-868. The
proteins can be labeled with fluorescent chromophore groups by
conventional procedures such as those disclosed in U.S. Pat. Nos.
3,940,475, 4,289,747, and 4,376,110. One group of fluorescers
having a number of the desirable properties described above is the
xanthene dyes, which include the fluoresceins and rhodamines.
Another group of fluorescent compounds are the naphthylamines. Once
labeled with a fluorophore or chromophore, the protein can be used
to detect the presence or localization of the plasma kallikrein in
a sample, e.g., using fluorescent microscopy (such as confocal or
deconvolution microscopy).
[0850] Histological Analysis.
[0851] Immunohistochemistry can be performed using the proteins
described herein. For example, in the case of an antibody, the
antibody can be synthesized with a label (such as a purification or
epitope tag), or can be detectably labeled, e.g., by conjugating a
label or label-binding group. For example, a chelator can be
attached to the antibody. The antibody is then contacted to a
histological preparation, e.g., a fixed section of tissue that is
on a microscope slide. After an incubation for binding, the
preparation is washed to remove unbound antibody. The preparation
is then analyzed, e.g., using microscopy, to identify if the
antibody bound to the preparation.
[0852] Of course, the antibody (or other polypeptide or peptide)
can be unlabeled at the time of binding. After binding and washing,
the antibody is labeled in order to render it detectable.
[0853] Protein Arrays.
[0854] The plasma kallikrein binding protein can also be
immobilized on a protein array. The protein array can be used as a
diagnostic tool, e.g., to screen medical samples (such as isolated
cells, blood, sera, biopsies, and the like). Of course, the protein
array can also include other binding proteins, e.g., that bind to
plasma kallikrein or to other target molecules.
[0855] Methods of producing polypeptide arrays are described, e.g.,
in De Wildt et al., 2000, Nat. Biotechnol. 18:989-994; Lueking et
al., 1999, Anal. Biochem. 270:103-111; Ge, 2000, Nucleic Acids Res.
28, e3, I-VII; MacBeath and Schreiber, 2000, Science 289:1760-1763;
WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be
spotted at high speed, e.g., using commercially available robotic
apparati, e.g., from Genetic MicroSystems or BioRobotics. The array
substrate can be, for example, nitrocellulose, plastic, glass,
e.g., surface-modified glass. The array can also include a porous
matrix, e.g., acrylamide, agarose, or another polymer.
[0856] For example, the array can be an array of antibodies, e.g.,
as described in De Wildt, supra. Cells that produce the proteins
can be grown on a filter in an arrayed format. Polypeptide
production is induced, and the expressed polypeptides are
immobilized to the filter at the location of the cell. A protein
array can be contacted with a labeled target to determine the
extent of binding of the target to each immobilized polypeptide.
Information about the extent of binding at each address of the
array can be stored as a profile, e.g., in a computer database. The
protein array can be produced in replicates and used to compare
binding profiles, e.g., of a target and a non-target.
[0857] FACS (Fluorescence Activated Cell Sorting).
[0858] The plasma kallikrein binding protein can be used to label
cells, e.g., cells in a sample (e.g., a patient sample). The
binding protein is also attached (or attachable) to a fluorescent
compound. The cells can then be sorted using fluorescence activated
cell sorter (e.g., using a sorter available from Becton Dickinson
Immunocytometry Systems, San Jose Calif.; see also U.S. Pat. Nos.
5,627,037; 5,030,002; and 5,137,809). As cells pass through the
sorter, a laser beam excites the fluorescent compound while a
detector counts cells that pass through and determines whether a
fluorescent compound is attached to the cell by detecting
fluorescence. The amount of label bound to each cell can be
quantified and analyzed to characterize the sample.
[0859] The sorter can also deflect the cell and separate cells
bound by the binding protein from those cells not bound by the
binding protein. The separated cells can be cultured and/or
characterized.
[0860] In Vivo Imaging.
[0861] Also featured is a method for detecting the presence of
plasma kallikrein expressing tissues in vivo. The method includes
(i) administering to a subject (e.g., a patient having, e.g., a
plasma kallikrein associated disease or condition) an anti-plasma
kallikrein antibody, conjugated to a detectable marker; (ii)
exposing the subject to a means for detecting said detectable
marker to the plasma kallikrein expressing tissues or cells. For
example, the subject is imaged, e.g., by NMR or other tomographic
means.
[0862] Examples of labels useful for diagnostic imaging include
radiolabels such as .sup.131I, .sup.111In, .sup.123I, .sup.99mTc,
.sup.32P, .sup.125I, .sup.3H, .sup.14C, and .sup.188Rh, fluorescent
labels such as fluorescein and rhodamine, nuclear magnetic
resonance active labels, positron emitting isotopes detectable by a
positron emission tomography ("PET") scanner, chemiluminescers such
as luciferin, and enzymatic markers such as peroxidase or
phosphatase. Short range radiation emitters, such as isotopes
detectable by short range detector probes can also be employed. The
protein can be labeled with such reagents; for example, see Wensel
and Meares, 1983, Radioimmunoimaging and Radioimmunotherapy,
Elsevier, New York for techniques relating to the radiolabeling of
antibodies and D. Colcher et al., 1986, Meth. Enzymol. 121:
802-816.
[0863] The binding protein can be labeled with a radioactive
isotope (such as .sup.14C, .sup.3H, .sup.35S, .sup.125I, .sup.32P,
.sup.131I). A radiolabeled binding protein can be used for
diagnostic tests, e.g., an in vitro assay. The specific activity of
a isotopically-labeled binding protein depends upon the half life,
the isotopic purity of the radioactive label, and how the label is
incorporated into the antibody.
[0864] In the case of a radiolabeled binding protein, the binding
protein is administered to the patient, is localized to cells
bearing the antigen with which the binding protein reacts, and is
detected or "imaged" in vivo using known techniques such as
radionuclear scanning using e.g., a gamma camera or emission
tomography. See e.g., A. R. Bradwell et al., "Developments in
Antibody Imaging", Monoclonal Antibodies for Cancer Detection and
Therapy, R. W. Baldwin et al., (eds.), pp 65 85 (Academic Press
1985). Alternatively, a positron emission transaxial tomography
scanner, such as designated Pet VI located at Brookhaven National
Laboratory, can be used where the radiolabel emits positrons (e.g.,
.sup.11C, .sup.18F, .sup.15O, and .sup.13N).
[0865] MRI Contrast Agents.
[0866] Magnetic Resonance Imaging (MRI) uses NMR to visualize
internal features of living subject, and is useful for prognosis,
diagnosis, treatment, and surgery. MRI can be used without
radioactive tracer compounds for obvious benefit. Some MRI
techniques are summarized in EP-A-0 502 814. Generally, the
differences related to relaxation time constants T1 and T2 of water
protons in different environments are used to generate an image.
However, these differences can be insufficient to provide sharp
high resolution images.
[0867] The differences in these relaxation time constants can be
enhanced by contrast agents. Examples of such contrast agents
include a number of magnetic agents paramagnetic agents (which
primarily alter T1) and ferromagnetic or superparamagnetic (which
primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTA
chelates) can be used to attach (and reduce toxicity) of some
paramagnetic substances (e.g., Fe.sup.+3, Mn.sup.+2, Gd.sup.+3).
Other agents can be in the form of particles, e.g., less than 10 mm
to about 10 nM in diameter). Particles can have ferromagnetic,
antiferromagnetic, or superparamagnetic properties. Particles can
include, e.g., magnetite (Fe.sub.3O.sub.4),
.gamma.-Fe.sub.2O.sub.3, ferrites, and other magnetic mineral
compounds of transition elements. Magnetic particles may include:
one or more magnetic crystals with and without nonmagnetic
material. The nonmagnetic material can include synthetic or natural
polymers (such as sepharose, dextran, dextrin, starch and the
like.
[0868] The plasma kallikrein binding protein can also be labeled
with an indicating group containing of the NMR active .sup.19F
atom, or a plurality of such atoms inasmuch as (i) substantially
all of naturally abundant fluorine atoms are the .sup.19F isotope
and, thus, substantially all fluorine containing compounds are NMR
active; (ii) many chemically active polyfluorinated compounds such
as trifluoracetic anhydride are commercially available at
relatively low cost; and (iii) many fluorinated compounds have been
found medically acceptable for use in humans such as the
perfluorinated polyethers utilized to carry oxygen as hemoglobin
replacements. After permitting such time for incubation, a whole
body MRI is carried out using an apparatus such as one of those
described by Pykett, 1982, Sci. Am. 246:78 88 to locate and image
tissues expressing plasma kallikrein.
[0869] The following examples provide further illustration and are
not limiting.
EXAMPLES
Example 1
[0870] We have discovered several antibody inhibitors and binders
of plasma kallikrein (pKal). The most potent of these have been
further characterized and shown to have apparent inhibition
constants (K.sub.i,app)<10 nM, to be specific pKal inhibitors
with respect to other tested serine proteases, and to not bind
prekallikrein. Amino acid sequences of the CDRs for the inhibitors
and the binders are shown in Tables 1 and 2, respectively.
TABLE-US-00004 TABLE 1 CDR Amino Acid Sequences, ELISA Signal, and
Apparent Inhibition Constant of Antibody Inhibitors of PKal (SEQ ID
NOs: 8-223) LV-CDR2 LV-CDR1 (SEQ ID LV-CDR3 Human pKal Human pKal
(SEQ ID NOs: NOs: (SEQ ID Initial Name ELISA (T/B) (Ki, app nM)
8-43) 44-79) NOs: 80-115 M6-D09 39.9 5.9 RASQSIRNYLN AASTLQS
QQLSGYPHT M7-B04 4.1 54 TGTNSDVGNYNLVS EVNKRPS CSYAGNRNFYV M7-E07
45.7 36 SGDKLGDKYAC QDSKRPS QAWDSSTGV M8-A09 5.4 105 SGDKLGNKYAY
QDNNRPS QAWDSRTVV M10-F10 39.2 <100 nM RASQSISVYLN GASNLQF
QQTFSLFT M10-H05 42.2 18 RASQSVSSSYLA GASSRAT QQYGSSPFT M12-D05
48.5 5.2 SGDQLGDKYVG QDTKRPS QAWDTSTAG M27-E05 41.3 16 SGDKLGDKYAC
QDSKRPS QAWDSSTGV M28-B11 33.3 5.5 SGDQLGDKYVG QDTKRPS QAWDTSTAG
M29-D09 47.5 0.7 SGNKLGDKYVA QDTKRPS QAWDSSIVI M29-E09 28.8 11
SGDNLGNKYNS QDTKRPS QAWDGNVV M35-G04 11.1 2.9 RASQSVSSYLA DASNRAT
QQRSNWPRGFT M38-F02 33.5 14 SGEKLGDKYVS EDSRRPS QAWDSSTAI M41-A11
28.0 13 SGDKLGDKYTS QDIKRPS QAWDSPNARV M73-D06 4.0 <100 nM
SGSSSNIGSNTVS NDHRRPS SAWDDSLNGVV M76-D01 11.2 <100 nM
RSSQSLSDDGNTYLD TLSYRAS MQGTHWPPT M110-C12 2.4 <100 nM
RSSLSLLHSNGYNYLD LSSTRAS MQPLETPPT M137-E12 4.5 79 SGNNSNFGSNTVT
SDSRRPS AAWDDSLNGV M142-H08 29.9 0.2 RASQPIDNYLN AASRLQS QQSYTVPYT
M145-D01 6.2 1.1 RASQSVSSYLA DASNRAT QQRSNWPRGFT M145-D11 40.0 0.79
SGDKLGDKYTS QDIKRPS QAWDSPNARV M146-E12 49.6 2.2 RASGDIGNALG
DASTLQS LQGYNYPRT M152-A12 19. <100 nM RASQSISSYLS AASSLQS
QQSISIPRT M160-G12 38.3 17 RASQGISSYLA AASTLQS QQLNSYPLT M161-C11
41.8 0.3 SGDKLGDKYVS QDTKRPS QAWDSSTYV M162-A04 11.4 4.8
RASQSISSWLA KASTLES QQYNTYWT X67-B03 nd 2.1 RASQPIDNYLN AASRLQS
QQSYTVPYT X67-C03 nd 0.7 RASQPIDNYLN AASRLQS QQSYTVPYT X67-C09 nd
8.6 RASQPIDNYLN AASRLQS QQSYTVPYT X67-D03 nd 0.1 RASQPIDNYLN
AASRLQS QQSYTVPYT X67-E04 nd 1.3 RASQPIDNYLN AASRLQS QQSYTVPYT
X67-F01 nd 0.9 RASQPIDNYLN AASRLQS QQSYTVPYT X67-F10 nd 1.3
RASQPIDNYLN AASRLQS QQSYTVPYT X67-G04 nd 0.35 RASQPIDNYLN AASRLQS
QQSYTVPYT X67-H04 nd 3.6 RASQPIDNYLN AASRLQS QQSYTVPYT X81-B01 nd
0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HV-CDR1 (SEQ ID HV-CDR2 HV-CDR3
Initial Name NOs: 116-151) (SEQ ID NOs: 152-187) (SEQ ID NOs:
188-223) M6-D09 FYYMV VIYPSGGITVYADSVKG DKWAVMPPYYYYAMDV M7-B04
WYSMV SISPSGGLTNYADSVKG HTAARPFYYYYMDV M7-E07 WYLMI
YIYPSGGFTYYADSVKG TEGPLSWGYGMDV M8-A09 TYFML SIYPSGGNTVYADSVKG
AASPVRNYYYYGMDV M10-F10 FYNMN SISPSGGETNYADSVKG GGGAYRNNWWGGFDI
M10-H05 PYNMY SIRPSGGGTVYADSVKG GFIAARWYYFDY M12-D05 WYTMV
RIYPSGGWTKYADSVKG EGLLWFGENAFDI M27-E05 WYLMI YIYPSGGFTYYADSVKG
TEGPLSWGYGMDV M28-B11 WYTMV RIYPSGGWTKYADSVKG EGLLWFGENAFDI M29-D09
WYTMV YIYPSGGATFYADSVKG GSYDYIWGFYSDH M29-E09 WYEMG
SIYSSGGGTMYADSVKG NPQYSGYDRSLSDGAFDI M35-G04 YYHMS
VISPSGGSTKYADSVKG GGSSDYAWGSYRRPYYFDY M38-F02 YYMMV
YIYSSGGHTVYADSVKG DLFLYDFWSKGAFDI M41-A11 HYRMS SIYPSGGRTVYADSVKG
DKFEWRLLFRGIGNDAFDI M73-D06 RYEMY SISSSGGPTAYADSVKG
GTPKWELLLRSIYIENAFDI M76-D01 FYAMH GIVPSGGRTHYADSVKG DSSGSPNPLFDY
M110-C12 YYEMD GISSSGGHTAYADSVKG ERRSSSRARYYYGMDV M137-E12 DYRMQ
VIVPSGGNTMYADSVKG GGPGSSIAARRAPTGYYGMDV M142-H08 AYSMI
YIRPSGGRTTYADSVKG GGLLLWFRELKSNYFDY M145-D01 YYHMS
VISPSGGSTKYADSVKG GGSSDYAWGSYRRPYYFDY M145-D11 HYRMS
SIYPSGGRTVYADSVKG DKFEWRLLFRGIGNDAFDI M146-E12 RYIMH
SISPSGGLTSYADSVKG EFENAYHYYYYGMDV M152-A12 PYFMG GIGPSGGSTTYADSVKG
EGPPYSSGWYRGLRQYHFDY M160-G12 HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTSYFDY M161-C11 DYAMK SISSSGGVTQYADSVKG
EEDYSSSWYSRRFDYYYGMDV M162-A04 HYIMM GIYSSGGITVYADSVKG
RRTGIPRRDAFDI X67-B03 AYSMI YIRPSGGRTTYADSVKG GGLLLWSRELKSNYFDY
X67-C03 AYSMI YIRPSGGRTTYADSVKG GGLLLWFMELKSNYFDY X67-C09 AYSMI
YIRPSGGRTTYADSVKG GGLLLWGRELKSNYFDY X67-D03 AYSMI YIRPSGGRTTYADSVKG
GGLLLWNRELKSNYFDY X67-E04 AYSMI YIRPSGGRTTYADSVKG GGLLLWDRELKSNYFDY
X67-F01 AYSMI YIRPSGGRTTYADSVKG GGLLLWQRELKSNYFDY X67-F10 AYSMI
YIRPSGGRTTYADSVKG GGLLLWTRELKSNYFDY X67-G04 AYSMI YIRPSGGRTTYADSVKG
GGLLLWARELKSNYFDY X67-H04 AYSMI YIRPSGGRTTYADSVKG GGLLLWERELKSNYFDY
X81-B01 HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY Abbreviations
used: "T/B" is the ELISA signal obtained using of the "target"
(biotinylated plasma kallikrein) divided by the ELISA signal of the
"background" (streptavidin); both of which were coated on
microtiter plates. "nd" is not determined. The symbol "q" refers to
the amber suppressible stop codon (TAG), which is translated as
glutamine (Q) in strains of E. coli such as the TG1 cells that were
used to express the Fab fragments.
TABLE-US-00005 Amino acid sequences of light chain (LC) and heavy
chain (HC) variable domain of pKal antibody inhibitors are shown
below (SEQ ID NOs: 224-293 numbered in order from top to bottom).
