U.S. patent application number 17/498112 was filed with the patent office on 2022-01-20 for method of treating atherosclerosis.
This patent application is currently assigned to CSL Limited. The applicant listed for this patent is CSL Limited. Invention is credited to Yung-Chih CHEN, Hamid HOSSEINI, Con PANOUSIS, Karlheinz PETER.
Application Number | 20220017638 17/498112 |
Document ID | / |
Family ID | 1000005880276 |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220017638 |
Kind Code |
A1 |
PANOUSIS; Con ; et
al. |
January 20, 2022 |
METHOD OF TREATING ATHEROSCLEROSIS
Abstract
The present disclosure relates to a method of treating or
preventing atherosclerosis in a subject by administering an
inhibitor of FXII.
Inventors: |
PANOUSIS; Con; (Bundoora,
AU) ; PETER; Karlheinz; (Hawthorn East, AU) ;
HOSSEINI; Hamid; (Glen Waverly, AU) ; CHEN;
Yung-Chih; (Boxhill South, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CSL Limited |
Parkville |
|
AU |
|
|
Assignee: |
CSL Limited
|
Family ID: |
1000005880276 |
Appl. No.: |
17/498112 |
Filed: |
October 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16090861 |
Oct 3, 2018 |
11174321 |
|
|
PCT/AU2017/050297 |
Apr 6, 2017 |
|
|
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17498112 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/31 20130101;
A61K 2039/505 20130101; C07K 14/811 20130101; C07K 14/43568
20130101; C07K 14/8135 20130101; C07K 2317/565 20130101; C07K
14/765 20130101; A61K 2039/54 20130101; A61K 47/60 20170801; C07K
2319/30 20130101; C12N 9/6424 20130101; A61K 38/57 20130101; C07K
2319/00 20130101; C07K 14/81 20130101; C07K 16/36 20130101; A61P
9/10 20180101; A61K 2039/545 20130101; C07K 2317/76 20130101; C07K
14/4703 20130101 |
International
Class: |
C07K 16/36 20060101
C07K016/36; A61K 47/60 20060101 A61K047/60; A61P 9/10 20060101
A61P009/10; A61K 38/57 20060101 A61K038/57; C07K 14/765 20060101
C07K014/765; C07K 14/81 20060101 C07K014/81; C07K 14/435 20060101
C07K014/435; C07K 14/47 20060101 C07K014/47; C12N 9/64 20060101
C12N009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 6, 2016 |
EP |
16164009.9 |
Claims
1. A method of treating atherosclerosis in a subject, comprising
administering to the subject an effective amount of an anti-Factor
XII (FXII) antibody or antigen binding fragment thereof comprising:
(i) a V.sub.H comprising: (a) a sequence set forth in SEQ ID NO: 6;
or (b) a CDR1 comprising a sequence set forth in SEQ ID NO: 8; a
CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3
comprising a sequence set forth in SEQ ID NO: 12; or (c) a CDR1
comprising a sequence set forth in SEQ ID NO: 8; a CDR2 comprising
a sequence set forth in SEQ ID NO: 9; and a CDR3 comprising a
sequence set forth in SEQ ID NO: 11; and/or (ii) a V.sub.L
comprising: (a) a sequence set forth in SEQ ID NO: 7; or (b) a CDR1
comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising
a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a
sequence set forth in SEQ ID NO: 16; or (c) a CDR1 comprising a
sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence
set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set
forth in SEQ ID NO: 15.
2. The method of claim 1, wherein the anti-FXII antibody or antigen
binding fragment thereof comprises: (i) a V.sub.H comprising a
sequence set forth in SEQ ID NO: 18 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 19 or (ii) a heavy chain
comprising a sequence set forth in SEQ ID NO: 20 and a light chain
comprising a sequence set forth in SEQ ID NO: 21.
3. The method of claim 1, wherein the anti-FXII antibody is an IgG
antibody.