M6-D09 LC QDIQMTQSPS SLSASVGDRV TITCRASQSI RNYLNWYQQK PGKAPNLLIY
AASTLQSGVP 60 ARFSGSGSGT DFTLTISSLQ PEDFATYYCQ QLSGYPHTFG QGTKLEIK
108 M6-D09 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS FYYMVWVRQA
PGKGLEWVSV IYPSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARDK WAVMPPYYYY AMDVWGQGTT 120 VTVSSASTKG PSVFPLAPSS KS 142
M7-B04 LC QSALTQPASV SGSPGQSITI SCTGTNSDVG NYNLVSWYQQ HPGEAPKLLI
YEVNKRPSGV 60 SNRFSGSKSG NTASLTISGL QAEDEADYLC CSYAGNRNFY
VFGAGTKVTV L 111 M7-B04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS
WYSMVWVRQA PGKGLEWVSS ISPSGGLTNY 60 ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TAVYYCARHT AARPFYYYYM DVWGKGTTVT 120 VSSASTKGPS
VFPLAPSSKS 140 M7-E07 LC QSELTQPPSV SVSPGQTASI TCSGDKLGDK
YACWYQQKPG QSPVLVIYQD SKRPSGIPER 60 FSGSNSGNTA TLTISGTQAM
DEADYYCQAW DSSTGVFGGG TKLTVL 106 M7-E07 HC EVQLLESGGG LVQPGGSLRL
SCAASGFTFS WYLMIWVRQA PGKGLEWVSY IYPSGGFTYY 60 ADSVKGRFTI
SRDNSKNTLY LQMNSLRAED MAVYYCARTE GPLSWGYGMD VWGQGTTVTV 120
SSASTKGPSV FPLAPSSKS 139 M8-A09 LC QCELTQPPSE SVSPGQTANI TCSGDKLGNK
YAYWYQQKPG QSPVLVIYQD NNRPSGIPER 60 FSGSNSGNTA TLTISGTQAI
DEANYYCQAW DSRTVVFGGG TKLTVL 106 M8-A09 HC EVQLLESGGG LVQPGGSLRL
SCAASGFTFS TYFMLWVRQA PGKGLEWVSS IYPSGGNTVY 60 ADSVKGRFTI
SRDNSKNTLY LQMNSLRAED TAVYYCARAA SPVRNYYYYG MDVWGQGTTV 120
TVSSASTKGP SVFPLAPSSK S 141 M10-F10 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI SVYLNWYQHK PGKAPKLLIY GASNLQFGVP 60 SRFSGSGYGT
DFTLTISSLQ PEDFATYHCQ QTFSLFTFGG GTKVEIK 107 M10-F10 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS FYNMNWVRQA PGKGLEWVSS ISPSGGETNY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG GAYRNNWWGG FDIWGLGTMV
120 TVSSASTKGP SVFPLAPSSK S 141 M10-H05 LC QDIQMTQSPG TLSLSPGERA
TLSCRASQSV SSSYLAWYQQ KPGQAPRLLI YGASSRATGI 60 PDRFSGSGSG
TDFTLTISRL EPEDFAVYYC QQYGSSPFTF GPGTKVDIK 109 M10-H05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYNMYWVRQA PGKGLEWVSS IRPSGGGTVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGGF IAARWYYFDY
WGQGTLVTVS 120 SASTKGPSVF PLAPSSKS 138 M12-D05 LC QSVLTQPPSV
SVSPGQTATI TCSGDQLGDK YVGWYQQKPG QSPILVIYQD TKRPSGIPER 60
FSGSNSGNTA TLTISGTHTV DEAHYYCQAW DTSTAGFGGG TKLTVL 106 M12-D05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMVWVRQA PGKGLEWVSR IYPSGGWTKY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAREG LLWFGENAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M27-E05 LC QSELTQPPSV
SVSPGQTASI TCSGDKLGDK YACWYQQKPG QSPVLVIYQD SKRPSGIPER 60
FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSSTGVFGGG TKLTVL 106 M27-E05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYLMIWVRQA PGKGLEWVSY IYPSGGFTYY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARTE GPLSWGYGMD
VWGQGTTVTV 120 SSASTKGPSV FPLAPSSKS 139 M28-B11 LC QSVLTQPPSV
SVSPGQTATI TCSGDQLGDK YVGWYQQKPG QSPILVIYQD TKRPSGIPER 60
FSGSNSGNTA TLTISGTHTV DEAHYYCQAW DTSTAGFGGG TKLTVL 106 M28-B11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMVWVRQA PGKGLEWVSR IYPSGGWTKY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAREG LLWFGENAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M29-D09 LC QSALTQPPTV
SVSPGQTARI TCSGNKLGDK YVAWYQQKPG QSPMLVIYQD TKRPSRVSER 60
FSGSNSANTA TLSISGTQAL DEADYYCQAW DSSIVIFGGG TRLTVL 106 M29-D09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMVWVRQA PGKGLEWVSY IYPSGGATFY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAMGS YDYIWGFYSD
HWGQGTLVTV 120 SSASTKGPSV FPLAPSSKS 139 M29-E09 LC QYELTQPPSV
SVSPGQTATI TCSGDNLGNK YNSWYQQKPG QSPLLVIYQD TKRPSAIPER 60
FSGSNSGNTA TLTISGTQAM DEADYYCQAW DGNVVFGGGT KLTVL 105 M29-E09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYEMGWVRQA PGKGLEWVSS IYSSGGGTMY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARNP QYSGYDRSLS
DGAFDIWGQG 120 TMVTVSSAST KGPSVFPLAP SSKS 144 M35-G04 LC QDIQMTQSPA
TLSLSPGERA TLSCRASQSV SSYLAWYQQK PGQAPRLLIY DASNRATGIP 60
ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRSNWPRGFT FGPGTKVDIK 110 M35-G04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYHMSWVRQA PGKGLEWVSV
ISPSGGSTKY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
SSDYAWGSYR RPYYFDYWGQ 120 GTLVTVSSAS TKGPSVFPLA PSSKS 145 M38-F02
LC QSVLTQPPSV SVSPGQTASI TCSGEKLGDK YVSWYQQKPG QSPSLVICED
SRRPSGIPER 60 FSGSNSGNTA TLTISGAQPM DEADYYCQAW DSSTAIFGPG TKVTVL
106 M38-F02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYMMVWVRQA
PGKGLEWVSY IYSSGGHTVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARDL FLYDFWSKGA FDIWGQGTMV 120 TVSSASTKGP SVFPLAPSSK S 141
M41-A11 LC QSVLTQPPSV SVSPGQTASI TCSGDKLGDK YTSWYQQRPG QSPVLVIYQD
IKRPSGIPER 60 FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSPNARVFGS GTKVTVL
107 M41-A11 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYRMSWVRQA
PGKGLEWVSS IYPSGGRTVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKDK FEWRLLFRGI GNDAFDIWGQ 120 GTMVTVSSAS TKGPSVFPLA PSSKS
145 M73-D06 LC QSELTQPPSA SETPGQRVTI SCSGSSSNIG SNTVSWFQQL
PGSAPRLLIY NDHRRPSGVP 60 DRFSGSKSGT SASLVISGLQ SQDEADYYCS
AWDDSLNGVV FGGGTKLTVL 110 M73-D06 HC EVQLLESGGG LVQPGGSLRL
SCAASGFTFS RYEMYWVRQA PGKGLEWVSS ISSSGGPTAY 60 ADSVKGRFTI
SRDNSKNTLY LQMNSLRAED TAMYYCAKGT PKWELLLRSI YIENAFDIWG 120
QGTMVTVSSA STKGPSVFPL APSSKS 146 M76-D01 LC QDIVMTQTPP SLPVNPGEPA
SISCRSSQSL SDDGNTYLDW YLQRPGQSPQ LLIHTLSYRA 60 SGVPDRFSGS
GSGTDFTLKI SRVEAEDVGV YYCMQGTHWP PTFGQGTKVE IK 112 M76-D01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS FYAMHWVRQA PGKGLEWVSG IVPSGGRTHY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATDS SGSPNPLFDY
WGQGTLVTVS 120 SASTKGPSVF PLAPSPKS 138 M110-C12 LC QDIQMTQSPL
SLSVTPGEPA SISCRSSLSL LHSNGYNYLD WYVQRPGQSP QLLMYLSSTR 60
ASGVPDRFSG SGSGTDFTLE ISRVEAEDVG VYYCMQPLET PPTFGGGTKV EIK 113
M110-C12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYEMDWVRQA PGKGLEWVSG
ISSSGGHTAY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARER
RSSSRARYYY GMDVWGQGTT 120 VTVSSASTKG PSVFPLAPSS KS 142 M137-E12 LC
QSVLIQPPSV SGIPGQRVTI SCSGNNSNFG SNTVTWYQQL PGTAPKLLIY SDSRRPSGVP
60 DRFSGSRSDT SASLAISGLQ SEDEAEYHCA AWDDSLNGVF GGGTKLTVL 109
M137-E12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYRMQWVRQA PGKGLEWVSV
IVPSGGNTMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
PGSSIAARRA PTGYYGMDVW 120 GQGTTVTVSS ASTKGPSVFP LAPSSKS 147
M142-H08 LC QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY
AASRLQSGVP 60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR
108 M142-H08 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA
PGKGLEWVSY IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARGG LLLWFRELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143
M145-D01 LC QDIQMTQSPA TLSLSPGERA TLSCRASQSV SSYLAWYQQK PGQAPRLLIY
DASNRATGIP 60 ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRSNWPRGFT
FGPGTKVDIK 110 M145-D01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS
YYHMSWVRQA PGKGLEWVSV ISPSGGSTKY 60 ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TAVYYCARGG SSDYAWGSYR RPYYFDYWGQ 120 GTLVTVSSAS
TKGPSVFPLA PSSKS 145 M145-D11 LC QSVLTQPPSV SVSPGQTASI TCSGDKLGDK
YTSWYQQRPG QSPVLVIYQD IKRPSGIPER 60 FSGSNSGNTA TLTISGTQAM
DEADYYCQAW DSPNARVFGS GTKVTVL 107
M145-D11 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYRMSWVRQA PGKGLEWVSS
IYPSGGRTVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDK
FEWRLLFRGI GNDAFDIWGQ 120 GTMVTVSSAS TKGPSVFPLA PSSKS 145 M146-E12
LC QDIQMTQSPS SLSASVGDRV TITCRASGDI GNALGWYQQK PGKAPRLLIS
DASTLQSGVP 60 LRFSGSGSGT EFTLTISSLQ PEDFATYYCL QGYNYPRTFG QGTKLEIR
108 M146-E12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYIMHWVRQA
PGKGLEWVSS ISPSGGLTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAREF ENAYHYYYYG MDVWGQGTTV 120 TVSSASTKGP SVFPLAPSSK S 141
M152-A12 LC QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLSWYQQR PGKAPNLLIY
AASSLQSGVP 60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSISIPRTFG QGTKVEVK
108 M152-A12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYFMGWVRQA
PGKGLEWVSG IGPSGGSTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAREG PPYSSGWYRG LRQYHFDYWG 120 QGTLVTVSSA STKGPSVFPL APSSKS
146 M160-G12 LC QDIQMTQSPS FLSASVGDRV TITCRASQGI SSYLAWYQQK
PGKAPKLLIY AASTLQSGVP 60 SRFSGSGSGT EFTLTISSLQ PEDFATYYCQ
QLNSYPLTFG GGTKVEIK 108 M160-G12 HC EVQLLESGGG LVQPGGSLRL
SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY 60 ADSVKGRFTI
SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120
VTVSSASTKG PSVFPLAPSS KS 142 M161-C11 LC QSALTQPPSV SVSPGQTASI
TCSGDKLGDK YVSWYQQRPG QSPVLVIYQD TKRPSGIPER 60 FSGSNSGNTA
TLTISGTQAV DEADYYCQAW DSSTYVFGGG TKVTVL 106 M161-C11 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS DYAMKWVRQA PGKGLEWVSS ISSSGGVTQY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREE DYSSSWYSRR FDYYYGMDVW
120 GQGTTVTVSS ASTKGPSVFP LAPSSKS 147 M162-A04 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M162-A04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 X67-B03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWSRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-C03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWMRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-C09 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWGRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-D03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-D03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWNRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-E04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-E04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWDRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-F01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWQRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-F10 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWTRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-G04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWARELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-H04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-H04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWERELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 Note: X81-B01 is a
germilined IgG derived from X63-G06 which is shown in Table 7.
TABLE-US-00006 TABLE 2 CDR Amino Acid Sequences and ELISA Signal of
Antibody Binders of PKal (SEQ ID NOs: 294-743) Human HV-CDR1 pKal
LV-CDR1 LV-CDR2 LV-CDR3 (SEQ ID HV-CDR2 HV-CDR3 Initial ELISA (SEQ
ID NOs: (SEQ ID (SEQ ID NOs: NOs: (SEQ ID (SEQ ID Name (T/B)
294-368) NOs: 369-443) 444-518) 519-593) NOs: 594-668) NOs:
669-743) M6-A06 11.7 RASQSISMYLN GTSSLQS QQSYSAPWT LYQMT
GIWPSGGFTDYADSVKG VSTAVADNDY M6-A08 23.4 RASQRISFYLN GASSLQS
QQTFSTPNT PYPMQ SISSSGGMTEYADSVKG DDYGGKGGAFDI M6-D03 15.5
RASQSISSYLN AASSLQS QQSYSTLWT KYFMG VIGSSGGWTSYADSVKG VSTAVADNDY
M6-D08 16 RASQSISSYLN GASSLQS QQSYTRWT RYHMV SISPSGGWTNYADSVKG
EMATIAGQFDP M6-G05 18.5 RASQSISTYLN NAFSMER QQSYTTPTT RYRMV
SIYPSGGMTAYADSVKG DAVGIGDAFDI M8-C04 44.7 SGDKLGDKYTS QDSKRPS
QAWDSSTV YYPMQ YIYPSGGLTSYADSVKG LFYGSGSVGFEY M8-D05 11.9
RASQDISSWLV DASNLQS QQADGFPLT LYNMN SISPSGGFTDYADSVKG DLDLGILDY
M8-E06 8.8 RASQSISSYLN AASSLQS QQSYSTLMYT HYFMT SIVPSGGMTQYADSVKG
DSYSSSWFDI M8-G09 28.2 RASQGVSYYLA GASSLQS QQYNTYPPT LYEML
VIYPSGGYTDYADSVKG SFSGFGEIDY M8-H04 3.3 RASQYISTYLN GTSSLQS
QQSFTTPFT GYWMG SISSSGGWTQYADSVKG DDEIAAGGAFDI M9-A03 14.4
RASQNIDIYLN GAYNLQS QQSYGTPV GYFMM SIYSSGGYTDYADSVKG EVAGTYAFDI
M9-A08 5.5 RASQRISTYLN GASSLQS QQSYNTPRT AYEMW YIGSSGGSTSYADSVKG
GNSSSFDAFDI M9-C08 10.9 RASQSISIYVN AASSLQR QQSFSTPLT HYGMV
YIVPSGGLTYYADSVKG VDYTGDGLGY M9-C10 7.8 RASQGISSYLN GASSLQS
QESYSTLFT LYPMQ SIGSSGGMTFYADSVKG EVGAAGFAFDI M9-D08 35.9
RASRTISFYLN GGSSLHS QQSFSSPWT WYKMM SIYPSGGWTNYADSVKG GSPWGDDAFDI
M9-E04 18.8 RASQSISGYLN AASNLQT QQSHTPPKT EYDMM SIGSSGGMTYYADSVKG
DQVAAAAIDY M9-F08 10.9 RASQSISSYLN AASSLQS QQSYSTPPYT PYAMT
VIYPSGGFTDYADSVKG ASGSYLDAFDI M9-F09 7 RASQSISSYLN AASSLQS
QQTYTTPWT SYPMG RISSSGGMTIYADSVKG DDWNVGMDV M9-F10 8.4 RASQSINTYLN
AASTLES QQSYSTPYT DYDME SISPSGGSTIYADSVKG QGLLTAFDI M9-G08 4.8
RASQSISSYLN AASSLQS QQSYSTPIT YYTML SIYPSGGFTMYADSVKG VDTAMAMIDY
M9-H02 3.5 RASRSIATYLN GASTLQS QQSFSDPYT AYMMI VIYPSGGVTMYADSVKG
GTVGASDAFDI M9-H03 4.4 SGDKLGNRYTS QDNKRPS QALDSNTYV WYSMG
YIVPSGGYTMYADSVKG DPGVSYYYYGMDV M9-H04 16.1 RASQSISSYLN AASSLQS
QQSYSTPPT AYTMW SIWPSGGSTFYADSVKG TYDSSAGEVDY M10-A03 33.7
RASQRISFYLN GASSLQS QQTFSTPNT PYPMQ SISSSGGMTEYADSVKG DDYGGKGGAFDI
M10-A12 20.8 RASRDISVYLN GASSLQS QQSYSIPFT LYLMH SIYSSGGFTTYADSVKG
DTDYGMDV M10-B09 14.1 RASQSISTYLN GASSLQS QQSFSTPWT WYEMS
RIWPSGGVTMYADSVKG TSITTVGMDV M10-C11 5.3 RASQSISIYLN AASTLQS
QQSHSIPPT MYPMM YISPSGGMTDYADSVKG VAGSSDAFDI M10-D11 6.4
RSSQSLLHSNGYNYLD LGSNRAS MQALQTPLT AYPMN RISSSGGNTSYADSVKG GYLGY
M10-E06 32.8 RASQSISTYLN GASSLQS QQSYSDPYT LYRMF SIWSSGGPTMYADSVKG
EYPSTYYFDY M10-F09 4.8 RASQTIDDDLI AASSLQS QQSYNIPRT NYDMM
YISPSGGFTRYADSVKG DIYYYNWGPSHYFDS M10-G09 7.1 RASQSISGYIN AASSLQS
QQYVSYPFT QYGMQ SIRSSGGATRYADSVKG DGYYDSSGYPDY M11-A10 25
RASQSIDTYLN DASNL QHYLYAPYS NYWMM GIGSSGGFTSYADSVKG GSYSDYGVFES
M11-E01 11.7 RASQSISSYLN AASSLQS QQSYSTPPT TYEMY GIGSSGGMTMYADSVKG
EQPGIAALQF M11-E04 43.2 RASQSISIYLT GAATLQT QQTFSLPRT MYHMN
GIVSSGGVTFYADSVKG ITTVTTGGAFDI M11-E05 41.4 RTSQTINNYLN ATHTLES
QQSFAFPYT WYTMG WIYFGGLTTYADSVKG LGGPLDAFDI M11-E06 12.6
RASRGIGTYLN AASSLET QESFTNVYN QYAMH SIYPSGGFTLYADSVKG GGWLAGGELLN
M11-G09 23.6 RTSQGINHYLN AASELQT QQTYTSPYT LYNMT YIYPSGGGTHYADSVKG
DTGFWSADAFDI M11-G12 4.9 RASQTISVYVN GASSLQS QQSYSIPFT QYPMN
SISSSGGFTTYADSVKG EEQQGGFDY M12-A08 40.4 RASQSISRYLN AASTLET
QQSYSTPYT WYYMG WIVSSGGLTLYADSVKG TTVTTGDAFDI M12-B04 18
RASQGIRNDLG AASILQS LQDYEYPLT LYSMY RIRPSGGGTVYADSVKG DPLYSSGDV
M12-C09 7 RASQSIGIYLN GASSLQS QHSYSTPFT SYAMV SIGSSGGFTLYADSVKG
MNLGGGDAFDI M12-C10 8.3 SGDKLGEKYVS QDNKRPS QAWDSYTVV DYEMH
GISPSGGKTQYADSVKG DLKWGGRGSPDWYFDL M12-D10 9.9 RASQSISSYLN AASSLQS
QQSYSTPPT NYPMD SISSSGGWTNYADSVKG DTSGSYLGFDY M12-E06 48
RASQSISTYLN GAFSLQS QQSHSTPPT QYKML GIGPSGGLTAYADSVKG APWFGELGMDV
M27-A10 3.2 RASQSISAYLN YGVGSLQS QQGYTTPVT WYRMD SIWPSGGLTSYADSVKG
GWAPGGDAFDI M27-B01 33.1 RASQSISSYLN AASSLQS QQSYSTPYT DYTMW
SISSSGGITFYADSVKG SADTAMGGAFDI M27-B12 2.3 SGDKLGDEYAA QDRKRPS
QAWGKRNVV WYQMM SISPSGGITEYADSVKG DRSSGWYYYGMDV M27-E03 35.9
RASQSISSYLN AASSLQS QQSYSTPRT SYMMH GIYPSGGWTDYADSVKG LVAGLDAFDI
M27-F04 10.5 RASQSISSYLN AASSLQS QQSYSTPPT WYPMT SIGPSGGQTIYADSVKG
EYGDYGGGFDP M27-F11 10 RASQGISSYLA AASSLQS QQSYNTLRT SYHMM
SIYPSGGATMYADSVKG DGYHYGDYTYFQH M27-G01 31.4 RASQSISTYLN GASSLQS
QQSYSDPYT LYRMF SIWSSGGPTMYADSVKG EYPSTYYFDY M27-G04 4.1
RASQRISYYLT AASSLES QQAFSTPFT AYYMV YISPSGGQTQYADSVKG EAISSSSFDY
M27-G09 2.2 RTRQSISNYLN AASSLQS QQSYDIPFT EYDMA YIVSSGGFTSYADSVKG
WAGWIAAADY M27-H10 12.4 RASQSISNYLN AASSLQS QQSYSTPQT AYQMA
VIYSSGGYTDYADSVKG HNWNDGAFDI M28-A01 19 RASQSISSYLN AASSLQS
QQSYSTLT WYAMH GIYSSGGYTKYADSVKG DLSNGDDVFDI M28-C03 2.2
RASQSINFYLN VASSLES LQSYSAPYT YYQMG SIYPSGGMTDYADSVKG GSPWGDDAFDI
M28-D02 3.7 RTSRRIGTYLN GASSLQS QQSFSSPWT WYPMQ YIYPSGGGTDYADSVKG
SSGWLGDAFDI M28-D12 41.6 RASQSIATYLN AASSLQS QQSYSTRET WYTMH
VIYPSGGPTSYADSVKG DGSGSYLGFDY M28-E01 41 RASQSISSYLN AASSLQS
QQTYTTPWT SYPMG RISSSGGMTIYADSVKG DDWNVGMDV M28-E11 29.3
RASQDISNWLA AASSLQT QQSYSLPWT LYDMT GISSSGGVTIYADSVKG
TYYYDSSGYADAFDI M28-F01 1.5 RASQSINTYLN AASTLES QQSYSTPPT VYLMH
GISPSGGYTQYADSVKG PGGLDAFDI M28-F05 31.4 RASQSISSYLN AASSLQS
QQSYSTPLT RYIMW GIYSSGGYTQYADSVKG ELEGLGGFDY M28-F07 33 RASQGISSWLA
ATSGLQS QQAKSFPLT DYTMY SIVPSGGHTLYADSVKG DHLSSWYGGFFDY M29-C07 5.2
RASQSISSYLN AASSLQS QQSYSTRYT GYDMM VISSSGGNTAYADSVKG ESSGLYYFDY
M29-D10 23.6 RASQSITIYLN GASNLHS QQSYDTPLT WYPMY SIGSSGGPTPYADSVKG
WADYGGSLDY M29-E02 2 SGSSSNIGNNAVS YDDLLPS AAWDDSLNGFV RYPMM
VIYPSGGDTFYADSVKG GDDYLWEAAVY M29-G08 40.4 RASQNIGNDVA HASTRAY
QQFYDWPAHT YYHMW GISPSGGFTFYADSVKG DYYYDSSGYSPLGY M29-G10 16.4
RASQSISIYLN GASQLES QQSYNVPYT FYKMI SISSSGGSTQYADSVKG DRVDLGYLDY
M74-A07 8.6 RTSQNINTYLN GVSSLHR QQSYSSPWT QYLMM SIYPSGGYTSYADSVKG
VSTAVADNDY M76-F02 6.4 RASQTIDNYLH DASSLQS QQSYDTPQYT LYDMN
GISPSGGQTMYADSVKG QPMISAFDI M76-G02 10.3 RASQSISSYLN AASSLQS
QQSYSTPPWT LYAMW YISSSGGFTSYADSVKG YRVGVAATDY M76-G06 11.8
RASQSISTYLN AASSLQS QQSYSTPHT GYIMH WIYPSGGWTEYADSVKG DAPGVGAIDY
M76-H02 13.4 RASQDISVYLN GGASLQS QQSYSLPFT MYWMQ YIYPSGGPTKYADSVKG
PSGSYGDAFDI M77-C07 16.1 RASQNISSYLN AASSLQS QQSYSTPRT LYIMG
GIYPSGGFTMYADSVKG ESSGVAAPDY M77-H04 7.6 RSSQSLLHSRGYNYLD LGSNRAS
MQALQRRT YYTMI GIRSSGGGTRYADSVKG DGSRYSYGSIYYYYGMDA Abbreviations
used: "T/B" is the ELISA signal obtained using of the "target"
(biotinylated plasma kallikrein) divided by the ELISA signal of the
"background" (streptavidin); both of which were coated on
microtiter plates. "nd" is not determined. The symbol "q" refers to
the amber suppressible stop codon (TAG), which is translated as
glutamine (Q) in strains of E. coli such as the TG1 cells that were
used to express the Fab fragments.