4. The method of claim 1, wherein the anti-FXII antibody or antigen
binding fragment thereof is linked to a fusion partner, wherein the
fusion partner comprises polyethylene glycol (PEG) or a half-life
enhancing polypeptide, wherein the half-life enhancing polypeptide
is selected from the group consisting of albumin, afamin,
alpha-fetoprotein, vitamin D binding protein, human albumin, an
immunoglobulin, and an Fc of an IgG.
5. The method of claim 4, wherein the half-life enhancing
polypeptide is linked to the anti-FXII antibody or antigen binding
fragment thereof 4via a linker.
6. The method of claim 5, wherein the anti-FXII antibody or antigen
binding fragment thereof is a fusion protein comprising human
albumin linked to the anti-FXII antibody or antigen binding
fragment thereof via a linker peptide.
7. The method of claim 1, wherein the anti-FXII antibody or antigen
binding fragment thereof is administered to the subject
intravenously or subcutaneously or intrathecally.
8. The method of claim 1, wherein the anti-FXII antibody or antigen
binding fragment thereof is administered to the subject: (i) in a
single dose; or (ii) in a plurality of doses; or (iii) as a
continuous dose.
9. The method of claim 1, wherein the anti-FXII antibody or antigen
binding fragment thereof is administered to the subject at a
concentration from about 0.01 to about 100 mg/kg body weight.
10. The method of claim 1, wherein the subject is at risk of
developing atherosclerosis and/or suffers from diabetes and/or
obesity.
11. A kit for use in the treatment or prevention of atherosclerosis
in a subject, comprising: (a) an anti-FXII antibody or antigen
binding fragment thereof comprising (i) a V.sub.H comprising: (A) a
sequence set forth in SEQ ID NO: 6; or (B) a CDR1 comprising a
sequence set forth in SEQ ID NO: 8; a CDR2 comprising a sequence
set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set
forth in SEQ ID NO: 12; or (C) a CDR1 comprising a sequence set
forth in SEQ ID NO: 8; a CDR2 comprising a sequence set forth in
SEQ ID NO: 9; and a CDR3 comprising a sequence set forth in SEQ ID
NO: 11; and/or (ii) a V.sub.L comprising: (A) a sequence set forth
in SEQ ID NO: 7; or (B) a CDR1 comprising a sequence set forth in
SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO:
14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or
(c) a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2
comprising a sequence set forth in SEQ ID NO: 14; and a CDR3
comprising a sequence set forth in SEQ ID NO: 15; (b) instructions
for using the kit in treating or preventing atherosclerosis in the
subject; and (c) optionally, at least one further therapeutically
active compound or drug.
12. An anti-Factor XII (FXII) antibody or antigen binding fragment
thereof, wherein the anti-FXII antibody comprises: (i) a V.sub.H
comprising a sequence set forth in SEQ ID NO: 18 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 19; or (ii) a heavy
chain comprising a sequence set forth in SEQ ID NO: 20 and a light
chain comprising a sequence set forth in SEQ ID NO: 21.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/090,861, filed on Oct. 3, 3018, which is a
national stage entry under 35 U.S.C. .sctn. 371 of International
Application No. PCT/AU2017/050297, filed on Apr. 6, 2017, which
claims priority to European Patent Application No. 16164009.9,
filed on Apr. 6, 2016. The contents of these applications are each
incorporated herein by reference in their entirety.
FIELD
[0002] The present disclosure relates to a method of treating or
preventing atherosclerosis in a subject.
INTRODUCTION
[0003] Atherosclerosis is a major health burden in developed
countries and is the leading cause of mortality and morbidity
worldwide and represents a major economic burden to health care
systems. Epidemic proportions of obesity and diabetes mellitus have
considerably increased the numbers of atherosclerosis-associated
complications, such as myocardial infarction (MI) and stroke.
[0004] Atherosclerosis is a chronic inflammatory disease with
specific, localized manifestations at the arterial wall. The
development of atherosclerosis is driven by inflammatory processes
involving a plethora of cells and factors belonging to the innate
and/or adaptive immune system.
[0005] The role of inflammation in the development of
atherosclerosis has been studied. For example, blood leukocytes,
mediators of host defenses and inflammation, localize in the
earliest lesions of atherosclerosis. Elevation of inflammatory
markers has been shown to predict outcome of patients with acute
coronary syndromes, independently of myocardial damage. In
addition, low-grade chronic inflammation, as indicated by levels of
the inflammatory marker C-reactive protein, has been shown to
correlate with risk of atherosclerotic complications.
[0006] Atherosclerotic lesions, or atherosclerotic plaques, are
separated into two broad categories: stable and unstable (also
called vulnerable). Typically, stable atherosclerotic plaques,
which tend to be asymptomatic, are rich in extracellular matrix and
smooth muscle cells, whilst unstable plaques are rich in
macrophages and foam cells, and are usually fragile and prone to
rupture. Instability of atherosclerotic plaques is driven by
inflammatory processes.
[0007] Atherosclerotic plaques can become unstable and rupture
without warning, resulting in the formation of an occlusive
arterial thrombus. Ruptured plaques are heavily infiltrated by
inflammatory cells, such as macrophages, which secrete proteolytic
enzymes including plasminogen activators, cathepsins and matrix
metalloproteinases, and fibrotic tissue. Ruptured atherosclerotic
plaques manifest clinically as myocardial infarction and stroke. As
a consequence, many patients suffer a sudden cardiac death or fatal
stroke.
[0008] The most common treatment options for atherosclerotic
lesions include administration of lipid-lowering drugs such as
statins (e.g., Lovastatin, Pravastatin, Rosuvastatin, Simvastatin,
Atorvastatin, and Fluvastatin) and blood-thinning drugs
(anticoagulants like e.g., Aspirin, Clopidogrel, Prasugrel,
Ticagrelor, Warfarin and Heparin). However, on-going use of the
latter therapeutics increases the risk of dangerous bleeding.
[0009] The role of clotting factors in atherosclerosis has also
been studied. For example, Schnerb et al., (Arterioscler Thromb
Vasc Biol., 36: 475-481, 2016) studied atherosclerosis development
in mice lacking apolipoprotein E and Factor XI (FXI). The authors
found atherogenesis was slowed in FXI deficient mice. However,
these studies are based on animals that have lacked FXI since birth
and do not study the effects of inhibiting the protein following or
during development of atherosclerosis.
[0010] Therefore, there is a need in the art for improved
treatments for atherosclerosis and its thrombotic
complications.
SUMMARY
[0011] In producing the present invention, the inventors studied
the effects of inhibiting Factor XII (FXII) in mouse models of
atherosclerosis, e.g., atherosclerotic plaque formation and/or
atherosclerotic plaque instability and rupture. The inventors
studied the effects of FXII inhibition based on their understanding
that this protein has effects on numerous biological pathways, some
of which may be involved in atherosclerosis, e.g., inflammation.
The inventors found that inhibition of FXII not only slows
progression of atherosclerosis, but also attenuates development of
atherosclerotic lesions, reduces lesion size and stabilizes
unstable atherosclerotic plaques as evidenced by a reduction in
inflammatory cell accumulation in the lesion, increase in collagen
deposition and reduction in necrotic core area. The inventors
demonstrated these effects by administering an inhibitor of FXII to
accepted animal models of atherosclerosis, i.e., that mimic the
situation observed in humans. The experimental evidence includes
the findings that FXII inhibitors are useful in the treatment
and/or prevention of atherosclerosis by attenuating the progression
and development of atherosclerotic legions, reducing arterial
inflammation and stabilizing plaques.
[0012] The findings by the inventors provide the basis for methods
for treating or preventing atherosclerosis in a subject by
inhibiting FXII. The findings by the inventors also provide the
basis for or an inhibitor of FXII for use in treating or preventing
atherosclerosis in a subject.