TABLE-US-00007 Amino acid sequences of light chain (LC) and heavy
chain (HC) variable domain of pKal antibody binders are shown
below. (SEQ ID NOs: 744-893 numbered in order from top to bottom)
M6-A06 LC QDIQMTQSPS SLSASVGDSV TISCRASQSI SMYLNWYQHK PGKAPKLLIY
GTSSLQSGVP 60 SRFSGSGPGG TDFTLTISSL QPEDFATYYC QQSYSAPWTF GQGTKVEIK
109 M6-A06 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYQMTWVRQA
PGKGLEWVSG IWPSGGFTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARVS TAVADNDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M6-A08
LC QDIQMTQSPS SLSASVGDRV TITCRASQRI SFYLNWFQQK PGKAPNLLIY
GASSLQSGVP 60 SRFSGSGSGT DFTLTISSLQ PKDFGTYYCQ QTFSTPNTFG QGTKLEIK
108 M6-A08 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMQWVRQA
PGKGLEWVSS ISSSGGMTEY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARDD YGGKGGAFDI WGQGTMVTVS 120 SASTKGPSVF PLAPSSKS 138 M6-D03
LC QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY
AASSLQSGVP 60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLWTFG QGTKVEIK
108 M6-D03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS KYFMGWVRQA
PGKGLEWVSV IGSSGGWTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARVS TAVADNDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M6-D08
LC QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYqQK PGKAPKLLIY
GASSLQSGVP 60 SRFSGSGSGT DFTLTISSLQ PEDSATYYCQ QSYTRWTFGQ GTKVEIK
107 M6-D08 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYHMVWVRQA
PGKGLEWVSS ISPSGGWTNY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAMYYCAREM ATIAGQFDPW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKS 137 M6-G05
LC QDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQLK PGKAPKLLIY
NAFSMERGVP 60 STISGSGSGT DFTLTISSLQ PEDFATYYCQ QSYTTPTTFG QGTKVEIK
108 M6-G05 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMVWVRQA
PGKGLEWVSS IYPSGGMTAY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCARDA VGIGDAFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M8-C04
LC QSALTQPPSV SVSPGQTASI TCSGDKLGDK YTSWHQQKPG QSPVLVIYQD
SKRPSGIPER 60 FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSSTVFGGGT RLTVL 105
M8-C04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYPMQWVRQA PGKGLEWVSY
IYPSGGLTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLF
YGSGSVGFEY WGQGTLVTVS 120 SASTKGPSVF PLAPSSKS 138 M8-D05 LC
QDIQMTQSPS FVSASVGDRV TITCRASQDI SSWLVWYQQK PGKGPKLLIY DASNLQSGVP
60 SRFSGGGSGT HFTLTISSLQ PEDFATYYCQ QADGFPLTFG GGTKVEMK 108 M8-D05
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYNMNWVRQA PGKGLEWVSS
ISPSGGFTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL
DLGILDYWGQ GTLVTVSSAS 120 TKGPSVFPLA PSSKS 135 M8-E06 LC QDIQMTQSPS
SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLMYTF GQGTKLEIK 109 M8-E06 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYFMTWVRQA PGKGLEWVSS IVPSGGMTQY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDS YSSSWFDIWG
QGTMVTVSSA 120 STKGPSVFPL APSSKS 136 M8-G09 LC QDIQMTQSPS
SLSASVGDTV TITCRASQGV SYYLAWFQQK PGKAPKSLIY GASSLQSGVP 60
SKFSGSGSGT VFTLTISSLQ PDDFATYYCQ QYNTYPPTFG QGTRLDIK 108 M8-G09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYEMLWVRQA PGKGLEWVSV IYPSGGYTDY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARSF SGFGEIDYWG
QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M8-H04 LC QDIQMTQSPS
SLSASIGDRV TITCRASQYI STYLNWYEQK PGKAPKLLIY GTSSLQSGVP 60
SRFSGSGSGT EFSLTISSLQ PEDFATYYCQ QSFTTPFTFG QGTKLEIK 108 M8-H04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYWMGWVRQA PGKGLEWVSS ISSSGGWTQY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARDD EIAAGGAFDI
WGQGAMVTVS 120 SASTKGPSVF PLAPSSKS 138 M9-A03 LC QDIQMTQSPS
SLSASLGDRV TITCRASQNI DIYLNWYQQT PGKAPKLLIY GAYNLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFGTYYCQ QSYGTPVFGQ GTKLEIK 107 M9-A03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYFMMWVRQA PGKGLEWVSS IYSSGGYTDY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREV AGTYAFDIWG
QGTMVTVSSA 120 STKGPSVFPL APSSKS 136 M9-A08 LC QDIQMTQSPS
SLSASVGDRV TVTCRASQRI STYLNWYQQK PGKAPKLLIS GASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PDDFATYYCQ QSYNTPRTFG QGTKVEIR 108 M9-A08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYEMWWVRQA PGKGLEWVSY IGSSGGSTSY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTGGN SSSFDAFDIW
GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M9-C08 LC QDIQMTQSPS
SLSASVGDRV TITCRASQSI SIYVNWYQQK PGKAPNLLIF AASSLQRGVP 60
SRFSGSGSGA DFTLTISSLQ PEDFATYYCQ QSFSTPLTFG GGTKVEIK 108 M9-C08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYGMVWVRQA PGKGLEWVSY IVPSGGLTYY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVD YTGDGLGYWG
QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M9-C10 LC QDIQMTQSPS
SLSASVGDRV TITCRASQGI SSYLNWYQQK PGNAPNLLIY GASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ ESYSTLFTFG PGTTVEIK 108 M9-C10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYPMQWVRQA PGKGLEWVSS IGSSGGMTFY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTREV GAAGFAFDIW
GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M9-D08 LC QDIQMTQSPS
SLSASVGDRV TLTCRASRTI SFYLNWYQQK AGKAPELLIY GGSSLHSGVP 60
SRFSGSGSGT DFSLTISNLQ PEDIAVYYCQ QSFSSPWTFG QGTKVEIK 108 M9-D08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYKMMWVRQA PGKGLEWVSS IYPSGGWTNY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGS PWGDDAFDIW
GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M9-E04 LC QDIQMIQSPS
SLSASVGDRV TITCRASQSI SGYLNWYQQR SGKAPKLLIF AASNLQTGVP 60
SRFSGSGSGT DFTLTINNLQ PEDFATYYCQ QSHTPPKTFG PGTKVDIK 108 M9-E04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS EYDMMWVRQA PGKGLEWVSS IGSSGGMTYY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDQ VAAAAIDYWG
QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M9-F08 LC QDIQMTqSPS
SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPYTF GQGTKLEIK 109 M9-F08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYAMTWVRQA PGKGLEWVSV IYPSGGFTDY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARAS GSYLDAFDIW
GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M9-F09 LC QDIQMTQSPS
SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60
SKFSGSGSGT DYTLTISSLQ PEDFATYYCQ QTYTTPWTFG QGTKVEIK 108 M9-F09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMGWVRQA PGKGLEWVSR ISSSGGMTIY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD WNVGMDVWGQ
GTTVTVSSAS 120 TKGPSVFPLA PSSKS 135 M9-F10 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI NTYLNWYQQK PGKAPKVLIH AASTLESGVP 60 SRFSGSGSGT
DFTLTISSLQ PEDFATYYCQ QSYSTPYTFG QGTKLEVR 108 M9-F10 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS DYDMEWVRQA PGKGLEWVSS ISPSGGSTIY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARQG LLTAFDIWGQ GTMVTVSSAS
120 TKGPSVFPLA PSSKS 135 M9-G08 LC QDIQMTQSPS SLSASVGDRV TITCRASQSI
SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60 SRFSGSGSGT DFTLTISSLQ
PEDFATYYCQ QSYSTPITFG GGTKVEIK 108 M9-G08 HC EVQLLESGGG LVQPGGSLRL
SCAASGFTFS YYTMLWVRQA PGKGLEWVSS IYPSGGFTMY 60 ADSVKGRFTI
SRDNSKNTLY LQMNSLRAED TAVYYCARVD TAMAMIDYWG QGTLVTVSSA 120
STKGPSVFPL APSSKS 136 M9-H02 LC QDIQMTQSPS SLSASVGDRV IITCRASRSI
ATYLNWYQQK PGKAPNLLIF GASTLQSGVP 60 SRFSGSGSGT DFTLTISDLQ
PEDFATYYCQ QSFSDPYTFG QGTNLEMK 108
M9-H02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYMMIWVRQA PGKGLEWVSV
IYPSGGVTMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGT
VGASDAFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M9-H03 LC
QYELTQAPSV SVAPGQTASI TCSGDKLGNR YTSWYQQKPG QSPVLVIFQD NKRPSGIPER
60 FSGSNSGNTA TLTISGTQAM DEADYYCQAL DSNTYVFGTG TKVTVL 106 M9-H03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYSMGWVRQA PGKGLEWVSY IVPSGGYTMY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP GVSYYYYGMD
VWGQGTTVTV 120 SSASTKGPSV FPLAPSSKS 139 M9-H04 LC QDIQMTQSPS
SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTRLEIK 108 M9-H04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYTMWWVRQA PGKGLEWVSS IWPSGGSTFY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARTY DSSAGEVDYW
GQGTLVTVSS 120 ASTKGPSVFP LAPSSKS 137 M10-A03 LC QDIQMTQSPS
SLSASVGDRV TITCRASQRI SFYLNWFQQK PGKAPNLLIY GASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PKDFGTYYCQ QTFSTPNTFG QGTKLEIK 108 M10-A03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMQWVRQA PGKGLEWVSS ISSSGGMTEY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD YGGKGGAFDI
WGQGTMVTVS 120 SASTKGPSVF PLAPSSKS 138 M10-A12 LC QDIQMTQSPL
SLSAFVGDRV TITCRASRDI SVYLNWYQLK SGKAPKLLIY GASSLQSGVP 60
SRFSGSGSGT DFTLTITSLQ PEDFATYYCQ QSYSIPFTFG GGTKVETK 108 M10-A12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYLMHWVRQA PGKGLEWVSS IYSSGGFTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDT DYGMDVWGQG
TTVTVSSAST 120 KGPSVFPLAP SSKS 134 M10-B09 LC QDIQMTQSPS SLSASVGDGV
TITCRASQSI STYLNWYQQR PGKAPKLLIY GASSLQSGVP 60 SRFSGSGSGT
DFTLTISSLQ REDFATYYCQ QSFSTPWTFG QGTRVEIK 108 M10-B09 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS WYEMSWVRQA PGKGLEWVSR IWPSGGVTMY 60
ADSVKGRFTI SRDNSKNTLY LqMNSLRAED TAVYYCTRTS ITTVGMDVWG QGTTVTVSSA
120 STKGPSVFPL APSSKS 136 M10-C11 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI SIYLNWYQQK PEKAPKLLIF AASTLQSGVP 60 SRFSGSGSGT
DFTLTISNLQ PEDFATYYCQ QSHSIPPTFG LGTKVEVK 108 M10-C11 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS MYPMMWVRQA PGKGLEWVSY ISPSGGMTDY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARVA GSSDAFDIWG QGTMVTVSSA
120 STKGPSVFPL APSSKS 136 M10-D11 LC QDIQMTQSPL SLPVTPGEPA
SISCRSSQSL LHSNGYNYLD WYLQKPGQSP QLLIYLGSNR 60 ASGVPDRFSG
SGSGTDFTLK ISRVEAEDVG VYYCMQALQT PLTFGPGTKV HIK 113 M10-D11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYPMNWVRQA PGKGLEWVSR ISSSGGNTSY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCALGY LGYWGQGTLV
TVSSASTKGP 120 SVFPLAPSSK S 131 M10-E06 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI STYLNWYQQK PGKAPKLLIY GASSLQSGVP 60 SRFSGSGSGT
DFTLTISSLQ PEDFTIYYCQ QSYSDPYTFG QGTKLDIK 108 M10-E06 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS LYRMFWVRQA PGKGLEWVSS IWSSGGPTMY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREY PSTYYFDYWG QGTLVTVSSA
120 STKGPSVFPL APSSKS 136 M10-F09 LC QDIQMTQSPS SLSASVGDRV
TITCRASQTI DDDLIWYQQK PGRAPKLLIY AASSLQSGVP 60 SRFSGSGSGT
DFTLTITSLQ PEDFATYYCQ QSYNIPRTFG QGTKLESK 108 M10-F09 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS NYDMMWVRQA PGKGLEWVSY ISPSGGFTRY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAKDI YYYNWGPSHY FDSWGQGTLV
120 TVSSASTKGP SVFPLAPSSK S 141 M10-G09 LC QDIQMTQSPS SLSASVGDSV
TITCRASQSI SGYINWYQQK AGKAPKLLIY AASSLQSGVP 60 SRFSGSGSGT
HFTLTISSLQ PEDFATYYCQ QYVSYPFTFG PGTKVDIK 108 M10-G09 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS QYGMQWVRQA PGKGLEWVSS IRSSGGATRY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG YYDSSGYPDY WGQGTLVTVS
120 SASTKGPSVF PLAPSSKS 138 M11-A10 LC QDIQMTQSPS SLSASVGDRV
AITCRASQSI DTYLNWYQQK PGKAPKLLIY DASNLEIGVP 60 SRFSGSGSGT
DFTFIINSLQ PEDVATYYCQ HYLYAPYSFG QGTKLEIK 108 M11-A10 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS NYWMMWVRQA PGKGLEWVSG IGSSGGFTSY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKGS YSDYGVFESW GQGTLVTVSS
120 ASTKGPSVFP LAPSSKS 137 M11-E01 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60 SRFSGSGSGT
DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTKVEIK 108 M11-E01 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS TYEMYWVRQA PGKGLEWVSG IGSSGGMTMY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCAREQ PGIAALQFWG QGTLVTVSSA
120 STKGPSVFPL APSSKS 136 M11-E04 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI SIYLTWYQHR PGKAPNLLIY GAATLQTGVP 60 SRFSGSGSGT
DFTLTIRGLQ PEDFATYYCQ QTFSLPRTFG QGTKLEIK 108 M11-E04 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS MYHMNWVRQA PGKGLEWVSG IVSSGGVTFY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIT TVTTGGAFDI WGQGTMVTVS
120 SASTKGPSVF PLAPSSKS 138 M11-E05 LC QDIQMTQSPS SLSASVGDTV
TITCRTSQTI NNYLNWYQQR PGEAPKVLIY ATHTLESGVP 60 SRFSGSGSGT
DFTLTIGSLQ PEDFATYYCQ QSFAFPYTFG QGTKVEIT 108 M11-E05 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS WYTMGWVRQA PGKGLEWVSW IYFGGLTTYA 60
DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARLGG PLDAFDIWGQ GTMVTVSSAS
120 TKGPSVFPLA PSSKS 135 M11-E06 LC QDIQMTQSPS SLSASIGDRV
TISCRASRGI GTYLNWYQQH AGKAPKLLIR AASSLETGVP 60 PRFSGSGSGT
DFTLTISSLQ SDDFATYYCQ ESFTNVYNFG QGTKLEIK 108 M11-E06 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS QYAMHWVRQA PGKGLEWVSS IYPSGGFTLY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARGG WLAGGELLNW GQGTLVTVSS
120 ASTKGPSVFP LAPSSKS 137 M11-G09 LC QDIQMTQSPS SLSASVGDRV
TITCRTSQGI NHYLNWYQQK PGKAPKILVF AASELQTGVP 60 SRFSGTGSGT
SYTLTITSLQ PEDVATYYCQ QTYTSPYTFG QGTKLEVK 108 M11-G09 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS LYNMTWVRQA PGKGLEWVSY IYPSGGGTHY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDT GFWSADAFDI WGQGTMVTVS
120 SASTKGPSVF PLAPSSKS 138 M11-G12 LC QDIQMTQSPS SLSAFVGDRV
SITCRASQTI SVYVNWYQHK SGQAPKLLIY GASSLQSGVP 60 SRFSGSGSGT
DFTLTISSLQ PEDFATYFCQ QSYSIPFTFG GGTDVQIR 108 M11-G12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS QYPMNWVRQA PGKGLEWVSS ISSSGGFTTY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREE QQGGFDYWGQ GTLVTVSSAS
120 TKGPSVFPLA PSSKS 135 M12-A08 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI SRYLNWYQQK PGKAPKLLIY AASTLETGVP 60 SRFSGSGSGT
DFTLTITTLQ PEDFVIYYCQ QSYSTPYTFG QGTKLEIK 108 M12-A08 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS WYYMGWVRQA PGKGLEWVSW IVSSGGLTLY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARTT VTTGDAFDIW GQGTMVTVSS
120 ASTKGPSVFP LAPSSKS 137 M12-B04 LC QDIQMTQSPS SLSASVGDRV
TITCRASQGI RNDLGWYQHK PGKAPKLLIY AASILQSGVP 60 SRFSGTASGT
DFTLTISSLQ PEDFATYFCL QDYEYPLTFG GGTKLDIK 108 M12-B04 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS LYSMYWVRQA PGKGLEWVSR IRPSGGGTVY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP LYSSGDVWGQ GTTVTVSSAS
120 TKGPSVFPLA PSSKS 135 M12-C09 LC QDIQMTQSPS SLSASVGDRV
TITCRASQSI GIYLNWYHQK PGKAPNLLIY GASSLQSGVP 60 SRFSGSGSGT
DFTLTISSLQ PGDFATYYCQ HSYSTPFTFG GGTKVEIK 108 M12-C09 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS SYAMVWVRQA PGKGLEWVSS IGSSGGFTLY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASMN LGGGDAFDIW GQGTMVTVSS
120 ASTKGPSVFP LAPSSKS 137 M12-C10 LC QSALTQPPSV SVSPGQTASI
TCSGDKLGEK YVSWYQQKPG QSPVVVIYQD NKRPSGIPER 60
FSGSNSGNTA TLTISGTQAV DEADYYCQAW DSYTVVFGGG SKLTVLGQPK 110 M12-C10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYEMHWVRQA PGKGLEWVSG
ISPSGGKTQY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL
KWGGRGSPDW YFDLWGRGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M12-D10 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG GGTKVEIK 108 M12-D10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYPMDWVRQA PGKGLEWVSS
ISSSGGWTNY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATDT
SGSYLGFDYW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKS 137 M12-E06 LC
QDIQMTQSPS SLSASVGDRV SITCRASQSI STYLNWYQHK PGKAPTLLIY GAFSLQSGVP
60 SRFSGSGSGT DFALTISSLQ PEDFATYYCQ QSHSTPPTFG QGTRVEIK 108 M12-E06
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYKMLWVRQA PGKGLEWVSG
IGPSGGLTAY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARAP
WFGELGMDVW GQGTTVTVSS 120 ASTKGPSVFP LAPSSKS 137 M27-A10 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SAYLNWYQQK PGKAPQLLMY GVGSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYFCQ QGYTTPVTFG GGTKVEIK 108 M27-A10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYRMDWVRQA PGKGLEWVSS
IWPSGGLTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGW
APGGDAFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M27-B01 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPYTFG QGTKLEIK 108 M27-B01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYTMWWVRQA PGKGLEWVSS
ISSSGGITFY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARSA
DTAMGGAFDI WGQGTMVTVS 120 SASTKGPSVF PLAPSSKS 138 M27-B12 LC
QYELTQPPAV SVSPGQTATI TCSGDKLGDE YAAWYQQKPG QSPVLVIYQD RKRPSGIPER
60 FSGSNFGNTA TLTITGTQVM DEADYYCQAW GKRNVVFGGG TKLTVL 106 M27-B12
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYQMMWVRQA PGKGLEWVSS
ISPSGGITEY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR
SSGWYYYGMD VWGQGTTVTV 120 SSASTKGPSV FPLAPSSKS 139 M27-E03 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPRTFG QGTKVEIK 108 M27-E03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYMMHWVRQA PGKGLEWVSG
IYPSGGWTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARLV
AGLDAFDIWG QGTMVTVSSA 120 STKGPSVFPL APSSKS 136 M27-F04 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTKVEIK 108 M27-F04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMTWVRQA PGKGLEWVSS
IGPSGGQTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTTEY
GDYGGGFDPW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKS 137 M27-F11 LC
QDIQMTQSPS FLSASVGDRV TITCRASQGI SSYLAWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYNTLRTFG PGTKVDLK 108 M27-F11
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYHMMWVRQA PGKGLEWVSS
IYPSGGATMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARDG
YHYGDYTYFQ HWGQGTLVTV 120 SSASTKGPSV FPLAPSSKS 139 M27-G01 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQQK PGKAPKLLIY GASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFTIYYCQ QSYSDPYTFG QGTKLDIK 108 M27-G01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYRMFWVRQA PGKGLEWVSS
IWSSGGPTMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREY
PSTYYFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M27-G04 LC
QDIQMTQSPS SLSASVGDRV TITCRASQRI SYYLTWYQQK PGKVPKLLIY AASSLESGVP
60 SRFSGSGSGT DFTLTISNLQ PEDFATYYCQ QAFSTPFTFG GGTKVEIK 108 M27-G04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYYMVWVRQA PGKGLEWVSY
ISPSGGQTQY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREA
ISSSSFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M27-G09 LC
QDIQMTQSPS SVSASVGDRI TITCRTRQSI SNYLNWYQQK PGEPPKLLIF AASSLQSGVP
60 SRFSGSGTGT EFTLTISSLQ PEDLAIYYCQ QSYDIPFTFG QGTKLEIK 108 M27-G09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS EYDMAWVRQA PGKGLEWVSY
IVSSGGFTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTTWA
GWIAAADYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M27-H10 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SNYLNWYQQK PGKAPKFLIY AASSLQSGVP
60 SRFSGSGSGT DFTLSISSLQ PEDFATYYCQ QSYSTPQTFG QGTKVEMK 108 M27-H10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYQMAWVRQA PGKGLEWVSV
IYSSGGYTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARHN
WNDGAFDIWG QGTMVTVSSA 120 STKGPSVFPL APSSKS 136 M28-A01 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLTFGG GTKVEIK 107 M28-A01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYAMHWVRQA PGKGLEWVSG
IYSSGGYTKY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL
SNGDDVFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M28-C03 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI NFYLNWYQQK PGKAPKLLIY VASSLESGVP
60 SRFSGSASGT EFTLTISSLQ PEDFATYYCL QSYSAPYTFG QGTKVEIT 108 M28-C03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYQMGWVRQA PGKGLEWVSS
IYPSGGMTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGS
PWGDDAFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M28-D02 LC
QDIqMTQSPS SLSASEGDMV TITCRTSRRI GTYLNWYQQK PGKAPKLLIY GASSLQSGVP
60 SRFSGSGSGT DFTLTVSSLQ PEDVGTYYCQ QSFSSPWTFG PGTKVEIK 108 M28-D02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMQWVRQA PGKGLEWVSY
IYPSGGGTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCATSS
GWLGDAFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M28-D12 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI ATYLNWYQQK PGRAPKLLIY AASSLQSGVP
60 SRFVGGGSGS GTHFTLTISS LQPEDFATYY CQQSYSTRET FGQGTKVEIK 110
M28-D12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMHWVRQA PGKGLEWVSV
IYPSGGPTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARDG
SGSYLGFDYW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKS 137 M28-E01 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SKFSGSGSGT DYTLTISSLQ PEDFATYYCQ QTYTTPWTFG QGTKVEIK 108 M28-E01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMGWVRQA PGKGLEWVSR
ISSSGGMTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD
WNVGMDVWGQ GTTVTVSSAS 120 TKGPSVFPLA PSSKS 135 M28-E11 LC
QDIQMTQSPS SVSASVGDRV TINCRASQDI SNWLAWYQQK PGKAPNLLIY AASSLQTGAP
60 SRFSGSGSGT DFTLTISSLQ PEDFGTYVCQ QSYSLPWTFG LGTKVEVR 108 M28-E11
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYDMTWVRQA PGKGLEWVSG
ISSSGGVTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARTY
YYDSSGYADA FDIWGQGTMV 120 TVSSASTKGP SVFPLAPSSK S 141 M28-F01 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI NTYLNWYQQK PGKAPKVLIH AASTLESGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTKVEIK 108 M28-F01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYLMHWVRQA PGKGLEWVSG
ISPSGGYTQY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPG
GLDAFDIWGQ GTMVTVSSAS 120 TKGPSVFPLA PSSKS 135 M28-F05 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPLTFG GGTKVEIK 108 M28-F05
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYIMWWVRQA PGKGLEWVSG
IYSSGGYTQY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCAREL
EGLGGFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M28-F07 LC
QDIQMTQSPS SVSASVGDRV TITCRASQGI SSWLAWYQQK PGKAPKLLIY ATSGLQSGVP
60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QAKSFPLTFG GGTRVEIK 108 M28-F07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYTMYWVRQA PGKGLEWVSS IVPSGGHTLY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDH LSSWYGGFFD
YWGQGTLVTV 120 SSASTKGPSV FPLAPSSKS 139 M29-C07 LC QDIQMTQSPS
SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTRYTFG QGTKLEIK 108 M29-C07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYDMMWVRQA PGKGLEWVSV ISSSGGNTAY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARES SGLYYFDYWG
QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M29-D10 LC QDIQMTQSPS
SLSASVGDTV SITCRASQSI TIYLNWYQHK PGKAPNLLIY GASNLHSGVP 60
SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYDTPLTFG GGTKVEIK 108 M29-D10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMYWVRQA PGKGLEWVSS IGSSGGPTPY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARWA DYGGSLDYWG
QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M29-E02 LC QSVLTQPPSV
SEAPRQRVTI SCSGSSSNIG NNAVSWYQQL PGKAPKLLIY YDDLLPSGVS 60
DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSLNGFV FGTGTKVTVL 110 M29-E02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYPMMWVRQA PGKGLEWVSV
IYPSGGDTFY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASGD
DYLWEAAVYW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKS 137 M29-G08 LC
QDIQMTQSPA TLSASPGETV TLSCRASQNI GNDVAWYRQR PGQAPRLLIH HASTRAYGIP
60 ARLRGSGSAT EFTLTITSLE PEDFAIYYCQ QFYDWPAHTF ALGTRLEIKR 110
M29-G08 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYHMWWVRQA PGKGLEWVSG
ISPSGGFTFY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY
YYDSSGYSPL GYWGQGTLVT 120 VSSASTKGPS VFPLAPSSKS 140 M29-G10 LC
QDIQMTQSPS SLSSSVGDSA TITCRASQSI SIYLNWYQQK PGKAPKILIY GASQLESGVP
60 SRFSGSGSGT DFTLTVSGLQ PEDFATYWCQ QSYNVPYTFG QGTKLEIK 108 M29-G10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS FYKMIWVRQA PGKGLEWVSS
ISSSGGSTQY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR
VDLGYLDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M74-A07 LC
QDIQMTQSPS SLSASVRDRV TITCRTSQNI NTYLNWYYQA PGRAPKLLIF GVSSLHRGVS
60 SRFSGSGDGT EFTLTISSLQ PEDIGTYFCQ QSYSSPWTFG QGTKVEIK 108 M74-A07
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYLMMWVRQA PGKGLEWVSS
IYPSGGYTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVS
TAVADNDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M76-F02 LC
QDIQMTQSPS SLSASVGDRV TITCRASQTI DNYLHWYQQK PGKAPKVLIH DASSLQSGVP
60 PRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYDTPQYTF GQGTKLEIK 109
M76-F02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYDMNWVRQA PGKGLEWVSG
ISPSGGQTMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARQP
MISAFDIWGQ GTMVTVSSAS 120 TKGPSVFPLA PSSKS 135 M76-G02 LC
QDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPWTF GQGTKVEIK 109
M76-G02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYAMWWVRQA PGKGLEWVSY
ISSSGGFTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARYR
VGVAATDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M76-G06 LC
QDIQMTQSPS SLSASVRDRV TITCRASQSI STYLNWYQQK PGEAPKLLVF AASSLQSGVP
60 SRFSGSGSGT DFTLSISSLQ PEDFATYYCQ QSYSTPHTFG QGAKVEIK 108 M76-G06
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYIMHWVRQA PGKGLEWVSW
IYPSGGWTEY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDA
PGVGAIDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M76-H02 LC
QDIQMTQSPS SLSASEGDRV TITCRASQDI SVYLNWYQMK SGKAPKLLIY GGASLQSGVP
60 ARFSGSGYGT DFTLTITDLR PEDFATYYCQ QSYSLPFTFG GGTKVEIK 108 M76-H02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS MYWMQWVRQA PGKGLEWVSY
IYPSGGPTKY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPS
GSYGDAFDIW GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M77-C07 LC
QDIQMTQSPS TLSASVGDRV TITCRASQNI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP
60 SRFSGSGSGT DFTLTISSLQ PEDFATYSCQ QSYSTPRTFG QGTKVEIK 108 M77-C07
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYIMGWVRQA PGKGLEWVSG
IYPSGGFTMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARES
SGVAAPDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKS 136 M77-H04 LC
QDIQMTQSPL SLPVTPGEPA SISCRSSQSL LHSRGYNYLD WYLQKPGQSP QLLIYLGSNR
60 ASGVPDRFSG SGSGTDFTLK ISRVEAEDVG VYYCMQALQR RTFGQGTKLE IK 112
M77-H04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYTMIWVRQA PGKGLEWVSG
IRSSGGGTRY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDG
SRYSYGSIYY YYGMDAWGQG 120 TTVTVSSAST KGPSVFPLAP SSKS 144
Example 2: Lead Antibody Inhibitors
[0871] Antibodies were selected as lead plasma kallikrein
inhibitors on the basis of apparent inhibition constant
(K.sub.i,app), specificity with respect to lack of inhibition of
other serine proteases, inhibition of bradykinin generation, and
lack of binding to plasma prekallikrein (Table 3). Plasma
kallikrein circulates in the plasma as an inactive zymogen
(prekallikrein) at a concentration of approximately 500 nM.
Antibodies that bound prekallikrein may be rendered inaccessible
towards active plasma kallikrein inhibition and could substantially
increase the in vivo dose required for efficacy. Therefore, a
surface plasmon resonance (SPR) assay was used to identify
antibodies that do not bind prekallikrein (data not shown).
Specifically, human IgGs (X81-B01, M162-A04 (R84-H05); M160-G12
(R84-D02); and M142-H08) were captured on a CM5 chip using an
anti-human Fc surface and 100 nM of plasma kallikrein or 100 nM or
500 nM prekallikrein. The prekallikrein was treated with
aprotinin-sepharose to remove active plasma kallikrein. The
prekallikrein used for X81-B01 was buffer exchanged into the exact
preparation of SPR running buffer (HEPES buffered saline) to avoid
the refractive index shift that was observed with three other
antibodies that were tested: M162-A04 (R84-H05); M160-G12
(R84-D02); and M142-H08.
[0872] Of the antibodies listed in Table 3, only M142-H08 inhibits
human plasma kallikrein with a subnanomolar K.sub.i,app. However,
when M142-H08 was produced as an IgG it was found to be cleaved in
the CDR3 of the heavy chain. Consequently, we decided to undertake
two approaches to improve the affinity: 1) affinity maturation of
M162-A04 and M160-G12 using a novel form of light chain shuffling
called ROLIC (Rapid Optimization of Light Chains) (see, e.g., WO
2009/102927 and U.S. 2009-0215119); and 2) sequence optimization of
M142-H08 in order to prevent the cleavage of the IgG that occurs
while retaining the binding and inhibitor properties of
M142-H08.
TABLE-US-00008 TABLE 3 Top Ranking Antibody Inhibitors of PKal
Before Affinity Maturation or Sequence Optimization Criteria
M162-A04 M160-G12 M142-H08.sup.a K.sub.i,app human pKal 2 nM (as an
5.6 nM (as an 0.6 nM (as a IgG) IgG) Fab) K.sub.i,app rodent pKal 2
nM (mouse <1 nM ~1 nM (mouse and rat) (mouse) and rat) Binds
prekallikrein? No No No Specific inhibitor with respect to Yes Yes
Yes fXIa, plasmin, and trypsin Inhibits bradykinin generation Yes
Yes Yes .sup.aWhen M142-H08 was produced as an IgG it was
determined to be cleaved in the CDR3 of its heavy chain
(GGLLLWFR-ELKSNYFDY (SEQ ID NO: 894)).
Example 3: Sequence Optimization of M142-H08
[0873] Of the antibodies listed in Table 3, only M142-H08 inhibits
human pKal with a subnanomolar K.sub.i,app. However, when M142-H08
was produced as an IgG it was found to be cleaved in the CDR3 of
the heavy chain. M142-H08 was found by mass spectrometry to be
cleaved after the arginine in the "WFR" sequence of the HC-CDR3
sequence (GGLLLWFRELKSNYFDY (SEQ ID NO:894)). This cleavage
suggests that a protease from the cells used to express the
antibody (both CHO and 293T human kidney cells) is enzymatically
cleaving the antibody at a single specific site. We mutated the
HC-CDR3 sequence of M142-H08 in order to identify amino acid
substitutions that prevent the cleavage of the IgG that occurs
while retaining the binding and inhibitor properties of M142-H08.
Previous experience with similarly "clipped" antibodies suggested
that focusing simply on the putative P1 position (protease subsite
1, see Table 4) may not be sufficient to identify antibodies that
retain potent inhibition of the target enzyme while not being
clipped by a host cell protease. Therefore, we created a small
library of single point mutations in the region around the cleavage
site in order to identify variants of M142-H08 that are not clipped
but are still potent pKal inhibitors. We refer to this library as
the "CDR3 by Design" library. The small library was constructed
using a PCR primer that contains the randomized codon NNK at either
the P3, the P2, the P1, or the P1' site. This results in a small
library where each of the 4 positions may contain any of the 20
amino acids (20+20+20+20=80 members). Using PCR, this library was
cloned into the M142-H08 Fab sequence in the pMid21 vector, which
is a standard phagemid vector.
TABLE-US-00009 TABLE 4 Primer sequences Primer Name Sequence N (SEQ
ID P3 P2 P1 P1' P2' NO: 895) G G L L L W F R E L K S N Y
559A.P1.top GGC GGT CTA TTA CTA TGG TTC NNK GAG CTG 20 (SEQ ID AAG
TCT AAC TAC NO: 896) 559A.P2.top GGC GGT CTA TTA CTA TGG NNK AGG
GAG CTG 20 (SEQ ID AAG TCT AAC TAC NO: 897) 559A.P3.top GGC GGT CTA
TTA CTA NNK TTC AGG GAG CTG 20 (SEQ ID AAG TCT AAC TAC NO: 898)
559A.P1p.top GGC GGT CTA TTA CTA TGG TTC AGG NNK CTG 20 (SEQ ID AAG
TCT AAC TAC NO: 899)
[0874] By DNA sequencing, we recovered 61 of the possible 80
antibodies (Table 5). These antibodies were produced as Fab
fragments in small scale (.about.20 .mu.g) and tested for
inhibition against human pKal in an in vitro protease cleavage
assay using Pro-Phe-Arg-aminomethylcoumarin as the synthetic
peptide substrate. The Fabs that were found to be inhibitors of
human pKal were subcloned into our pBRH1f vector (a vector for
transient expression of IgGs in 293T cells) for conversion to full
length human IgG1 antibodies. Five antibodies were then expressed
in 293T cells and purified by protein A sepharose chromatography.
The antibodies were analyzed by SDS-PAGE to determine which of the
inhibitory mutants are not cleaved by the host cell protease(s)
(data not shown). The cleaved antibodies (559A-X67-G05,
559A-X67-H01, 559A-X67-G09) had an extra band that migrated between
the 38 and the 49 kDa molecular weight marker. This band is absent
in the 559A-X67-H04 and 559A-X67-D03 antibodies, which indicates
that these antibodies are intact.
[0875] K.sub.i,app values were determined by steady state enzyme
kinetics for those that were shown by SDS-PAGE to be not cleaved
(Table 5). Interestingly, the P2 position was the only position
where amino acid substitutions yielded intact antibody inhibitors
of pKal. Of the 14 different mutations that were recovered at the
P3 position (Table 5), only one mutant (W to L) was found to be a
pKal inhibitor as a Fab but it was subsequently shown to be clipped
as an IgG. None of the 16 different mutations at the P1 position
(Table 5) were found to be pKal inhibitors. Eight of the 15
different mutations at the P1' position were found to be inhibitors
of pKal as a Fab but all were clipped as an IgG. Consequently, only
mutations at the P2 position led to antibody inhibitors that were
not clipped during expression. Of the 16 different mutations that
were recovered at the P2 position (Table 5), eight mutants were
found to be a pKal inhibitor as a Fab but it was subsequently shown
to be clipped as an IgG. Four mutants at the P2 position were found
to have subnanomolar K.sub.i,app values: X67-G04 (F to A), X67-C03
(F to M), X67-F01 (F to Q) and X67-D03 (F to N). The antibody with
the highest potency is X67-D03 (K.sub.i,app=0.1 nM). The two
antibodies shown in Table 6 were not cleaved when expressed as IgGs
and were found to inhibit pKal with a subnanomolar K.sub.i,app.
[0876] DNA and amino acid sequence alignments of the light chains
of nongermlined (X63-G06) and germlined, codon optimized (X81-B01)
versions of the same antibody discovered using ROLIC affinity
maturation are shown in FIGS. 4 and 5, respectively. DNA and amino
acid sequence alignments of the heavy chains of nongermlined
(X63-G06) and germlined, codon optimized (X81-B01) versions of the
same antibody discovered using ROLIC affinity maturation are shown
in FIGS. 6 and 7, respectively.
TABLE-US-00010 TABLE 5 HV-CDR3 Sequences Obtained from "CDR3 by
Design" Library* Ki, app Inhibit Intact as an Mutation HV-CDR3 as a
as an IgG Site Antibody I.D. (SEQ ID NOs: 900-961) Fab? IgG? (nM)
Parental X69-C09 GGLLLWFRELKSNYFDY Yes No 0.2 P3 X68-E07
GGLLLAFRELKSNYFDY No n/a n/a P3 X68-E12 GGLLLCFRELKSNYFDY No n/a
n/a P3 X68-A03 GGLLLDFRELKSNYFDY No n/a n/a P3 X68-E03
GGLLLEFRELKSNYFDY No n/a n/a P3 X68-A12 GGLLLGFRELKSNYFDY No n/a
n/a P3 X68-D11 GGLLLKFRELKSNYFDY No n/a n/a P3 X68-E01
GGLLLLFRELKSNYFDY Yes No n/a P3 X68-F05 GGLLLMFRELKSNYFDY No n/a
n/a P3 X68-D10 GGLLLPFRELKSNYFDY No n/a n/a P3 X68-F10
GGLLLQFRELKSNYFDY No n/a n/a P3 X68-G01 GGLLLRFRELKSNYFDY No n/a
n/a P3 X68-G05 GGLLLSFRELKSNYFDY No n/a n/a P3 X68-F12
GGLLLTFRELKSNYFDY No n/a n/a P3 X68-H04 GGLLLVFRELKSNYFDY No n/a
n/a P2 X67-G04 GGLLLWARELKSNYFDY Yes Yes 0.35 P2 X67-G01
GGLLLWCRELKSNYFDY No n/a n/a P2 X67-E04 GGLLLWDRELKSNYFDY Yes Yes
1.3 P2 X67-H04 GGLLLWERELKSNYFDY Yes Yes 3.6 P2 X67-C09
GGLLLWGRELKSNYFDY Yes Yes 8.6 P2 X67-B04 GGLLLWKRELKSNYFDY Yes No
n/a P2 X67-G09 GGLLLWLRELKSNYFDY Yes No n/a P2 X67-C03
GGLLLWMRELKSNYFDY Yes Yes 0.7 P2 X67-D03 GGLLLWNRELKSNYFDY Yes Yes
0.1 P2 X67-B05 GGLLLWPRELKSNYFDY No n/a n/a P2 X67-F01
GGLLLWQRELKSNYFDY Yes Yes 0.9 P2 X67-G05 GGLLLWRRELKSNYFDY Yes No
n/a P2 X67-B03 GGLLLWSRELKSNYFDY Yes Yes 2.1 P2 X67-F10
GGLLLWTRELKSNYFDY Yes Yes 1.3 P2 X67-H01 GGLLLWWRELKSNYFDY Yes No
n/a P2 X67-F08 GGLLLWYRELKSNYFDY Yes No n/a P1 X66-E09
GGLLLWFAELKSNYFDY No n/a n/a P1 X66-B05 GGLLLWFCELKSNYFDY No n/a
n/a P1 X66-D03 GGLLLWFEELKSNYFDY No n/a n/a P1 X66-H04
GGLLLWFFELKSNYFDY No n/a n/a P1 X66-H02 GGLLLWFGELKSNYFDY No n/a
n/a P1 X66-C11 GGLLLWFHELKSNYFDY No n/a n/a P1 X66-A07
GGLLLWFKELKSNYFDY No n/a n/a P1 X66-C03 GGLLLWFLELKSNYFDY No n/a
n/a P1 X66-G05 GGLLLWFMELKSNYFDY No n/a n/a P1 X66-F10
GGLLLWFPELKSNYFDY No n/a n/a P1 X66-E04 GGLLLWFQELKSNYFDY No n/a
n/a P1 X66-F01 GGLLLWFSELKSNYFDY No n/a n/a P1 X66-H11
GGLLLWFTELKSNYFDY No n/a n/a P1 X66-C02 GGLLLWFVELKSNYFDY No n/a
n/a P1 X66-F09 GGLLLWFWELKSNYFDY No n/a n/a P1 X66-G08
GGLLLWFYELKSNYFDY No n/a n/a P1' X69-D08 GGLLLWFRALKSNYFDY No n/a
n/a P1' X69-B02 GGLLLWFRCLKSNYFDY No n/a n/a P1' X69-D09
GGLLLWFRGLKSNYFDY Yes No n/a P1' X69-D02 GGLLLWFRHLKSNYFDY No n/a
n/a P1' X69-A12 GGLLLWFRKLKSNYFDY No n/a n/a P1' X69-F05
GGLLLWFRLLKSNYFDY Yes No n/a P1' X69-B08 GGLLLWFRNLKSNYFDY Yes No
n/a P1' X69-A10 GGLLLWFRPLKSNYFDY No n/a n/a P1' X69-A09
GGLLLWFRQLKSNYFDY Yes No n/a P1' X69-E05 GGLLLWFRRLKSNYFDY No n/a
n/a P1' X69-F09 GGLLLWFRSLKSNYFDY Yes No n/a P1' X69-F01
GGLLLWFRTLKSNYFDY Yes No n/a P1' X69-C12 GGLLLWFRVLKSNYFDY Yes No
n/a P1' X69-E01 GGLLLWFRWLKSNYFDY Yes No n/a P1' X69-H10
GGLLLWFRYLKSNYFDY No n/a n/a *All of these antibodies are single
point mutations of the M142-H08 sequence.