[0013] In other words, the inventors provide the basis for or an
inhibitor of Factor XII for use in treating or preventing
atherosclerosis in a subject by (i) preventing atherosclerotic
plaques formation in a subject and/or (ii) stabilizing vulnerable
atherosclerotic plaques in a subject and/or (iii) preventing
atherosclerotic plaque rupture in a subject.
[0014] For example, the present disclosure provides a method for
treating atherosclerosis in a subject comprising administering to
the subject an inhibitor of FXII. In another example, the
disclosure provides a method for preventing atherosclerosis in a
subject, the method comprising administering to the subject an
inhibitor of FXII.
[0015] In an alternative example, the present disclosure provides
an inhibitor of FXII for use in treating atherosclerosis in a
subject. In another example, the disclosure provides an inhibitor
of FXII for use in preventing atherosclerosis in a subject.
[0016] The inventors have also found that they can reduce the
progression of atherosclerotic lesions in a subject. Accordingly,
the present disclosure additionally provides a method for or an
inhibitor of FXII for use in reducing the progression of
atherosclerosis in a subject. For example, the present disclosure
provides a method for or an inhibitor of FXII for use in reducing
the risk of or preventing atherosclerotic plaque rupture in a
subject. In one example, the present disclosure provides a method
for or an inhibitor of FXII for use in stabilization of unstable
atherosclerotic plaques. In one example, the present disclosure
provides a method for or an inhibitor of FXII for use in preventing
atherosclerotic plaques formation.
[0017] In one example, the inhibitor of FXII is a direct inhibitor.
In one example, the inhibitor of FXII binds to FXII and/or FXIIa.
In one example, the inhibitor of FXII binds to FXII and/or FXIIa
and inhibits the activity of FXII and/or FXIIa. For example, the
inhibitor of FXII binds to FXIIa and inhibits the activity of
FXIIa. In another example, the inhibitor of FXII binds to FXII and
inhibits FXII activation. In one example, the activity of FXII
and/or FXIIa is inhibited by at least about 50%. For example, the
activity of FXII and/or FXIIa is inhibited by about 60%, or about
70%, or about 80%, or about 85%, or about 90%, or about 95%, or
about 99%, or about 100%. Methods for determining the activity of
FXII and/or FXIIa are known in the art and/or described herein.
[0018] In one example, the inhibitor of FXII is a serine protease
inhibitor. For example, the FXII inhibitor is Infestin-4. In
another example, the FXII inhibitor is SPINK-1. In a further
example, the FXII inhibitor is an Infestin-4 or SPINK-1
variant.
[0019] In one example, the inhibitor of FXII is not a serine
protease inhibitor. For example, the inhibitor of FXII is not
Infestin-4. For example, the inhibitor of FXII is not a variant of
Infestin-4. In one example, the inhibitor of FXII is not SPINK-1.
For example, the inhibitor of FXII is not a variant of SPINK-1.
[0020] In one example, the method of or the inhibitor of FXII for
use in the present disclosure comprises administering an inhibitor
of FXII, wherein the inhibitor comprises: [0021] (i) the wild type
Infestin-4 polypeptide sequence (SEQ ID NO: 1), or a polypeptide
sequence comprising: [0022] (a) SEQ ID NO: 1 modified to contain
1-5 amino acid mutations outside of N-terminal amino acid positions
2-13 of SEQ ID NO: 1; and/or [0023] (b) an identity of at least 70%
to SEQ ID NO: 1 and retaining six conserved cysteine residues from
SEQ ID NO: 1; or [0024] (ii) a wild-type SPINK-1 polypeptide
sequence (SEQ ID NO: 2), or a polypeptide sequence comprising:
[0025] (a) SEQ ID NO: 2 mutated to replace N-terminal amino acid
positions 2-13 with the N-terminal amino acids 2-13 of SEQ ID NO:
1; and optionally further modified to contain 1-5 additional amino
acid mutations that increase the homology of the polypeptide
sequence to sequence of SEQ ID NO: 1; and/or [0026] (b) an identity
of at least 70% to SEQ ID NO: 2 and retaining six conserved
cysteine residues from SEQ ID NO: 2; and/or [0027] (iii) one of
SPINK-1 mutants K1 (SEQ ID NO: 3), K2 (SEQ ID NO: 4), or K3 (SEQ ID
NO: 5).