TABLE-US-00011 Amino acid sequences of light chain (LC) and heavy
chain (HC) variable domain of pKal antibodies with designed HC
CDR3s are shown below. (SEQ ID NOs: 962-1085) X68-E07 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLAFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-E12 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLCFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-A03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-A03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLDFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-E03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLEFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-A12 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-A12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLGFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-D11 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-D11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLKFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-E01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLLFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-F05 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-F05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLMFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-D10 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-D10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLPFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-F10 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-F10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLQFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-G01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-G01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLRFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-G05 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-G05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNILY LQMNSLRAED TAVYYCARGG LLLSFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-F12 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-F12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLTFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X68-H04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-H04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLVFRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-G04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWARELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-G01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWCRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-E04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-E04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWDRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-H04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-H04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWERELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-E09 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-E09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFAELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-B05 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-B05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFCELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-D03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108
X66-D03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFEELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-H04 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-H04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFFELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-H02 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-H02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMHSLRAED TAVYYCARGG
LLLWFGELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-C11 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-C11
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFHELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-A07 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-A07
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFKELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-C03 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-C03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFLELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-G05 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-G05
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFMELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-F10 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-F10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFPELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-E04 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-E04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFQELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-D08 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-D08
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRALKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-B02 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-B02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRCLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-C09 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-C09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRELKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-D09 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-D09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRGLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-D02 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-D02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRHLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-A12 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-A12
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRKLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-F05 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-F05
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRLLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-B08 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-B08
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRNLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-A10 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-A10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRPLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-A09 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-A09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRQLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-E05 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-E05
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRRLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-F09 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60 SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-F09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY
IRPSGGRTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG
LLLWFRSLKS NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-F01 LC
QDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP
60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-F01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRTLKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-C12 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-C12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRVLKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-E01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-E01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRWLKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X69-H10 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-H10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRYLKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-F01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-F01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFSELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-H11 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-H11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFTELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-C02 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-C02 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFVELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-F09 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-F09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFWELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X66-G08 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-G08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFYELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-C09 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWGRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-B04 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWKRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-G09 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWLRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-C03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWMRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-D03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-D03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWNRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-B05 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWPRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-F01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWQRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-G05 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWRRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-B03 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWSRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-F10 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWTRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-H01 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-H01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWWRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143 X67-F08 LC QDIQMTQSPS
SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP 60
SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWYRELKS
NYFDYWGQGT 120 LVTVSSASTK GPSVFPLAPS SKS 143
TABLE-US-00012 TABLE 7 CDR Amino Acid Sequences of Affinity Matured
Antibody Inhibitors of pKal Discovered using ROLIC (SEQ ID NOs:
1093-1113) LV-CDR1 LV-CDR2 LV-CDR3 HV-CDR1 HV-CDR2 HV-CDR3 Initial
Ki, app (SEQ ID NOs: (SEQ ID NOs: (SEQ ID NOs: (SEQ ID (SEQ ID (SEQ
ID Name (nM) 1093-1098) 1099-1104) 1105-1110) NO: 1111) NO: 1112)
NO: 1113) X59-C07 6.1 RAGRSISTYVN AASSLQS QQSQSTPYT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTSYFDY X60-D01 2.0 RASQIVSSRYLA GAASRAT
QQTYSSPFT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY X63-G10 9.0
RASQSISNYLN AASSLQS QQSYTSPYT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTSYFDY X64-F04 1.9 RASQIVSSNYLA GASNRAT QQSFNIPYT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTSYFDY X63-G06 0.4 (Fab) RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY
X81-B01.sup.a 0.2 (IgG) RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTSYFDY .sup.aX81-B01 is the codon
optimized and germlined version of X63-G06 as a full length human
IgG produced in HEK 293T cells.
TABLE-US-00013 TABLE 6 CDR Amino Acid Sequences of Optimized
Antibody Inhibitor of pKal Based on M142-H08 (SEQ ID NOs:
1086-1092) Ki, app LV-CDR1 LV-CDR2 LV-CDR3 HV-CDR1 HV-CDR3.sup.a
Initial (nM) of (SEQ ID (SEQ ID (SEQ ID (SEQ ID HV-CDR2 (SEQ ID
NOs: 1091 Name IgG NO: 1086) NO: 1087) NO: 1088) NO: 1089) (SEQ ID
NO: 1090) and 1092) X67-D03 0.1 RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI
YIRPSGGRTTYADSVKG GGLLLWNRELKSNYFDY X67-G04 0.35 RASQPIDNYLN
AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWARELKSNYFDY
.sup.aThe F to N substitution (in bold) in the CDR3 of the M142-H08
gives X67-D03, an IgG that is not cleaved during expression and is
a potent inhibitor of human. Similarly, the F to A substitution
gives X67-G04, which is also not cleaved.
TABLE-US-00014 Amino acid sequences of light chain (LC) and heavy
chain (HC) variable domain of affinity matured antibody inhibitors
of pKal discovered using ROLIC are shown below (SEQ ID NOs:
1114-1123). X59-C07 LC QDIQMTQSPS SLSASVGDRV TVTCRAGRSI STYVNWYQQK
PGKAPKLLIY AASSLQSGVP 60 SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ
QSQSTPYTFG QGTKLEVK 108 X59-C07 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS
HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG
PSVFPLAPSS KS 142 X60-D01 LC QDIQMTQSPG TLSLSPGERA TLSCRASQIV
SSRYLAWYQQ RPGQAPRLLI YGAASRATGI 60 PDRFSGSGSG TDFTLTISSL
QAEDFATYYC QQTYSSPFTF GQGTKMEIK 109 X60-D01 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL
120 VTVSSASTKG PSVFPLAPSS KS 142 X63-G06 LC QDIQMTQSPG TLSLSPGERA
TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60 PDRFSGTGYG
TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 X63-G06 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS
YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 X63-G10 LC QDIQMTQSPD
SLSASVGDRV TITCRASQSI SNYLNWYQQK PGKAPKLLIY AASSLQSGVP 60
SRFSGSGSGT DFTLTISGLQ PEDFASYYCQ QSYTSPYTFV QGTKLEIKRT 110 X63-G10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 X64-F04 LC
QDIQMTQSPA TLSLSPGERA TLSCRASQIV SSNYLAWYQQ KPGQAPRLLI YGASNRATGI
60 PDRFSGSGSG TEFTLTISSL QSEDFAIYYC QQSFNIPYTF GQGTRVDIK 109
X64-F04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142
X81-B01 is the germlined IgG produced in HEK 293T cells version of
the X63-G06 Fab, as indicated above. X101-A01 (aka DX-2922) is the
germlined IgG produced in CHO cells version of the X63-G06 Fab
Example 4: Affinity Maturation
[0877] In addition to optimizing the sequence of the clipped
antibody (M142-H08), we also performed affinity maturation on two
of the antibodies identified by phage display (M162-A04 and
M160-G12). Both of these antibodies inhibit human pKal with single
digit nanomolar potency, appear specific to pKal, and do not bind
prekallikrein (Table 3). We first performed a novel form of light
chain shuffling called ROLIC (Rapid Optimization of Light Chains)
on M162-A04 and M160-G12 (see, e.g., WO 2009/102927 and U.S.
2009-0215119). From the screening of the antibodies discovered by
ROLIC we identified one antibody with subnamolar potency (X63-G06)
that shared the same heavy chain as M160-G12. We then constructed
HV-CDR3 spiking affinity maturation libraries based on CDR3
sequences in M162-A04 and X63-G06 (described below).
[0878] Affinity Maturation by ROLIC.
[0879] We used ROLIC to affinity mature the two leads from Table 3
that were not cleaved (M162-A04 and M160-G12). This process
identified one antibody that inhibits pKal with a subnanomolar
K.sub.i,app (Table 7). X63-G06 inhibits pKal with a K.sub.i,app of
approximately 0.4 nM as a Fab fragment. When this antibody was
converted to an IgG that is germlined and sequenced optimized for
CHO cell expression (X81-B01) it was found to inhibit pKal with a
K.sub.i,app of approximately 0.2 nM.
Example 5: Affinity Maturation of Heavy Chain CDR1/2 and CDR3
[0880] We used two additional affinity maturation strategies to
identify highly potent antibodies based on two different parental
antibody inhibitor leads: M162-A04 and X63-G06. One approach was to
generate libraries that shuffled the CDR1/2 of the HC of two
different parental antibody inhibitor leads (M162-A04 and X63-G06)
against additional CDR1/2 diversity. Another approach was to create
heavy chain CDR3 spiking libraries based on these leads.
[0881] The 82 antibodies that were discovered based on improvements
in M162-A04 due to modifications in either the CDR1/2 or CDR3
region are shown in Table 8. Inhibition screening with 10 nM
antibody (as Fab fragments) revealed that there were 33 antibodies
that inhibited pKal activity by over 90%. Several antibodies were
shown to be subnanomolar inhibitors of human pKal.
[0882] The 62 antibodies that were discovered based on improvements
in X63-G06 due to modifications in either the CDR1/2 or CDR3 region
are shown in Table 9. Inhibition screening with 10 nM antibody (as
Fab fragments) revealed that there were 24 antibodies that
inhibited pKal activity by over 90%. Several antibodies were shown
to be subnanomolar inhibitors of human pKal.
TABLE-US-00015 TABLE 8 Sequences of Antibodies Obtained from CDR1/2
and CDR3 Spiking Affinity Maturation Libraries Based on M162-A04
(SEQ ID NOs: 1124-1372) HV- human CDR1 pKal (SEQ Ki, LV-CDR1
LV-CDR2 LV-CDR3 ID HV-CDR2 HV-CDR3 Antibody % inhibition app (SEQ
ID (SEQ ID (SEQ ID NOs: (SEQ ID NOs: (SEQ ID NOs: I.D. at 10 nM
(nM) NO: 1124) NO: 1125) NO: 1126) 1127-1208) 1209-1290) 1291-1372)
M202-A12 97.5 0.2 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG QRTGVPRRDSFNI M196-C06 97.2 0.1 RASQSISSWLA
KASTLES QQYNTYWT IYSMH SIYPSRGMTWYADSVKG RRTGIPRRDAFDI M198-F09
96.9 0.2 RASQSISSWLA KASTLES QQYNTYWT VYNMH SIYPSGGMTYYADSVKG
RRTGIPRRDAFDI M199-A08 96.4 0.06 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRIGVPRRDEFDI M202-C01 96.3 0.1 RASQSISSWLA
KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRWDDFDI M198-A06
96.1 0.4 RASQSISSWLA KASTLES QQYNTYWT IYSMH SIYSSGGPTKYADSVKG
RRTGIPRRDAFDI M200-D03 95.9 0.1 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRIGVPRRDSFDM M202-H03 95.7 0.1 RASQSISSWLA
KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRWDDFDI M201-A07
95.7 0.1 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG
RRTGVPRRDEFDI M197-A01 95.3 RASQSISSWLA KASTLES QQYNTYWT IYDMI
SIYPSGGNTSYADSVKG RRTGIPRRDAFDI M202-D09 95.0 0.4 RASQSISSWLA
KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRIGVPRRDSFDI M197-A09
94.9 0.6 RASQSISSWLA KASTLES QQYNTYWT VYNMH SIYPSGGMTTYADSVKG
RRTGIPRRDAFDI M198-G07 94.9 RASQSISSWLA KASTLES QQYNTYWT IYDMT
SIYPSGGQTIYADSVKG RRTGIPRRDAFDI M200-A10 94.3 0.3 RASQSISSWLA
KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDSFDI M197-H10
94.1 RASQSISSWLA KASTLES QQYNTYWT SYNMH SIVPSGGKTNYADSVKG
RRTGIPRRDAFDI M196-D12 94.1 0.2 RASQSISSWLA KASTLES QQYNTYWT RYSMR
VIYPSGGQTYYADSVKG RRTGIPRRDAFDI M197-A08 93.7 RASQSISSWLA KASTLES
QQYNTYWT IYSMQ SIGSSGGKTLYADSVKG RRTGIPRRDAFDI M198-B09 93.5
RASQSISSWLA KASTLES QQYNTYWT VYSMT SIGSSGGSTTYADSVKG RRTGIPRRDAFDI
M198-E09 93.1 RASQSISSWLA KASTLES QQYNTYWT IYDMN SIYPSGGRTRYADSVKG
RRTGIPRRDAFDI M202-B03 93.1 0.3 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRTGVPRRDDFDI M198-C10 93.0 RASQSISSWLA KASTLES
QQYNTYWT HYMGMN SIVPSGGWTQYADSVKG RRTGIPRRDAFDI M197-E12 93.0
RASQSISSWLA KASTLES QQYNTYWT TYTMR SIYPSGGKTQYADSVKG RRTGIPRRDAFDI
M198-F04 92.9 RASQSISSWLA KASTLES QQYNTYWT IYDMW SIRPSGGITKYADSVKG
RRTGIPRRDAFDI M197-H11 92.9 RASQSISSWLA KASTLES QQYNTYWT IYNMI
SIYPSGGWTTYADSVKG RRTGIPRRDAFDI M197-F01 92.6 RASQSISSWLA KASTLES
QQYNTYWT IYHMY SIGPSGGPTGYADSVKG RRTGIPRRDAFDI M198-E11 92.5
RASQSISSWLA KASTLES QQYNTYWT TYSMY SIYPSGGLTWYADSVKG RRTGIPRRDAFDI
M202-C09 92.3 0.3 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRIGVPRRDDFDI M198-H08 92.3 RASQSISSWLA KASTLES
QQYNTYWT IYDMY SIGPSGGPTAYADSVKG RRTGIPRRDAFDI M198-F08 91.8
RASQSISSWLA KASTLES QQYNTYWT VYSMW SISSSGGMTEYADSVKG RRTGIPRRDAFDI
M202-E06 91.5 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG
RRRGVPRRDDFDI M195-D12 90.8 RASQSISSWLA KASTLES QQYNTYWT IYGMF
GIGPSGGPTKYADSVKG RRTGIPRRDAFDI M197-F03 90.7 RASQSISSWLA KASTLES
QQYNTYWT IYSMF SIGPSGGVTHYADSVKG RRTGIPRRDAFDI M198-E02 90.3
RASQSISSWLA KASTLES QQYNTYWT IYSMY YIRPSGGNTKYADSVKG RRTGIPRRDAFDI
M198-A02 89.1 RASQSISSWLA KASTLES QQYNTYWT RYSMI SIWSSGGATEYADSVKG
RRTGIPRRDAFDI M202-A01 88.9 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRIGVPRRDAFDI M202-G03 88.3 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDSFEI M195-B12 87.7
RASQSISSWLA KASTLES QQYNTYWT KYWMY YIRPSGGQTYYADSVKG RRTGIPRRDAFDI
M198-A07 86.1 RASQSISSWLA KASTLES QQYNTYWT RYQMH WISPSGGITGYADSVKG
RRTGIPRRDAFDI M198-H02 85.8 RASQSISSWLA KASTLES QQYNTYWT PYNMY
WIVPGGVTKYADSVKG RRTGIPRRDAFDI M200-H07 85.4 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRNAFDN M201-H06 84.6
RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDAFDI
M202-F06 84.2 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG
RRTGVPRWDAFDI M195-C12 84.2 RASQSISSWLA KASTLES QQYNTYWT MYQMF
SISPGGGTQYADSVKG RRTGIPRRDAFDI M202-H05 84.0 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDVFDI M198-C05 83.9
RASQSISSWLA KASTLES QQYNTYWT RYKMY VIGPSGGATFYADSVKG RRTGIPRRDAFDI
M196-H03 83.9 RASQSISSWLA KASTLES QQYNTYWT RYVMW SISPSGDTHYADSVKG
RRTGIPRRDAFDI M200-E11 83.2 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRTGVPRRDAFDN M202-B04 81.9 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRSGVPRRDDFDI M202-A04 81.2
RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRKGIPRRDDFDI
M198-B12 80.7 RASQSISSWLA KASTLES QQYNTYWT KYSMA GIYPSGGRTLYADSVKG
RRTGIPRRDAFDI M198-A09 77.3 RASQSISSWLA KASTLES QQYNTYWT IYFMS
SIRSSGGPTWYADSVKG RRTGIPRRDAFDI M198-C06 76.5 RASQSISSWLA KASTLES
QQYNTYWT QYFMH YIYPSGGMTEYADSVKG RRTGIPRRDAFDI M198-C09 75.4
RASQSISSWLA KASTLES QQYNTYWT IYTMY SISPSGGWTYYADSVKG RRTGIPRRDAFDI
M195-B02 75.1 RASQSISSWLA KASTLES QQYNTYWT PYLMW YIGPSGGPTHYADSVKG
RRTGIPRRDAFDI M198-F12 74.6 RASQSISSWLA KASTLES QQYNTYWT IYTMM
SIWSSGGQTKYADSVKG RRTGIPRRDAFDI M201-H08 74.5 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDALDN M202-C02 74.3
RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRPGVPRRDAFDI
M198-C03 72.4 RASQSISSWLA KASTLES QQYNTYWT RYSMS GISPSGGETSYADSVKG
RRTGIPRRDAFDI M198-A08 72.3 RASQSISSWLA KASTLES QQYNTYWT WYMMQ
RISPSGGTTYADSVKG RRTGIPRRDAFDI M195-A02 71.3 RASQSISSWLA KASTLES
QQYNTYWT QYMMM GISSSGGHTDYADSVKG RRTGIPRRDAFDI M197-G10 67.6
RASQSISSWLA KASTLES QQYNTYWT VYAMR SIYPSGGKTWYADSVKG RRTGIPRRDAFDI
M195-G02 67.5 RASQSISSWLA KASTLES QQYNTYWT PYNMM SIWPSGGTTDYADSVKG
RRTGIPRRDAFDI M196-D02 66.2 RASQSISSWLA KASTLES QQYNTYWT VYSMH
VIGPSGGITLYADSVKG RRTGIPRRDAFDI M199-A11 65.4 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRRGIPRRDAFDI M200-F01 65.1
RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRMGIPRRNAFDI
M198-D12 63.5 0.7 RASQSISSWLA KASTLES QQYNTYWT LYVMY
YIVPSGGPTAYADSVKG RRTGIPRRDAFDI M197-C12 56.4 RASQSISSWLA KASTLES
QQYNTYWT PYDML YIVSSGGLTKYADSVKG RRTGIPRRDAFDI M198-G03 53.8
RASQSISSWLA KASTLES QQYNTYWT QYTMV WIYSSRANYADSVKG RRTGIPRRDAFDI
M199-B01 53.4 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG
RRTGIPRRDAFDN M202-A08 52.9 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRTGIPRWDAFDI M195-A12 51.7 RASQSISSWLA KASTLES
QQYNTYWT PYMMM GIYPSGGYTVYADSVKG RRTGIPRRDAFDI M202-E03 51.4
RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDAFEI
M196-G12 51.1 RASQSISSWLA KASTLES QQYNTYWT NYSMD RIYSSGGGTIYADSVKG
RRTGIPRRDAFDI M195-F12 45.5 RASQSISSWLA KASTLES QQYNTYWT HYVMM
YIVPSGGVTAYADSVKG RRTGIPRRDAFDI M200-B01 42.6 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDAFDS M198-H09 41.1
RASQSISSWLA KASTLES QQYNTYWT IYLMI YIGPSGGPTEYADSVKG RRTGIPRRDAFDI
M195-E12 38.0 RASQSISSWLA KASTLES QQYNTYWT YYIMF YISPSGGYTHYADSVKG
RRTGIPRRDAFDI M201-A06 36.8 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRTGIPRRDVFDI M202-A10 36.3 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDSFDI
M197-G11 19.2 RASQSISSWLA KASTLES QQYNTYWT TYAMV SIYPSGGITTYADSVKG
RRTGIPRRDAFDI M201-F11 15.7 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRSGIPRRDAFDI M198-A01 13.8 RASQSISSWLA KASTLES
QQYNTYWT PYTMI SISSSGGMTPYADSVKG RRTGIPRRDAFDI
TABLE-US-00016 Amino acid sequences of light chain (LC) and heavy
chain (HC) variable domain of pKal antibodies obtained from CDR1/2
and CDR3 spiking affinity maturation libraries based on M162-A04
(SEQ ID NOs: 1373-1536). M195-A02 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-A02 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS QYMMMWVRQA PGKGLEWVSG ISSSGGHTDY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M195-A12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-A12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS PYMMMWVRQA PGKGLEWVSG IYPSGGYTVY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M195-B02 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-B02 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS PYLMWWVRQA PGKGLEWVSY IGPSGGPTHY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M195-B12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-B12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS KYWMYWVRQA PGKGLEWVSY IRPSGGQTYY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M195-C12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-C12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS MYQMFWVRQA PGKGLEWVSS ISPGGGTQYA 60
DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS
120 SASTKGPSVF PLAPSSKS 138 M195-D12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-D12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS IYGMFWVRQA PGKGLEWVSG IGPSGGPTKY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M195-E12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-E12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS YYIMFWVRQA PGKGLEWVSY ISPSGGYTHY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M195-F12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-F12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS HYVMMWVRQA PGKGLEWVSY IVPSGGVTAY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M0195-G02 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-G02 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS PYNMMWVRQA PGKGLEWVSS IWPSGGTTDY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M196-C06 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-C06 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS IYSMHWVRQA PGKGLEWVSS IYPSRGMTWY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M196-D02 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-D02 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS VYSMHWVRQA PGKGLEWVSV IGPSGGITLY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M196-D12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-D12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYSMRWVRQA PGKGLEWVSV IYPSGGQTYY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M196-G12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-G12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS NYSMDWVRQA PGKGLEWVSR IYSSGGGTIY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M196-H03 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-H03 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS RYVMWWVRQA PGKGLEWVSS ISPSGDTHYA 60
DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS
120 SASTKGPSVF PLAPSSKS 138 M197-A01 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-A01 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS IYDMIWVRQA PGKGLEWVSS IYPSGGNTSY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M197-A08 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-A08 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS IYSMQWVRQA PGKGLEWVSS IGSSGGKTLY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M197-A09 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-A09 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS VYNMHWVRQA PGKGLEWVSS IYPSGGMTTY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M197-C12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-C12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS PYDMLWVRQA PGKGLEWVSY IVSSGGLTKY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M197-E12 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-E12 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS TYTMRWVRQA PGKGLEWVSS IYPSGGKTQY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M197-F01 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60 SRFSGSGSGT
EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-F01 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS IYHMYWVRQA PGKGLEWVSS IGPSGGPTGY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV
120 SSASTKGPSV FPLAPSSKS 139 M197-F03 LC QDIQMTQSPS TLSASVGDRV
TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-F03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMFWVRQA PGKGLEWVSS IGPSGGVTHY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M197-G10 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-G10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYAMRWVRQA PGKGLEWVSS IYPSGGKTWY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M197-G11 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-G11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYAMVWVRQA PGKGLEWVSS IYPSGGITTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M197-H10 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-H10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYNMHWVRQA PGKGLEWVSS IVPSGGKTNY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M197-H11 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-H11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYNMIWVRQA PGKGLEWVSS IYPSGGWTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-A01 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYTMIWVRQA PGKGLEWVSS ISSSGGMTPY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-A02 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A02 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMIWVRQA PGKGLEWVSS IWSSGGATEY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-A06 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A06 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMHWVRQA PGKGLEWVSS IYSSGGPTKY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-A07 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYQMHWVRQA PGKGLEWVSW ISPSGGITGY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-A08 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYMMQWVRQA PGKGLEWVSR ISPSGGTTYA
60 DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI
WGQGTMVTVS 120 SASTKGPSVF PLAPSSKS 138 M198-A09 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYFMSWVRQA PGKGLEWVSS IRSSGGPTWY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-B09 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-B09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYSMTWVRQA PGKGLEWVSS IGSSGGSTTY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-B12 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-B12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS KYSMAWVRQA PGKGLEWVSG IYPSGGRTLY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-C03 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMSWVRQA PGKGLEWVSG ISPSGGETSY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-C05 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C05 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS RYKMYWVRQA PGKGLEWVSV IGPSGGATFY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-C06 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C06 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYFMHWVRQA PGKGLEWVSY IYPSGGMTEY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-C09 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYTMYWVRQA PGKGLEWVSS ISPSGGWTYY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-C10 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYMGMNWVRQ APGKGLEWVS SIVPSGGWTQ
60 YADSVKGRFT ISRDNSKNTL YLQMNSLRAE DTAVYYCAYR RTGIPRRDAF
DIWGQGTMVT 120 VSSASTKGPS VFPLAPSSKS 140 M198-D12 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-D12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS LYVMYWVRQA PGKGLEWVSY IVPSGGPTAY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-E02 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-E02 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMYWVRQA PGKGLEWVSY IRPSGGNTKY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-E09 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-E09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMNWVRQA PGKGLEWVSS IYPSGGRTRY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-E11 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-E11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYSMYWVRQA PGKGLEWVSS IYPSGGLTWY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-F04 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F04 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMWWVRQA PGKGLEWVSS IRPSGGITKY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-F08 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYSMWWVRQA PGKGLEWVSS ISSSGGMTEY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-F09 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYNMHWVRQA PGKGLEWVSS IYPSGGMTYY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-F12 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F12 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYTMMWVRQA PGKGLEWVSS IWSSGGQTKY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-G03 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-G03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYTMVWVRQA PGKGLEWVSW IYSSRANYAD
60 SVKGRFTISR DNSKNTLYLQ MNSLRAEDTA VYYCAYRRTG IPRRDAFDIW
GQGTMVTVSS 120 ASTKGPSVFP LAPSSKS 137 M198-G07 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-G07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMTWVRQA PGKGLEWVSS IYPSGGQTIY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-H02 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-H02 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYNMYWVRQA PGKGLEWVSW IVPGGVTKYA
60 DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI
WGQGTMVTVS 120 SASTKGPSVF PLAPSSKS 138 M198-H08 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-H08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMYWVRQA PGKGLEWVSS IGPSGGPTAY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M198-H09 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-H09 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYLMIWVRQA PGKGLEWVSY IGPSGGPTEY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M199-A08 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M199-A08 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M199-A11 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M199-A11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR RGIPRRDAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M199-B01 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M199-B01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGIPRRDAFD
NWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M200-A10 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-A10 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDSFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M200-B01 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-B01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD
SWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M200-D03 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-D03 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAWRR IGVPRRDSFD
MWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M200-E11 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-E11 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDAFD
NWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M200-F01 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-F01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR MGIPRRNAFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M200-H07 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-H07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRNAFD
NWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M201-A06 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-A06 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDVFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M201-A07 LC QDIQMTQSPS
TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP 60
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-A07 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDEFD
IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M201-F11 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-F11
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
SGIPRRDAFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M201-H06 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-H06
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGVPRRDAFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M201-H08 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-H08
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGVPRRDALD NWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-A01 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
IGVPRRDAFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-A04 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
KGIPRRDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-A08 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A08
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGIPRWDAFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-A10 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A10
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR
TGIPRRDSFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-A12 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A12
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYQR
TGVPRRDSFN IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-B03 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-B03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGVPRRDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-B04 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-B04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
SGVPRRDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-C01 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-C01
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGVPRWDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-C02 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-C02
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
PGVPRRDAFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-C09 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-C09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
IGVPRRDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-D09 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-D09
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
IGVPRRDSFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-E03 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-E03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGIPRRDAFE IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-E06 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-E06
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
RGVPRRDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-F06 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-F06
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
TGVPRWDAFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-G03 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-G03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR
TGVPRRDSFE IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-H03 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-H03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR
TGVPRWDDFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139 M202-H05 LC
QDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP
60 SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-H05
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG
IYSSGGITVY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR
TGVPRRDVFD IWGQGTMVTV 120 SSASTKGPSV FPLAPSSKS 139
TABLE-US-00017 TABLE 9 Sequences of Antibodies Obtained from CDR1/2
and CDR3 Spiking Affinity Maturation Libraries Based on X63-G06
(SEQ ID NOs: 1537-1725) HV- CDR1 % inhi- human (SEQ bition pKal
LV-CDR1 LV-CDR2 LV-CDR3 ID HV-CDR2 HV-CDR3 Antibody at Ki, app (SEQ
ID (SEQ ID (SEQ ID NOs: (SEQ ID NOs: (SEQ ID NOs: I.D. 10 nM (nM)
NO: 1537) NO: 1538) NO: 1539) 1540-1601 1602-1663) 1664-1725)
M209-F04 97.6 0.09 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTSYLDq M209-C11 96.2 0.14 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VGQGIRGRSRTSYFAq M206-H08
96.0 0.17 RTSQFVNSNYLA GASSRAT QQSSRTPWT DYMMA SIVPSGGHTHYADSVKG
VARGIAARSRTSYFDY M210-C12 95.6 0.16 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VAQGIAARSRTSSVDq M208-F04 95.4 0.2
RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTSFFDY M206-B10 94.7 0.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT
qYLMA SIYPSGGWTKYADSVKG VARGIAARSRTSYFDY M208-H02 94.4 0.2
RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIASRSRTRYCDY M210-G04 94.2 0.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VATGIVARSRTRYFDq M210-H06 93.8 0.2
RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTRYFDY M208-E10 93.7 0.09 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VAQGISARSRTSYFDY M209-B09 93.5 0.2
RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VAQGIVARSRTSYLHq M209-C12 93.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VGRGIAARSRTSqLDY M208-G03 93.4 0.3 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYLDY M206-A06
93.0 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYMMG SISPSGGLTKYADSVKG
VARGIAARSRTSYFDY M210-H07 92.8 0.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VARGIAARSRTRYFDq M206-F01 92.6 0.2
RTSQFVNSNYLA GASSRAT QQSSRTPWT GYMMV RISPSGGPTIYADSVKG
VARGIAARSRTSYFDY M208-F10 92.5 0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDq M209-E02 92.4 0.3
RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTILLDq M208-C06 91.7 0.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSFIDY M205-D04 91.5 0.4
RTSQFVNSNYLA GASSRAT QQSSRTPWT TYKMq SISPSGGPTNYADSVKG
VARGIAARSRTSYFDY M210-G10 91.2 0.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT
HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYLDF M207-A04 90.9 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTRSFDY M210-B02
90.9 0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTSYFNq M208-B01 90.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTSFFDq M209-G07 89.8 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDT M204-A02
89.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT DYMMT YISPSGGLTSYADSVKG
VARGIAARSRTSYFDY M206-H01 87.6 RTSQFVNSNYLA GASSRAT QQSSRTPWT EYMMV
RISPSGGTTEYADSVKG VARGIAARSRTSYFDY M209-B11 87.3 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTRYIDq M206-F09
86.8 RTSQFVNSNYLA GASSRAT QQSSRTPWT VYMMS SIVPSGGSTTYADSVKG
VARGIAARSRTSYFDY M209-C02 86.8 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIAYRRRTSYFDY M208-G02 86.7 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIADRSRTSYSDY M205-C11
86.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT QYMMM RISPSGGSTLYADSVKG
VARGIAARSRTSYFDY M205-H08 85.9 RTSQFVNSNYLA GASSRAT QQSSRTPWT DYMMM
SIVPSGGHTqYADSVKG VARGIAARSRTSYFDY M210-H01 85.5 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRNSqQDY M209-D12
85.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTSYFDq M209-H09 85.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTVYFDH M204-E12 84.1 RTSQFVNSNYLA
GASSRAT QQSSRTPWT TYMMq YIGPSGGKTDYADSVKG VARGIAARSRTSYFDY M209-H03
82.6 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VAQGIAARSRTTqFDY M206-H05 82.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT GYKMq
SISPSGGITMYADSVKG VARGIAARSRTSYFDY M209-D03 80.4 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VGRGIAARSRTSFFDq M205-A02
80.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT TYLMA GIVSSGGRTLYADSVKG
VARGIAARSRTSYFDY M208-A10 78.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIAARSRTSqFDH M205-E11 78.2 RTSQFVNSNYLA
GASSRAT QQSSRTPWT NYTMG SISPSGGKTDYADSVKG VARGIAARSRTSYFDY M206-E02
77.6 RTSQFVNSNYLA GASSRAT QQSSRTPWT EYMMM VISPSGGQTHYADSVKG
VARGIAARSRTSYFDY M205-H01 77.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYTMQ
YISPSGGYTGYADSVKG VARGIAARSRTSYFDY M207-A02 76.6 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTINLDY M209-H07
76.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARqRTSYYDY M209-G01 74.8 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VAqGISGRSRLSYVDY M210-A06 74.8 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSqFDY M209-D02
74.7 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGITARSRTSYFDD M205-B04 71.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYDMI
SISSSGGTTKYADSVKG VARGIAARSRTSYFDY M203-A03 69.1 RTSQFVNSNYLA
GASSRAT QQSSRTPWT VYMMI SISPSGGQTTYADSVKG VARGIAARSRTSYFDY M209-E03
68.8 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
qARGIAARSRTSYFDY M207-A01 67.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGISARSRTSCFDY M206-C03 65.5 RTSQFVNSNYLA
GASSRAT QQSSRTPWT qYMMV SIYSSGGNTPYADSVKG VARGIAARSRTSYFDY M207-C05
61.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VGRGIAARSRTSYFDK M205-A12 58.8 RTSQFVNSNYLA GASSRAT QQSSRTPWT QYDMI
YISSSGGFTRYADSVKG VARGIAARSRTSYFDY M205-F03 58.6 RTSQFVNSNYLA
GASSRAT QQSSRTPWT SqQMV YISPSGGNTYYADSVKG VARGIAARSRTSYFDY M203-A01
51.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYLMA WIVPSGGYTEYADSVKG
VARGIAARSRTSYFDY M209-B01 47.0 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VARGIVARSRTSNFDq M208-D12 43.7 RTSQFVNSNYLA
GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG LARGIAARSRTSYqDI M206-H04
19.0 RTSQFVNSNYLA GASSRAT QQSSRTPWT SYMMV SISPSGGYTIqADSVKG
VARGIAARSRTSYFDY
TABLE-US-00018 Amino acid sequences of light chain (LC) and heavy
chain (HC) variable domain of pKal antibodies obtained from CDR1/2
and CDR3 spiking affinity maturation libraries based on X63-G06.
(SEQ ID NOs: 1726-1849) M203-A01 LC QDIQMTQSPG TLSLSPGERA
TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60 PDRFSGTGYG
TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M203-A01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYLMAWVRQA PGKGLEWVSW IVPSGGYTEY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS
YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M203-A03 LC QDIQMTQSPG
TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60
PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M203-A03
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYMMIWVRQA PGKGLEWVSS
ISPSGGQTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M204-A02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M204-A02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYMMTWVRQA PGKGLqWVSY
ISPSGGLTSY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M204-E12 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M204-E12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYMMqWVRQA PGKGLEWVSY
IGPSGGKTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-A02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-A02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYLMAWVRQA PGKGLEWVSG
IVSSGGRTLY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-A12 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-A12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYDMIWVRQA PGKGLEWVSY
ISSSGGFTRY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-B04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-B04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYDMIWVRQA PGKGLEWVSS
ISSSGGTTKY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-C11 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-C11 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS QYMMMWVRQA PGKGLEWVSR
ISPSGGSTLY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-D04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-D04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS TYKMqWVRQA PGKGLEWVSS
ISPSGGPTNY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-E11 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-E11 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYTMGWVRQA PGKGLEWVSS
ISPSGGKTDY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-F03 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-F03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS SqQMVWVRQA PGKGLEWVSY
ISPSGGNTYY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-H01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-H01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYTMQWVRQA PGKGLqWVSY
ISPSGGYTGY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M205-H08 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M205-H08 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYMMMWVRQA PGKGLEWVSS
IVPSGGHTqY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-A06 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-A06 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYMMGWVRQA PGKGLqWVSS
ISPSGGLTKY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-B10 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-B10 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS qYLMAWVRQA PGKGLEWVSS
IYPSGGWTKY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-C03 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-C03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS qYMMVWVRQA PGKGLEWVSS
IYSSGGNTPY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-E02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-E02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS EYMMMWVRQA PGKGLEWVSV
ISPSGGQTHY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-F01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-F01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYMMVWVRQA PGKGLEWVSR
ISPSGGPTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-F09 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-F09 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS VYMMSWVRQA PGKGLEWVSS
IVPSGGSTTY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-H01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-H01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS EYMMVWVRQA PGKGLEWVSR
ISPSGGTTEY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-H04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60
PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-H04
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYMMVWVRQA PGKGLEWVSS
ISPSGGYTIq 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-H05 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-H05 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS GYKMqWVRQA PGKGLEWVSS
ISPSGGITMY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M206-H08 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M206-H08 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS DYMMAWVRQA PGKGLEWVSS
IVPSGGHTHY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M207-A01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M207-A01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGISARSRTS CFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M207-A02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M207-A02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TALYYCARVA
RGIAARSRTI NLDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M207-A04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M207-A04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTR SFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M207-C05 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
R0121-D02 = M0207-C05 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS
HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TAVYYCARVG RGIAARSRTS YFDKWGQGTL 120 VTVSSASTKG
PSVFPLAPSS KS 142 M208-A10 LC QDIQMTQSPG TLSLSPGERA TLSCRTSQFV
NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60 PDRFSGTGYG TDFTLTISRL
EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-A10 HC EVQLLESGGG
LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS qFDHWGQGTL
120 VTVSSASTKG PSVFPLAPSS KS 142 M208-B01 LC QDIQMTQSPG TLSLSPGERA
TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60 PDRFSGTGYG
TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-B01 HC
EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY
60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS
FFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-C06 LC QDIQMTQSPG
TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60
PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-C06
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS FIDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-D12 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-D12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLA
RGIAARSRTS YqDIWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-E10 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-E10 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
QGISARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-F04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-F04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS FFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-F10 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-F10 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-G02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-G02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIADRSRTS YSDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-G03 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-G03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YLDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M208-H02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M208-H02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIASRSRTR YCDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-B01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-B01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIVARSRTS NFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-B09 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-B09 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
QGIVARSRTS YLHqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-B11 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-B11 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTR YIDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-C02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-C02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAYRRRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-C11 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60
PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-C11
HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAMVG
QGIRGRSRTS YFAqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-C12 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-C12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVG
RGIAARSRTS qLDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M0209-D02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-D02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGITARSRTS YFDDWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-D03 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-D03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVG
RGIAARSRTS FFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-D12 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-D12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATVA
RGIAARSRTS YFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-E02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-E02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTI LLDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-E03 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-E03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARqA
RGIAARSRTS YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-F04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-F04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YLDqWSQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-G01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-G01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
qGISGRSRLS YVDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-G07 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-G07 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YFDTWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-H03 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-H03 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
QGIAARSRTT qFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-H07 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-H07 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARqRTS YYDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M209-H09 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M209-H09 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTV YFDHWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-A06 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-A06 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS qFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-B02 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-B02 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASVA
RGIAARSRTS YFNqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-C12 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-C12 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
QGIAARSRTS SVDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-G04 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-G04 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
TGIVARSRTR YFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-G10 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-G10 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTS YLDFWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-H01 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-H01 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRNS qQDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-H06 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-H06 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTR YFDYWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142 M210-H07 LC
QDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI
60 PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109
M210-H07 HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY
ISPSGGHTIY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA
RGIAARSRTR YFDqWGQGTL 120 VTVSSASTKG PSVFPLAPSS KS 142
Example 6: In Vivo Testing of M162-A04 (IgG) and X101-A01
[0883] Bradykinin and other bioactive kinins have been previously
implicated in carrageenan-induced edema and inflammatory pain
(Sharma J. N. et al. (1998) Inflammopharmacology 6, 9-17; Asano M.
et al. (1997) Br J Pharmacol 122, 1436-1440; De Campos R. O. et al.
(1996) Eur J Pharmacol 316, 277-286). Plasma kallikrein and tissue
kallikrein 1 are the two primary kininogenases in mammals (Schmaier
A. H. (2008) Int Immunopharmacol 8, 161-165). M162-A04 (M162-A4)
(IgG), a specific plasma kallikrein inhibitor, was tested to
determine whether it would be effective in carrageenan induced
edema. The study design is outlined in Table 10. The route of
administration (ROA) for the vehicle (PBS), the antibody, and the
positive control (indomethacin) was intra-peritoneal (IP) and was
given 30 minutes prior to carrageenan injection (0.1 mL of a 2%
carrageenan solution). It is evident from FIG. 2 that antibody
doses at 10 mg/kg and above were equally effective in reducing
carrageenan-induced edema as the positive control (indomethacin).
However, the antibody was not effective in reducing
carrageenan-induced thermal hyperalgesia (FIG. 3). The reason for
the dissociation between effectiveness in edema and hyperalgesia
are not obvious but may be due to differences in the bioactivity of
different kinin metabolites. Lys-desArg9-bradykinin is the most
potent agonist of the B1 receptor, which is believed to be
primarily involved in pain hypersensitivity (Leeb-Lundberg L. M et
al. (2005) Pharmacol Rev 57, 27-77). This kinin metabolite is
generated by tissue kallikrein 1, not plasma kallikrein (Schmaier
A. H. (2008) Int Immunopharmacol 8, 161-165). This difference in
kinin generation and resulting bradykinin receptor activation may
account for the unexpected decoupling of edema and hyperalgesia in
this model.
[0884] Another pKal antibody inhibitor X101-A01 was also tested in
the CPE model using the study design shown in Table 10B. The data
obtained in FIG. 14 shows that X101-A01 inhibited edema in a
dose-dependent manner to an extent that is comparable to that of
the positive control (indomethacin).
TABLE-US-00019 TABLE 10A Carrageenan-Induced Paw Edema Study Design
to test M162-A04 Dose Timing Dose Group Number (mg/ relative to
Volume # of Rats Treatment kg) ROA carrageenan (mL/kg) 1 6 Vehicle
N/A IP T -30 20 minutes 2 6 559A- 3 IP T -30 20 M162-A4 minutes 3 6
559A- 10 IP T -30 20 M162-A4 minutes 4 6 559A- 30 IP T -30 20
M162-A4 minutes 5 6 Indomethacin 5 IP T -30 20 minutes
TABLE-US-00020 TABLE 10B Carrageenan-Induced Paw Edema Study Design
to Test X101- Dose Vol Group Treatment n (mg/Kg) ROA Timing *
(mL/Kg) 1 Vehicle 10 N/A IP -30 min 20 2 X101-A01 10 1 IP -30 min
20 3 X101-A01 10 3 IP -30 min 20 4 X101-A01 10 10 IP -30 min 20 5
X101-A01 10 30 IP -30 min 20 6 Indomethacin 10 5 IP -30 min 20
Example 7: Evaluation of Selected Antibody Inhibitors of Plasma
Kallikrein
[0885] Evaluation of selected optimized antibodies (X81-B01 and
X67-D03) is shown in Table 11. Neither antibody has any putative
deamidation, isomerization, or oxidation sites.
TABLE-US-00021 TABLE 11 Criteria X81-B01 (IgG) X67-D03 (IgG) <
nM Ki, app against human pKal 0.2 nM 0.1 nM < nM Ki, app against
rodent pKal mouse - 11 pM mouse - 0.7 nM rat - 0.14 nM rat - 0.34
nM prekallikrein binding no no Specific inhibitor with respect yes
yes to fXIa, plasmin, and trypsin Inhibits bradykinin generation
yes yes Inhibits pKal in presence of yes yes prekallikrein
Competition for binding with yes yes aprotinin Stability in human
serum yes nd* *not done; a parental form of this antibody was shown
to be stable in serum
Example 8: Epitope Mapping
[0886] The region of pKal bound by selected anti-pKal antibodies
was investigated using several methods. First, competition assays
were used to determine whether the antibodies competed for binding
to pKal with known active site-directed inhibitors. Second,
antibodies were grouped according to whether they were inhibitors
or just binders to pKal. Third, epitopes were investigated using
synthetic peptides and peptidic structures based on the sequence
and 3-dimensional structure of pKal. These peptidic structures are
called "CLIPS" (Chemically Linked Peptides on Scaffolds) and the
testing was performed by a fee for service company called
Pepscan.
[0887] Fourth, antibodies were tested for their ability to inhibit
pKal from other species, besides human, where the amino acid
sequence of pKal has been determined in order to identify amino
acids that may account for the differences in inhibition.
[0888] Competition Assays
[0889] Using a BIACORE.RTM. SPR assay antibodies of interest were
tested for competition with a known active site inhibitor of pKal.
EPI-KAL2 is potent (K.sub.i,app=0.1 nM) active site inhibitor of
pKal and a Kunitz domain inhibitor based on the first domain of
tissue factor pathway inhibitor (Markland (1996) Iterative
optimization of high-affinity protease inhibitors using phage
display. 2. Plasma kallikrein and thrombin. Biochemistry.