[0028] In one example, the inhibitor of FXII comprises the sequence
of the serine protease inhibitor Infestin-4. For example, the
inhibitor of FXII comprises the sequence set forth in SEQ ID NO:
1.
[0029] In one example, the inhibitor of FXII comprises a modified
Infestin-4. For example, the inhibitor of FXII comprises the
sequence set forth in SEQ ID NO: 1 modified to contain 1-5 amino
acid mutations outside of N-terminal amino acid positions 2-13 of
SEQ ID NO: 1.
[0030] In another example, the inhibitor of FXII comprises a
sequence with at least 70% identity to the sequence set forth in
SEQ ID NO: 1 and retaining six conserved cysteine residues from SEQ
ID NO: 1. For example, the inhibitor of FXII has an identity of
about 75% to SEQ ID NO: 1, or an identity of about 80% to SEQ ID
NO: 1, or an identity of about 85% to SEQ ID NO: 1, or an identity
of about 90% to SEQ ID NO: 1, or an identity of about 95% to SEQ ID
NO: 1, or an identity of about 98% to SEQ ID NO: 1, or an identity
of about 99% to SEQ ID NO: 1.
[0031] In one example, the inhibitor of FXII comprises the sequence
of the serine protease inhibitor SPINK-1. For example, the
inhibitor of FXII comprises the sequence set forth in SEQ ID NO:
2.
[0032] In another example, the inhibitor of FXII comprises the
sequence set forth in SEQ ID NO: 2 mutated to replace N-terminal
amino acid positions 2-13 with the N-terminal amino acids 2-13 of
SEQ ID NO: 1; and optionally further modified to contain 1-5
additional amino acid mutations that increase the homology of the
polypeptide sequence to sequence of SEQ ID NO: 1.
[0033] In another example, the inhibitor of FXII comprises a
sequence with at least 70% identity to the sequence set forth in
SEQ ID NO: 2 and retaining six conserved cysteine residues from SEQ
ID NO: 2. For example, the inhibitor of FXII has an identity of
about 75% to SEQ ID NO: 2, or an identity of about 80% to SEQ ID
NO: 2, or an identity of about 85% to SEQ ID NO: 2, or an identity
of about 90% to SEQ ID NO: 2, or an identity of about 95% to SEQ ID
NO: 2, or an identity of about 98% to SEQ ID NO: 2, or an identity
of about 99% to SEQ ID NO: 2.
[0034] In one example, inhibitor of FXII is a protein comprising a
variable region fragment (Fv). For example, the protein is selected
from the group consisting of: [0035] (i) a single chain Fv fragment
(scFv); [0036] (ii) a dimeric scFv (di-scFv); or [0037] (iii) a
diabody; [0038] (iv) a triabody; [0039] (v) a tetrabody; [0040]
(vi) a Fab; [0041] (vii) a F(ab').sub.2; [0042] (viii) a Fv; [0043]
(ix) one of (i) to (ix) linked to a constant region of an antibody,
Fc or a heavy chain constant domain (C.sub.H) 2 and/or C.sub.H3; or
[0044] (x) an antibody.
[0045] In one example, an inhibitor of FXII is an antibody. For
example, the antibody is an anti-FXII antibody. In another example,
the antibody is an anti-FXIIa antibody.
[0046] Exemplary antibodies are full-length and/or naked
antibodies.