35(24):8058-67). Kunitz domains are known active site inhibitors of
serine proteases, such as pKal.
[0890] The sequence of EPI-KAL2 is:
TABLE-US-00022 (SEQ ID NO: 1850)
EAMHSFCAFKADDGPCRAAHPRWFFNIFTRQCEEFSYGGCGGNQNRFESL EECKKMCTRD
(amino acids in italics are those that differ from TFPI)
[0891] As shown in FIGS. 8A-8B, the antibodies X81-B01 and X67-D03
were competed for binding to pKal in the presence or EPI-KAL2. This
result indicates that these antibodies either bind in vicinity of
the active site or allosteric changes in the conformation of the
pKal-EPI-KAL2 complex prevent antibody binding.
[0892] Antibody Binders Vs Inhibitors
[0893] As shown in Tables 1 and 2, all the unique antibodies
discovered by phage display were characterized as being either pKal
inhibitors or binders but not inhibitors. Antibodies that inhibit
the activity of pKal either bind near the active site and preclude
substrate interactions (competitive inhibitors) or that bind away
from the active site and induce allosteric changes in the structure
of the active site (noncompetitive inhibitors). Antibodies that
bind but do not inhibit pKal are unlikely to bind near the active
site and may bind the non-catalytic domain (i.e. the apple domain).
Table 12 categorizes selected antibodies as being either inhibitors
or binders of pKal. Also shown in Table 12 for the listed
antibodies, is a demonstration of whether they cross-react with
mouse pKal as inhibitors and whether they bind prekallikrein.
TABLE-US-00023 TABLE 12 Binding Properties of Selected Anti-pKal
Antibodies human mouse CLIPS Ki, app Ki, app Peptide(s) Number
Antibody Binding Category (nM) (nM) Identified 1 M6-A06 1) Binder
only no no C4 2 M6-D09 2) inhibitor, prekallikrein 5.9 3.9 C1, C5
binder, inhibits mouse and human pKal 3 M8-C04 1) Binder only no no
4 M8-G09 1) Binder only no no C1, C4, C6, C7 5 M29-D09 3)
inhibitor, does not bind 0.7 no C1, C4, prekallikrein, does not C7
inhibit mouse pKal 6 M35-G04 2) inhibitor, prekallikrein 2.9 8 C1,
C4 binder, inhibits mouse and human pKal 7 M145-D11 3) inhibitor,
does not bind 0.79 800 C1, C4 prekallikrein, weak inhibitor of
mouse pKal 8 M160-G12 4) inhibitor of both mouse 5 0.2 C2 and human
pKal, does not bind prekallikrein 9 X55-F01 4) inhibitor of both
0.4 2 C2, C3 mouse and human pKal, does not bind prekallikrein 10
X73-H09 4) inhibitor, does not bind 20 70 C6 prekallikrein, weak
inhibitor of human and mouse pKal 11 X81-B01 4) inhibitor of both
mouse 0.1 0.011 C2, C3, and human pKal, does not C5, C6 bind
prekallikrein 12 A2 5) Negative control, does No binding No binding
No not bind pKal, binds binding streptavidin C1-C7: peptides in
pKal identified by CLIPS epitope mapping (see FIGS. 9 and 10A-10C).
C1 corresponds to positions 55-67 of the catalytic domain, C2 to
positions 81-94, C3 to positions 101-108, C4 to positions 137-151,
C5 to positions 162-178, C6 to positions 186-197, and C7 to
positions 214-217.
[0894] Epitope Mapping Using CLIPS
[0895] The 11 anti-pKal antibodies listed in Table 12, plus one
negative control (A2) were tested for binding to 5000 different
synthetic CLIPS (Chemically Linked Peptides on Scaffolds) by
Pepscan as described below in the CLIP METHODS sections. This
analysis led to the identification of peptide regions in pKal that
are likely to be a part of the antibody epitope for each of the
tested antibodies (FIG. 9).
[0896] Clips Methods
[0897] The linear and CLIPS peptides were synthesized based on the
amino acid sequence of the target protein using standard
Fmoc-chemistry and deprotected using trifluoric acid with
scavengers. The constrained peptides were synthesized on chemical
scaffolds in order to reconstruct conformational epitopes, using
Chemically Linked Peptides on Scaffolds (CLIPS) technology
(Timmerman et al. (2007). For example, the single looped peptides
were synthesized containing a dicysteine, which was cyclized by
treating with alpha, alpha'-dibromoxylene and the size of the loop
was varied by introducing cysteine residues at variable spacing. If
other cysteines besides the newly introduced cysteines were
present, they were replaced by alanine. The side-chains of the
multiple cysteines in the peptides were coupled to CLIPS templates
by reacting onto credit-card format polypropylene PEPSCAN cards
(455 peptide formats/card) with a 0.5 mM solution of CLIPS template
such as 1,3-bis (bromomethyl) benzene in ammonium bicarbonate (20
mM, pH 7.9)/acetonitrile (1:1(v/v)). The cards were gently shaken
in the solution for 30 to 60 minutes while completely covered in
solution. Finally, the cards were washed extensively with excess of
H.sub.2O and sonicated in disrupt-buffer containing 1 percent
SDS/0.1 percent beta-mercaptoethanol in PBS (pH 7.2) at 70.degree.
C. for 30 minutes, followed by sonication in H.sub.2O for another
45 minutes. The binding of antibody to each peptide were tested in
a PEPSCAN-based ELISA. The 455-well credit card format
polypropylene cards containing the covalently linked peptides were
incubated with primary antibody solution for example consisting of
1 micrograms/mL diluted in blocking solution called SQ (4% horse
serum, 5% ovalbumin (w/v) in PBS/1% Tween or diluted in PBS e.g.,
20% SQ) overnight. After washing, the peptides were incubated with
a 1/1000 dilution of rabbit anti-human antibody peroxidase or
goat-anti-human FAB peroxidase for one hour at 25.degree. C. After
washing, the peroxidase substrate
2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2
microlitres of 3 percent H.sub.2O.sub.2 were added. After one hour,
the color development was measured. The color development was
quantified with a charge coupled device (CCD)--camera and an image
processing system (as firstly described in Slootstra et al.,
1996).
[0898] Data Calculation
[0899] Raw Data: Optical Density (Arbitrary OD Units)
[0900] The raw data are optical values obtained by a CCD-camera.
The values mostly range from 0 to 3000, a log scale similar to 1 to
3 of a standard 96-well plate elisa-reader. First the CCD-camera
makes a picture of the card before peroxidase coloring and then
again a picture after the peroxidase coloring. These two pictures
are subtracted from each other which results in the data which is
called raw-data. This is copied into the Peplab.TM. database. Then
the values are copied to excel and this file is labeled as raw-data
file. One follow-up manipulation is allowed. Sometimes a well
contains an air-bubble resulting in a false-positive value, the
cards are manually inspected and any values caused by an air-bubble
are scored as 0.
[0901] Normally assays are not done in replicate (only upon request
client request). Replicate tests are usually very similar. In
addition, the dataset of thousands of peptides contains many
peptides that are similar, thus results are never based on
recognition of one peptide but on families of similar peptides. If
one or a few peptides do not bind, or exhibit lower binding, in a
replicate experiment, a different epitope mapping is not normally
attributed.
[0902] Timmerman et al. (2007). Functional reconstruction and
synthetic mimicry of a conformational epitope using CLIPS.TM.
technology. J. Mol. Recognit. 20:283-99
[0903] Slootstra et al. (1996). Structural aspects of
antibody-antigen interaction revealed through small random peptide
libraries, Molecular Diversity, 1, 87-96.
Example 9: Analysis of pKal Sequences from Different Species
[0904] All available sequence of pKal were obtained from public
databases and aligned using ClustalW and regions were highlighted
based on solvent accessibility, contact with an active site Kunitz
inhibitor, and those peptides identified by CLIPS analysis (FIGS.
10A-10C). Citrated plasma from each of these species was obtained
and activated using a commercially available prekallikrein
activator (from Enzyme Research Laboratories) according to the
instructions of the manufacturer. Kallikrein activity was then
measured in each of the samples in the presence or absence of
X81-B01.
[0905] It was found that X81-B01 inhibited pKal from all the
species except for pig pKal. Since the CLIPS analysis identified
four peptides of pKal that X81-B01 binds to--C2 (positions 81-94),
C3 (positions 101-108), C5 (positions 162-178) and C6 (positions
186-197)--differences in the pig pKal sequence that correspond to
these peptides were examined to identify potential amino acids
changes that account for the lack of inhibition of pig pKal by
X81-B01. Peptides C2 and C3 are close in the sequence and are both
highly similar in sequence among the different species. However,
there is a difference at position 479. All the species except pig,
frog, and dog have a serine at position 479. The frog and dog pKal
sequence has an alanine and a threonine at position 479,
respectively; both of which are considered conservative
substitutions for a serine. In contrast, the pig pKal sequence has
a leucine at position 479, which is a considerably less
conservative substitution for a serine. Peptide C5 in pig pKal is
highly similar to the sequences from the other species. However, at
position 563, only in the pig pKal is a histidine present (bold in
FIG. 10C). This position in all the other species, except frog, is
a tyrosine. In the frog pKal, which is inhibited by X81-B01, this
position is a threonine. Peptide C6 in pig pKal is again highly
similar to the other sequences. However, only in the pig pKal
sequence is position 585 a glutamate (in bold in FIG. 10C). In all
the other species this position is an aspartate. This analysis may
indicate potentially critical residues in pKal that interact with
X81-B01.
Example 10: In Vitro and In Vivo Assays to Assess Efficacy of a
Plasma Kallikrein Binding Protein
[0906] Binding to Prekallikrein Vs. Kallikrein:
[0907] The advantage of an antibody inhibitor of pKal that does not
bind prekallikrein over an antibody that binds prekallikrein can be
demonstrated experimentally. For example, an in vitro experiment
can be designed to compare the potency of a pKal antibody inhibitor
that does not bind prekallikrein (e.g. DX-2922) with one that binds
prekallikrein (e.g. M6-D09) using an activated partial
thromboplastin time (APTT) plasma clotting time assay. The APTT
assay induces clotting in plasma by the addition of a reagent that
specifically activates the contact system component of the
intrinsic coagulation pathway, of which the activity of pKal is
involved. It is well known in the literature that the inhibition of
pKal or that a genetic deficiency in pKal leads to prolonged aPPT
(see e.g., Morishita, H., et al., Thromb Res, 1994. 73(3-4): p.
193-204; Wynne Jones, D., et al., Br J Haematol, 2004. 127(2): p.
220-3). An in vitro experiment can be performed to measure the
effect of spiking citrated human plasma with different
concentrations of either M6-D09 or DX-2922 on observed clotting
times induced using commercially available APTT reagents and a
coagulation analyzer (Table 13). It is expected that the observed
EC50 for APTT prolongation of M6-D09 will be significantly higher
than that of DX-2922 due to the binding of M6-D09 to the high
concentration prekallikrein (.about.500 nM) in the normal plasma
sample. Efficacy of the antibody inhibitor of pKal as demonstrated
by prolonging APTT supports the potential therapeutic use of the
antibody in treating or preventing cardiovascular disease
associated with aberrant clot formation, such as may be observed in
atherosclerosis, stroke, vasculitis, aneurism, and patients
implanted with ventricular assist devices.
TABLE-US-00024 TABLE 13 Study Design to Measure Effect of Antibody
Inhibitors of pKal on APTT Condition Observed Effect on APPT No
treatment, just plasma Normal Prekallikrein depleted plasma Maximum
prolongation control (commercially available) M6-D09 at low
concentration Normal M6-D09 at middle concentration Normal M6-D09
at high concentration Prolonged APTT DX-2922 at low concentration
Prolonged APTT DX-2922 at middle concentration Prolonged APTT
DX-2922 at high concentration Maximum prolongation
[0908] Efficacy in a Rat Model of Edema:
[0909] An in vivo experiment can also be conducted to demonstrate
the increased potency of an antibody inhibitor of pKal that does
not bind prekallikrein. The carrageenan-induced paw edema (CPE)
model of edema in rats is a common pharmacology model. A group of
rats will be treated with escalating doses of M6-D09 and DX-2922 by
intraperitoneal (IP) injection prior to injecting carrageenan (e.g.
0.1 mL of a 10% w/v solution) in the paws of the rats (Table 14).
It is expected that DX-2922 will be more effective in reducing the
observed paw swelling than M6-D09. Efficacy of the antibody
supports the therapeutic use of the antibody in various
inflammatory diseases that are associated with either swelling
(e.g. hereditary angioedema, stroke induced edema, brain edema) or
bradykinin mediated inflammation and pain (e.g. rheumatoid
arthritis, inflammatory bowel disease).
TABLE-US-00025 TABLE 14 Study Design to Observe Effect of Antibody
Inhibitors on CPE Example Dose Group Treatment (mg/Kg) Effect
Expected 1 Vehicle N/A Maximum swelling 2 Indomethacin 5 Maximum
reduction of (positive control) swelling 3 M6-D09 1 No effect on
swelling 4 M6-D09 3 No effect on swelling 5 M6-D09 10 Intermediate
reduction of swelling 6 DX-2922 1 No effect on swelling 7 DX-2922 3
Intermediate reduction of swelling 8 DX-2922 10 Maximum reduction
of swelling
[0910] Measuring Half-Life:
[0911] The pharmacokinetic properties of DX-2922 will be determined
in rats and cynomolgus monkeys in a study design outlined in Table
15. Serum will be collected at the times indicated following IV
injection of DX-2922. The concentration of DX-2922 will be
determined by ELISA and plotted versus time in order to obtain
pharmacokinetic parameters (clearance, half life, volume of
distribution, etc). It is expected that DX-2922 will have a
half-life greater than 5 days in cynomolgus monkeys that will scale
to a half life greater than 7 days in humans.
TABLE-US-00026 TABLE 15 A Pharmacokinetic study of plasma
kallikrein inhibitor pKal administered by intravenous and
subcutaneous routes to cynomolgus monkeys. Dosage Number of Treat-
Level Animals Group ment (mg/kg/day) Route (sex) Serum Collection 1
pKAL 20 IV 4 (M) Pre-dose 0.08, 0.25. 0.5, 1, 4, 24 hours Day 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 2 pKAL 20 IV 4 (M) Pre-dose
0.08, 0.25. 0.5, 1, 4, 24 hours Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14
Example 11: Epitope Mapping Using Amino Acid Mutations of pKal
[0912] Based on the epitope mapping studies described herein in
Example 8, we inspected the published 3 dimensional model in the
RCSB Protein Data Bank (available on the world wide web at
rcsb.org; pdb code 2ANY) and identified a collection of sets of
amino acids in surface accessible loops near the enzyme active site
that we reasoned could interact with the antibody binding resulting
in enzyme inhibition. These amino acids were substituted for
alanine and the catalytic domain of each of the mutant was
expressed in Pichia pastoris with a His tag fusion and purified by
IMAC. Four different mutant pKal mutants were synthesized and
tested:
[0913] Mutant 1: Amino acids S478, N481, S525, and K526 of the
human kallikrein sequence (Accession No. NP_00883.2) were mutated
to alanine. These amino acids were determined to be involved in
substrate recognition (S3 subsite).
[0914] Mutant 2: Amino acid residues R551, Q553, Y555, T558 and
R560 of the human kallikrein sequence (Accession No. NP_00883.2)
were mutated to alanine. It was determined that these residues are
involved in the active site substrate recognition (S1'
subsite).
[0915] Mutant 3: Amino acids D572, K575, and D577 of the human
kallikrein sequence (Accession No. NP_00883.2) were mutated to
alanine. These amino acid residues are involved in substrate
recognition (S1' subsite)
[0916] Mutant 4: Amino acids N395, S397 and S398 of the human
kallikrein sequence (Accession No. NP_00883.2) were mutated to
alanine. These residues are distal from the active site of plasma
kallikrein.
[0917] Three of the 4 mutants (Mutant 1, 2, and 4) have similar
activity to that of the wildtype catalytic domain of pKal. The
amino acid substitutions in Mutant 3 yielded an inactive protein
that was not recognized in SPR (Biacore) binding assays by any of
the tested anti-pKal antibodies.
[0918] The antibodies tested for inhibition of mutants 1, 2 and 4
are shown herein in Table 16. Based on the measured K.sub.i,app
values for the antibodies in Group 1 (i.e., antibodies that inhibit
human and mouse pKal but do not bind prekallikrein) it is evident
that this group of antibodies binds an epitope on pKal that
contains the amino acids that were mutated in Mutant 2 but were not
dependent on residues mutated in Mutants 1 or 4. In addition, the
interaction of plasma kallikrein binding proteins
X81-B01/X101-A01/DX-2922 and affinity matured derivative X115-B07
to kallikrein is adversely affected by the substitutions in Mutant
1. For an example of the differences in the ability of the
antibodies to bind prekallikrein see e.g., FIGS. 11A and 11B, which
compares prekallikrein the binding of DX-2922 (Group 1) to that of
M6-D09 (Group 3).
[0919] The antibodies in Group 2 (i.e., those that inhibit human
pKal not mouse pkal and do not bind prekallikrein) were not
significantly affected by the mutated amino acids indicating that
they make contact with alternate amino acids. The Group 2
antibodies are likely to bind near the active site, as they were
unable to bind pKal complexed with a Kunitz domain (EPI-KAL2),
which is known to bind at the active site of a serine protease.
Furthermore, one of the antibodies in Group 2 (M145-D11) is similar
to those in Group 1 in that it is unable to bind pKal in a Biacore
assay that is inactivated with the suicide inhibitor AEBSF
(4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), which is
a small molecule covalent inhibitor of trypsin-like serine
proteases (FIG. 12). However, the other antibody (M29-D09) assigned
to Group 2 was able to bind AEBSF inactivated pKal, indicating that
it may bind a different epitope than M145-D11 despite sharing
similar binding properties.
[0920] The antibodies in Group 3 inhibited human and mouse pKal but
bound prekallikrein. One of these antibodies, M6-D09, was unable to
bind pKal inactivated by either EPI-KAL2 or AEBSF, indicating that
this group of pKal inhibitors interacts with alternative amino
acids near the active site. The K.sub.i,app for M6-D09 increased
approximately 5-fold towards Mutant 2 (i.e., decreased potency of
M6-D09).
Example 12: Affinity Maturation
[0921] In addition to the affinity maturation described herein in
Examples 4 and 5, which involved optimization of the light chain we
attempted to further optimize affinity with libraries that vary
amino acids in the CDR1, CDR2, and CDR3 regions of the variable
heavy chain of two different parental anti-pKal antibodies. Both of
the antibodies selected for further optimization (X63-G06 and
M162-A04) exhibit desirable properties for further development as a
therapeutic antibody inhibitor of plasma kallikrein; properties
which include: a) complete inhibition of human and rodent plasma
kallikrein and b) no binding to prekallikrein. In some embodiments,
complete inhibition of human pKal is essential to block the
activity of plasma kallikrein in disease uses. Inhibition of rodent
pKal facilitates preclinical development including toxicity
assessment. The lack of binding to prekallikrein is a highly
desirable property for an antibody inhibitor of pKal to maximize
the bioavailability of the antibody therapeutic towards active pKal
target and to potentially reduce the dose required for
efficacy.
[0922] Affinity maturation was performed using 4 different phage
display libraries. For each parental antibody (e.g., I62-A04), a
library was constructed that contained varied amino acid positions
in both the CDR1 and the CDR2 of the heavy chain. An additional
library was constructed for each of the two parental antibodies
wherein positions in the CDR3 of the heavy chain were varied. Each
of these 4 phage display libraries were selected (panned) with
decreasing amounts of active pKal in each subsequent round in order
to obtain high affinity antibodies. To minimize the appearance of
prekallikrein binding in the selected antibody output libraries
were initially depleted against immobilized prekallikrein. After
screening as Fab fragments, we discovered the affinity matured
antibodies shown in Table 16 (i.e. the antibodies with the
identification number starting with "X115").
[0923] Four discovered antibodies (X115-B07, X115-D05, X115-E09,
and X115-H06) are derived from the DX-2922 parental antibody (also
known as X63-G06 as a Fab fragment, X81-B01 as an IgG produced in
293T cells, or X101-A01 as an IgG produced in CHO cells) were found
to be potent pKal inhibitors. For comparison the amino acid
sequence of DX-2922 is shown. It is evident that three of the
affinity matured antibodies (X115-B07, X115-E09, and X115-H06)
contain mutations in Hv-CDR3; whereas X115-D05 has a different
Hv-CDR1/CDR2. Four other discovered antibodies (X115-F02, X115-A03,
X115-D01, and X115-G04) are derived from the M162-A04 parental
antibody. All 8 affinity matured antibodies do not bind
prekallikrein.