[0047] In one example, the inhibitor of FXII is a protein that is
recombinant, chimeric, CDR grafted, , synhumanized, primatized,
deimmunized or human.
[0048] In one example, the antibody is an IgG antibody.
[0049] In one example, the anti-FXII antibody comprises a heavy
chain variable region (V.sub.H) comprising a sequence set forth in
SEQ ID NO: 6.
[0050] In one example, the anti-FXII antibody comprises a light
chain variable region (V.sub.L) comprising a sequence set forth in
SEQ ID NO: 7.
[0051] In one example, the anti-FXII antibody comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 6 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 7.
[0052] In one example, the anti-FXII antibody comprises a variable
region comprising the complementary determining regions (CDRs) of
the V.sub.H and/or the V.sub.L of SEQ ID NO: 6 and SEQ ID NO:
7.
[0053] In one example, the protein or antibody is any form of the
protein or antibody encoded by a nucleic acid encoding any of the
foregoing proteins or antibodies, such as a variant missing an
encoded C-terminal lysine residue, a deamidated variant and/or a
glycosylated variant and/or a variant comprising a pyroglutamate,
e.g., at the N-terminus of a protein and/or a variant lacking a
N-terminal residue.
[0054] In one example, the anti-FXII antibody comprises:
(i) a V.sub.H comprising: [0055] (a) a sequence set forth in SEQ ID
NO: 6; or [0056] (b) a CDR1 comprising a sequence set forth in SEQ
ID NO: 8; a CDR2 comprising a sequence set forth in SEQ ID NO: 10;
and a CDR3 comprising a sequence set forth in SEQ ID NO: 12; or
[0057] (c) a CDR1 comprising a sequence set forth in SEQ ID NO: 8;
a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and a CDR3
comprising a sequence set forth in SEQ ID NO: 11; and/or (ii) a VL
comprising: [0058] (a) a sequence set forth in SEQ ID NO: 7; or
[0059] (b) a CDR1 comprising a sequence set forth in SEQ ID NO: 13;
a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3
comprising a sequence set forth in SEQ ID NO: 16; or [0060] (c) a
CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2
comprising a sequence set forth in SEQ ID NO: 14; and a CDR3
comprising a sequence set forth in SEQ ID NO: 15.
[0061] In one example, the anti-FXII antibody comprises:
(i) a V.sub.H comprising: [0062] (a) a CDR1 comprising a sequence
set forth in SEQ ID NO: 8; [0063] (b) a CDR2 comprising a sequence
set forth in SEQ ID NO: 10; and [0064] (c) a CDR3 comprising a
sequence set forth in SEQ ID NO: 12; and/or (ii) a V.sub.L
comprising: [0065] (a) a CDR1 comprising a sequence as set forth in
SEQ ID NO: 13; [0066] (b) a CDR2 comprising a sequence as set forth
in SEQ ID NO: 14; and [0067] (c) a CDR3 comprising a sequence as
set forth in SEQ ID NO: 16.
[0068] In one example, the anti-FXII antibody comprises:
(i) a V.sub.H comprising: [0069] (a) a CDR1 comprising a sequence
set forth in SEQ ID NO: 8; [0070] (b) a CDR2 comprising a sequence
set forth in SEQ ID NO: 9; and [0071] (c) a CDR3 comprising a
sequence set forth in SEQ ID NO: 11; and/or (ii) a V.sub.L
comprising: [0072] (a) a CDR1 comprising a sequence as set forth in
SEQ ID NO: 13; [0073] (b) a CDR2 comprising a sequence as set forth
in SEQ ID NO: 14; and [0074] (c) a CDR3 comprising a sequence as
set forth in SEQ ID NO: 15.
[0075] In one example, the anti-FXII antibody comprises:
(i) a V.sub.H comprising [0076] (a) a CDR1 set forth in SEQ ID NO:
8; [0077] (b) a CDR2 set forth in SEQ ID NO: 10 wherein the X at
position 3 is D, the X at position 4 is I, the X at position 5 is
P, the X at position 6 is T, the X at position 7 is K, and the X at
position 8 is G; and [0078] (c) a CDR3 set forth in SEQ ID NO: 11;
and/or (ii) a V.sub.L comprising [0079] (a) a CDR1 set forth in SEQ
ID NO: 13; [0080] (b) CDR2 set forth in SEQ ID NO: 14; and [0081]
(c) a CDR3 set forth in SEQ ID NO: 15.
[0082] For example, the anti-FXII antibody comprises a V.sub.H
comprising a sequence set forth in SEQ ID NO: 18 and a V.sub.L
comprising a sequence set forth in SEQ ID NO: 19.
[0083] In one example, the anti-FXII antibody comprises lambda
light chain constant regions.
[0084] In one example, the anti-FXII antibody comprises IgG4 or
stabilized IgG4 constant regions. For example, the stabilized IgG4
constant regions comprise a proline at position 241 of the hinge
region according to the system of Kabat (Kabat et al., Sequences of
Proteins of Immunological Interest Washington D.C. United States
Department of Health and Human Services, 1987 and/or 1991).
[0085] In one example, the anti-FXII antibody comprises a heavy
chain comprising a sequence set forth in SEQ ID NO: 20 and a light
chain comprising a sequence set forth in SEQ ID NO: 21.
[0086] In one example, the anti-FXII antibody is within a
composition. For example, the composition comprises a protein
comprising an antibody variable region or a V.sub.H or a V.sub.L or
an antibody as described herein. In one example, the composition
additionally comprises one or more variants of the protein or
antibody. For example, that comprises a variant missing an encoded
C-terminal lysine residue, a deamidated variant and/or a
glycosylated variant and/or a variant comprising a pyroglutamate,
e.g., at the N-terminus of a protein and/or a variant lacking a
N-terminal residue, e.g., a N-terminal glutamine in an antibody or
V region and/or a variant comprising all or part of a secretion
signal. Deamidated variants of encoded asparagine residues may
result in isoaspartic, and aspartic acid isoforms being generated
or even a succinamide involving an adjacent amino acid residue.
Deamidated variants of encoded glutamine residues may result in
glutamic acid. Compositions comprising a heterogeneous mixture of
such sequences and variants are intended to be included when
reference is made to a particular amino acid sequence.
[0087] In one example, of any method or an inhibitor of FXII for
use described herein, the inhibitor of FXII is linked to a fusion
partner. For example, the fusion partner comprises polyethylene
glycol (PEG) or a half-life enhancing polypeptide.
[0088] In one example, the inhibitor of FXII is linked to the
fusion partner directly. In another example, the inhibitor of FXII
is linked to the fusion partner via a linker. For example, the
inhibitor of FXII is linked to a half-life enhancing polypeptide
directly. In another example, the inhibitor of FXII is linked to a
half-life enhancing polypeptide via a linker. In one example, the
inhibitor of FXII is linked to the PEG directly. In another
example, the inhibitor of FXII is linked to the PEG via a
linker.
[0089] In one example, the linker is an intervening peptidic
linker. For example, the linker is a cleavable linker.
[0090] In one example, the half-life enhancing polypeptide is
selected from the group consisting of albumin, afamin,
alpha-fetoprotein, vitamin D binding protein, human albumin, an
immunoglobulin, and an Fc of an IgG. For example, the half-life
enhancing polypeptide is albumin.
[0091] In one example, the inhibitor of FXII is a fusion protein
comprising human albumin linked to a FXII inhibitor via a linker
peptide.
[0092] In one example, the inhibitor of FXII is administered
parenterally. For example, the inhibitor of FXII is administered
intravenously, or subcutaneously, or intrathecal. In one example,
the inhibitor of FXII is administered subcutaneously. In another
example, the inhibitor of FXII is administered intravenously.