TABLE-US-00027 TABLE 16 Summary of Affinity Matured Anti-pKal
Antibodies Inhibition Constants (Ki, app) on Wild Type pKal
Catalytic Domain and Mutants 1, 2, and 4.sup.a. WT cat. Mutant
Mutant Domain 1 Ki, Mutant 2 4 Ki, Competes Competes Ki, app app
Ki, app app with with epi- Isolate (nM) (nM) (nM) (nM) AEBSF kal2
Characteristics DX-2922 0.22 14 20 0.25 y y inhibits human and
mouse pKal; does not bind pre-kallikrein 559A-X115-B07 0.13 4.7 47
0.14 y nd inhibits human and mouse pKal; (aff mat; X101-A01 does
not bind pre-kallikrein parent) 559A-X115-D05 nd nd nd nd y nd
inhibits human and mouse pKal; (aff mat; X101-A01 does not bind
pre-kallikrein parent) 559A-X115-E09 nd nd nd nd y nd inhibits
human and mouse pKal; (aff mat; X101-A01 does not bind
pre-kallikrein parent) 559A-X115-H06 nd nd nd nd y nd inhibits
human and mouse pKal; (aff mat; X101-A01 does not bind
pre-kallikrein parent) 559A-X115-A03 0.16 0.23 3.7 0.13 y nd
inhibits human and mouse pKal; (aff mat; M162-A04 does not bind
pre-kallikrein parent) 559A-X115-D01 0.18 0.26 2.5 0.12 y nd
inhibits human and mouse pKal; (aff mat; M162-A04 does not bind
pre-kallikrein parent) 559A-X115-F02 0.09 0.14 5.9 0.1 y y inhibits
human and mouse pKal; (aff mat; M162-A04 does not bind
pre-kallikrein parent) 559A-X115-G04 0.3 0.4 2.2 0.3 y y inhibits
human and mouse pKal; (aff mat; M162-A04 does not bind
pre-kallikrein parent) 559A-M29-D09 (sFab)) 0.24 0.27 0.34 0.39 nd
y inhibits human and mouse pKal; does not inhibit mouse pKal; does
not bind pre-kallikrein 559A-M145-D11 (sFab) 0.16 0.23 0.1 0.21 y y
inhibits human and mouse pKal; weakly inhibits mouse pKal; does not
bind pre-kallikrein 559A-M06-D09 2.5 3.4 13.5 2.9 y y inhibits
human and mouse pKal; binds pre-kallikrein 559A-M35-G04 0.8 0.09
1.1 0.8 nd nd inhibits human and mouse pKal; binds pre-kallikrein
.sup.aAntibodies were obtained from HV-CDR1/2 and HV-CDR3 affinity
maturation, purified and tested for inhibition of either wild type
pKal catalytic domain (Note, the antibodies inhibited full length
wild type pKal approximately equal to that of the wild type
catalytic domain).
TABLE-US-00028 TABLE 17 (SEQ ID NOs: 1851-1934 numbered down the
columns starting on the left): Full length pKal Ki, app Isolate
LV-CDR1 LV-CDR2 LV-CDR3 HV-CDR1 HV-CDR2 HV-CDR3 (nM) DX-2922
RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VARGIAARSRTSYFDY 0.2 X115-B07 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VGQGIRGRSRTSYFAQ 0.33 X115-D05 RTSQFVNSNYLA
GASSRAT QQSSRTPWT DYMMA SIVPSGGHTHYADSVKG VARGIAARSRTSYFDY 0.25
X115-E09 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG
VAQGIAARSRTSSVDQ 0.34 X115-H06 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT
YISPSGGHTIYADSVKG VAQGISARSRTSYFDY 0.35 M162-A04 RASQSISSWLA
KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDAFDI X115-A03
RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRIGVPRRDSFDM
0.16 X115-D01 RASQSISSWLA KASTLES QQYNTYWT IYSMH SIYPSRGMTWYADSVKG
RRTGIPRRDAFDI 0.18 X115-F02 RASQSISSWLA KASTLES QQYNTYWT HYIMM
GIYSSGGITVYADSVKG RRIGVPRRDEFDI 0.089 X115-G04 RASQSISSWLA KASTLES
QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDEFDI 0.6 M29-D09
SGNKLGDKYVA QDTKRPS QAWDSSIVI WYTMV YIYPSGGATFYADSVKG GSYDYIWGFYSDH
0.7 M145-D11 SGDKLGDKYTS QDIKRPS QAWDSPNARV HYRMS SIYPSGGRTVYADSVKG
DKFEWRLLFRGIGNDAFDI 0.79 M06-D09 RASQSIRNYLN AASTLQS QQLSGYPHT
FYYMV VIYPSGGITVYADSVKG DKWAVMPPYYYYAMDV 5.9 M35-G04 RASQSVSSYLA
DASNRAT QQRSNWPRGFT YYHMS VISPSGGSTKYADSVKG GGSSDYAWGSYRRPYYFDY 2.9
WT cat. Mutant Mutant Mutant Domain 1 2 4 Ki, app Ki, app Ki, app
Ki, app competes with competes Isolate (nM) (nM) (nM) (nM) AEBSF
with epi-kal2 Group DX-2922 0.22 14 20 0.25 y y 1 X115-B07 4.7 47
0.14 y nd 1 X115-D05 nd nd nd nd y nd 1 X115-E09 nd nd nd nd y nd 1
X115-H06 nd nd nd nd y nd 1 M162-A04 nd nd nd nd y y 1 X115-A03
0.16 0.23 3.7 0.13 y nd 1 X115-D01 0.18 0.26 2.5 0.12 y nd 1
X115-F02 0.09 0.14 5.9 0.1 y y 1 X115-G04 0.3 0.4 2.2 0.3 y y 1
M29-D09 0.24 0.27 0.34 0.39 n y 2 M145-D11 0.16 0.23 0.1 0.21 y y 2
M06-D09 2.5 3.4 13.5 2.9 y y 3 M35-G04 0.8 0.09 1.1 0.8 nd nd 3
[0924] Equilibrium K.sub.i,app Measurements.
[0925] Apparent Inhibition constants (K.sub.i,app values) were
measured by pre-incubating enzyme and inhibitor solutions prior to
initiating the reactions with substrate. Enzyme and inhibitor were
pre-incubated for 2 hours at 30.degree. C. in a 96-well plate by
adding 10 .mu.L of a 10.times. enzyme solution and 10 .mu.L of
10.times. inhibitor solutions to 70 .mu.L of reaction buffer.
Reactions were initiated by the addition of 10 .mu.L of a 10.times.
concentrated stock of substrate, and were monitored at 30.degree.
C. in a fluorescence plate reader with the excitation and emission
wavelengths set at 360 nm/460 nm, respectively. Kinetic data were
acquired by the increase in fluorescence, and initial rates for
each condition were plotted against the total inhibitor
concentration. The data was fit to the following equation for tight
binding inhibitors:
A = A 0 - A inh ( ( K i , app + Inh + E ) - ( K i , app + Inh + E )
2 - 4 Inh E 2 E ) Eqn . 1 ##EQU00002##
[0926] Where A=initial rate observed at each inhibitor
concentration; A.sub.o=initial rate observed in the absence of
inhibitor; A.sub.inh=initial rate observed for the enzyme inhibitor
complex; Inh=concentration of inhibitor; E=total enzyme
concentration (treated as a floated parameter); and
K.sub.i,app=apparent equilibrium inhibition constant.
[0927] Groups of Antibody Inhibitors.
[0928] Antibodies in Group 1 inhibit human and mouse pKal but do
not bind prekallikrein. Antibodies in Group 2 inhibit human but not
mouse pKal and do not bind prekallikrein. Antibodies in Group 3
inhibit human and mouse pKal but bind prekallikrein.
[0929] Biacore Competition Analysis with an Exemplary Kallikrein
Antibody, Epi-Kal2.
[0930] Epi-Kal2 is an antibody inhibitor of kallikrein that acts by
binding to the active site of kallikrein (for sequence see Example
8). The Biacore competition analysis is used herein as an assay to
determine whether a test kallikrein antibody binds to the same site
as epi-Kal2 and is assessed by measuring the competition (e.g.,
displacement) between epi-Kal2 and the test antibody for binding to
the active site.
[0931] Goat anti-human Fc fragment specific IgG or anti-human Fab
IgG was immobilized by amine coupling on a CM5 sensor chip at
immobilization densities of approximately 5000 RU. Anti-pKal
antibodies or sFabs were captured on their respective surfaces by
injecting a 50 nM solution of IgG/sFab for 1-2 minutes at 5 at
.mu.l/min. Human pKal (100 nM) or human pKal-ep-kal2 complex (100
nM hpKal that had been pre-incubated with 1 .mu.M epi-kal2 for 1
hour at room temperature) were injected over the captured IgGs or
sFabs for 5 minutes at 20-50 .mu.l/min followed by a 5-10 minute
dissociation phase. Binding responses were recorded at the end of
the association phase. Anti-pKal IgGs or sFabs were considered to
compete with epi-kal2 for binding to human pKal if binding of the
pKal-epi-kal2 complex to anti-pKal antibodies was significantly
reduced (>70%) as compared to an injection of hpKal only. The
sensor chip surface was regenerated with a pulse of 10 mM glycine
pH 1.5 at a flow rate of 100 .mu.min. Measurements were performed
at 25.degree. C. using HBS-P (10 mM HEPES pH 7.4, 150 mM NaCl and
0.005% surfactant P20) as the running buffer. Results from the
Biacore competition analysis for epi-Kal2 are shown herein in FIGS.
11A and 11B.
[0932] Biacore Competition Analysis with the Small Molecule
Kallikrein Inhibitor, AEBSF.
[0933] AEBSF (i.e., 4-(2-aminoethyl)benzene sulfonyl fluoride
hydrochloride) is a small molecule inhibitor of kallikrein. The
Biacore competition analysis is used herein to determine whether a
test antibody binds to the same site (or an overlapping site)
utilized by AEBSF for kallikrein inhibition.
[0934] Goat anti-human Fc fragment specific IgG or anti-human Fab
IgG was immobilized by amine coupling on a CM5 sensor chip at
immobilization densities of approximately 5000 RU. Anti-pKal IgGs
or sFabs were captured on their respective surfaces by injecting a
50 nM solution of IgG/sFab for 1-2 minutes at 5 at .mu.l/min. Human
pKal (100 nM) or human pKal-AEBSF complex (100 nM hpKal that had
been pre-treated with 1 mM AEBSF for 1 hour at room temperature)
were injected over the captured IgGs or sFabs for 5 minutes at
20-50 .mu.l/min followed by a 5-10 minute dissociation phase.
Binding responses were recorded at the end of the association
phase. Anti-pKal IgGs or sFabs were considered to compete with
AEBSF for binding to human pKal if binding of the pKal-AEBSF
complex to anti-pKal antibodies was significantly reduced (>70%)
as compared to an injection of hpKal only. The sensor chip surface
was regenerated with a pulse of 10 mM glycine pH 1.5 at a flow rate
of 100 .mu.l/min. Measurements were performed at 25.degree. C.
using HBS-P (10 mM HEPES pH 7.4, 150 mM NaCl and 0.005% surfactant
P20) as the running buffer. Results from the Biacore competition
analysis for AEBSF are shown herein in FIG. 12.
The Following are Sequences for the Light Chain Variable Regions
(LV), and Heavy Chain Variable Regions (HV) Regions for 8 Exemplary
Affinity Matured Anti-pKal Antibodies
TABLE-US-00029 [0935] (SEQ ID NOs: 1935-1990): 559A-M0029-D09-LV
QSALTQPPTVSVSPGQTARITCSGNKLGDKYVAWYQQKPGQSPMLVIYQD
TKRPSRVSERFSGSNSANTATLSISGTQALDEADYYCQAWDSSIVIFGGG TRLTVL
559A-M0145-D11-LV
QSVLTQPPSVSVSPGQTASITCSGDKLGDKYTSWYQQRPGQSPVLVIYQD
IKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSPNARVFGS GTKVTVL
559A-M0162-A04-LV
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPNLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG TKVEIK
559A-X0101-A01-LV
EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFG QGTKVEIK
559A-X0115-A03-LV
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG TKVEIK
559A-X0115-B07-LV
EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFG QGTKVEIK
559A-X0115-D01-LV
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG TKVEIK
559A-X0115-D05-LV
EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFG QGTKVEIK
559A-X0115-E09-LV
EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFG QGTKVEIK
559A-X0115-F02-LV
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG TKVEIK
559A-X0115-G04-LV
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG TKVEIK
559A-X0115-H06-LV
EIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFG QGTKVEIK
559A-M0006-D09-LV
DIQMTQSPSSLSASVGDRVTITCRASQSIRNYLNWYQQKPGKAPNLLIYA
ASTLQSGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQLSGYPHTFGQ GTKLEIK
559A-M0035-G04-LV
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRGFT FGPGTKVDIK
559A-M0029-D09-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMVWVRQAPGKGLEWVSY
IYPSGGATFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAMGS
YDYIWGFYSDHWGQGTLVTVSS 559A-M0145-D11-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYRMSWVRQAPGKGLEWVSS
IYPSGGRTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDK
FEWRLLFRGIGNDAFDIWGQGTMVTVSS 559A-M0162-A04-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSG
IYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRR
TGIPRRDAFDIWGQGTMVTVSS 559A-X0101-A01-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSY
ISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVA
RGIAARSRTSYFDYWGQGTLVTVSS 559A-X0115-A03-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSG
IYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAWRR
IGVPRRDSFDMWGQGTMVTVSS 559A-X0115-B07-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSY
ISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAMVG
QGIRGRSRTSYFAQWGQGTLVTVSS 559A-X0115-D01-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYSMHWVRQAPGKGLEWVSS
IYPSRGMTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRR
TGIPRRDAFDIWGQGTMVTVSS 559A-X0115-D05-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYMMAWVRQAPGKGLEWVSS
IVPSGGHTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVA
RGIAARSRTSYFDYWGQGTLVTVSS 559A-X0115-E09-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSY
ISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVA
QGIAARSRTSSVDQWGQGTLVTVSS 559A-X0115-F02-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSG
IYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRR
IGVPRRDEFDIWGQGTMVTVSS 559A-X0115-G04-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSG
IYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRR
TGVPRRDEFDIWGQGTMVTVSS 559A-X0115-H06-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSY
ISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVA
QGISARSRTSYFDYWGQGTLVTVSS 559A-M0006-D09-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYYMVWVRQAPGKGLEWVSV
IYPSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDK
WAVMPPYYYYAMDVWGQGTTVTVSS 559A-M0035-G04-HV
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYHMSWVRQAPGKGLEWVSV
ISPSGGSTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGG
SSDYAWGSYRRPYYFDYWGQGTLVTVSS 559A-M0029-D09 LV
CAGAGCGCTTTGACTCAGCCACCCACAGTGTCTGTGTCCCCAGGACAGAC
AGCCAGGATCACCTGCTCTGGAAATAAATTGGGGGATAAATATGTTGCCT
GGTATCAGCAGAAGCCAGGCCAGTCCCCTATGTTGGTCATCTATCAAGAT
ACTAAGCGCCCCTCAAGAGTTTCTGAGCGATTCTCTGGCTCCAACTCTGC
GAATACAGCCACTCTGTCCATCAGCGGGACCCAGGCTCTGGATGAGGCTG
ACTATTACTGTCAGGCGTGGGACAGCAGCATTGTGATCTTCGGCGGAGGG
ACCAGGCTGACCGTCCTA 559A-M0145-D11 LV
CAGAGCGTCTTGACTCAGCCACCCTCAGTGTCCGTGTCTCCAGGACAGAC
AGCCAGCATCACCTGCTCTGGAGATAAATTGGGGGATAAATATACTTCCT
GGTATCAGCAGAGGCCAGGCCAGTCCCCTGTATTGGTCATCTATCAAGAT
ATCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGG
GAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTG
ACTATTACTGTCAGGCGTGGGACAGTCCCAATGCGAGGGTCTTCGGATCT
GGGACCAAGGTCACCGTCCTA 559A-M0162-A04 LV
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCAGTAGTTGGTTGG
CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAG
GCGTCTACTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG
CAACTTATTACTGCCAACAGTATAATACTTATTGGACGTTCGGCCAAGGG
ACCAAGGTGGAAATCAAA 559A-X0101-A01 LV
GAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGA
GAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACC
TGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTAC
GGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGG
CTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACT
TCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGC
CAGGGCACCAAGGTGGAGATCAAG 559A-X0115-A03 LV
GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGA
CAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGG
CCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAG
GCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTC
CGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCG
CCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGC
ACCAAGGTGGAAATCAAG 559A-X0115-B07 LV
GAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGA
GAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACC
TGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTAC
GGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGG
CTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACT
TCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGC
CAGGGCACCAAGGTGGAGATCAAG 559A-X0115-D01 LV
GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGA
CAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGG
CCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAG
GCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTC
CGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCG
CCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGC
ACCAAGGTGGAAATCAAG 559A-X0115-D05 LV
GAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGA
GAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACC
TGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTAC
GGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGG
CTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACT
TCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGC
CAGGGCACCAAGGTGGAGATCAAG 559A-X0115-E09 LV
GAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGA
GAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACC
TGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTAC
GGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGG
CTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACT
TCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGC
CAGGGCACCAAGGTGGAGATCAAG 559A-X0115-F02 LV
GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGA
CAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGG
CCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAG
GCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTC
CGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCG
CCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGC
ACCAAGGTGGAAATCAAG 559A-X0115-G04 LV
GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGA
CAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGG
CCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAG
GCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTC
CGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCG
CCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGC
ACCAAGGTGGAAATCAAG 559A-X0115-H06 LV
GAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGA
GAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACC
TGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTAC
GGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGG
CTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACT
TCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGC
CAGGGCACCAAGGTGGAGATCAAG 559A-M0006-D09 LV
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTCGCAACTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGCT
GCATCCACTTTGCAAAGTGGGGTCCCAGCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACTATCAGCAGTCTGCAGCCTGAAGATTTTG
CAACTTATTACTGTCAACAGCTTAGTGGTTACCCCCACACTTTTGGCCAG
GGGACCAAGCTGGAGATCAAA 559A-M0035-G04 LV
CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGG
GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACT
TAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATT
TTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGCGGATTCACT
TTCGGCCCTGGGACCAAAGTGGATATCAAA 559A-M0029-D09 HV
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTGGTACACTA
TGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTAT
ATCTATCCTTCTGGTGGCGCTACTTTTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGATGGGTTCA
TATGATTACATTTGGGGATTTTATAGTGACCACTGGGGCCAGGGAACCCT GGTCACCGTCTCAAGC
559A-M0145-D11 HV
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCATTACCGTA
TGTCTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCT
ATCTATCCTTCTGGTGGCCGTACTGTTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAAAGATAAG
TTCGAGTGGAGGTTATTATTTCGCGGGATTGGAAATGATGCTTTTGATAT
CTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 559A-M0162-A04 HV
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCATTACATTA
TGATGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGT
ATCTATTCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGTACCGCCGG
ACTGGGATTCCAAGAAGAGATGCTTTTGATATCTGGGGCCAAGGGACAAT GGTCACCGTCTCAAGC
559A-X0101-A01 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGTCTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGA
TGACCTGGGTGCGCCAGGCTCCTGGCAAGGGCCTCGAATGGGTGTCCTAC
ATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTATCTGCAGATGA
ACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGTGGCC
AGAGGAATCGCCGCCAGGTCCCGGACCTCCTACTTCGACTACTGGGGCCA
GGGCACCCTGGTGACCGTGTCCTCC 559A-X0115-A03 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTTACCTTCTCCCACTACATCA
TGATGTGGGTGCGACAGGCTCCAGGCAAGGGCCTGGAATGGGTGTCCGGC
ATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCTGGCGGAGA
ATCGGCGTGCCCAGACGGGACTCCTTCGACATGTGGGGACAGGGCACCAT GGTGACAGTGTCCTCC
559A-X0115-B07 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGA
TGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTAC
ATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCG
GTTTACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCATGGTCGGC
CAGGGAATCCGGGGCAGATCCCGGACCTCCTACTTCGCCCAGTGGGGCCA
GGGCACCCTGGTGACAGTGTCCTCT 559A-X0115-D01 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGAC
TGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTC
ACCTTCTCCATCTACTCCATGCACTGGGTGCGACAGGCTCCAGGCAAGGG
CCTGGAATGGGTGTCCTCCATCTACCCCTCCCGGGGCATGACTTGGTACG
CCGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGAAC
ACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTA
CTACTGCGCCTACCGGCGGACCGGCATCCCTAGACGGGACGCCTTCGACA
TCTGGGGGCAGGGCACCATGGTGACAGTGTCCTCC 559A-X0115-D05 HV
GAGGTGCAATTGCTGGAATCCGGCGGTGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCGACTACATGA
TGGCCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCTCC
ATCGTGCCCTCTGGCGGCCACACCCACTACGCCGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGTGGCC
AGAGGAATCGCCGCCAGATCCCGGACCTCCTACTTCGACTACTGGGGCCA
GGGCACCCTGGTGACAGTGTCCTCC 559A-X0115-E09 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGA
TGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTAC
ATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCG
GTTTACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCCGGGTGGCC
CAGGGAATCGCCGCCAGATCCCGGACCTCCTCTGTGGATCAGTGGGGCCA
GGGCACCCTGGTGACAGTGTCCTCT 559A-X0115-F02 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACATCA
TGATGTGGGTGCGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGTCCGGC
ATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCG
GTTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCTACCGGCGG
ATCGGCGTGCCCAGACGGGACGAGTTCGACATCTGGGGGCAGGGCACCAT GGTGACAGTGTCCTCC
559A-X0115-G04 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCTCACTACATTA
TGATGTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGGC
ATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCTACAGACGG
ACCGGCGTGCCCAGACGGGACGAGTTCGATATCTGGGGGCAGGGCACCAT GGTGACAGTGTCCTCC
559A-X0115-H06 HV
GAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTC
CCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGA
TGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTAC
ATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCG
GTTTACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGA
ACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCCGGGTGGCC
CAGGGAATCTCCGCCAGATCCCGGACCTCCTACTTCGATTACTGGGGCCA
GGGCACCCTGGTGACAGTGTCCTCT 559A-M0006-D09 HV
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTTTTACTATA
TGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTATCCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATAAA
TGGGCGGTGATGCCCCCCTACTACTACTACGCTATGGACGTCTGGGGCCA
AGGGACCACGGTCACCGTCTCAAGC 559A-M0035-G04 HV
GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTC
TTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTATTACCATA
TGTCTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTT
ATCTCTCCTTCTGGTGGCTCTACTAAGTATGCTGACTCCGTTAAAGGTCG
CTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGA
ACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGCGGT
TCGAGCGATTACGCTTGGGGGAGTTATCGTCGACCCTACTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180037666A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180037666A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References