[0093] In one example of any method described herein, the inhibitor
of FXII is administered to the subject in one or more doses. For
example, the inhibitor of FXII is administered to the subject:
(i) in a single dose; or (ii) in a plurality of doses; or (iii) as
a continuous infusion or application.
[0094] In one example, the inhibitor of FXII is administered to the
subject in a single dose.
[0095] In one example, the inhibitor of FXII is administered to the
subject in a plurality of doses. For example, the inhibitor of FXII
is administered to the subject as two doses, or three doses, or
four doses, or five doses or more. For example, administration of
each dose of the inhibitor of FXII is separated by a period of
hours. For example, administration of each dose of the inhibitor of
FXII is separated by the period of about 1 hour, or about 2 hours,
or about 3 hours, or about 4 hours, or about 6 hours, or about 8
hours, or about 12 hours, or about 16 hours, or about 20 hours, or
about 24 hours.
[0096] For example, administration of each dose of the inhibitor of
FXII is separated by a period of days. For example, administration
of each dose of the inhibitor of FXII is separated by the period of
about 1 day, or about 2 days, or about 3 days, or about 4 days, or
about 5 days, or about 6 days, or about 7 days.
[0097] In one example, administration of each dose of the inhibitor
of FXII is separated by at least 14 days or at least 28 days.
[0098] For example, administration of each dose of the inhibitor of
FXII is separated by a period of weeks. For example, administration
of each dose of the inhibitor of FXII is separated by the period of
about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks,
or about 5 weeks, or about 6 weeks.
[0099] In one example, administration of each dose of the inhibitor
of FXII is separated by at least one month.
[0100] In one example, the length of time between administrations
of the inhibitor of FXII is the same throughout the course of
administration. In one example, the length of time between
administrations of the inhibitor of FXII is different throughout
the course of administration. For example, the inhibitor of FXII is
administered on a weekly basis at the commencement of therapy and
then on a monthly basis following a predetermined number of doses.
In one example, the length of time between administrations of the
inhibitor of FXII is variable.
[0101] In one example, the inhibitor of FXII is administered to the
subject as a continuous dose. For example, the inhibitor of FXII is
administered to the subject as a continuous infusion over a period
of time. For example, the inhibitor of FXII is administered over a
period of between about 1 minute to about 24 hours. For example,
the inhibitor of FXII is administered over a period of about 10
minutes to about 12 hours, or about 10 minutes to about 6 hours, or
about 10 minutes to about 5 hours, or about 10 minutes to about 4
hours, or about 10 minutes to about 3 hours, or about 10 minutes to
about 2 hours, or about 10 minutes to about 1 hour, or about 30
minutes.
[0102] In one example, the inhibitor of FXII is administered a
plurality of times. For example, the inhibitor of FXII is
administered one or more times. For example, the inhibitor of FXII
is administered until the atherosclerosis is treated or prevented.
For example, the inhibitor of FXII is administered for a period of
days to months. For example, the inhibitor of FXII is administered
for about one day, or about 2 days, or about 3 days, or about 4
days, or about 5 days, or about 6 days, or about 1 week, or about 2
weeks, or about 4 weeks, or about six weeks, or about 2 months.
[0103] In one example, the inhibitor of FXII is administered in a
therapeutically or prophylactically effective amount. For example,
the inhibitor of FXII is administered to the subject at a dose of
about 0.01 mg/kg to about 1000 mg/kg. For example, the inhibitor of
FXII is administered at a dose of about 0.01 mg/kg bodyweight, or
about 0.1 mg/kg bodyweight, or about 1 mg/kg bodyweight, or about
50 mg/kg bodyweight, or about 100 mg/kg bodyweight, or about 200
mg/kg bodyweight, or about 500 mg/kg bodyweight, or about 1000
mg/kg bodyweight. For example, the inhibitor of FXII is
administered at a dose of about 0.001 mg/kg to about 100 mg/kg body
weight, or about 0.01 mg/kg to about 100 mg/kg, or about 0.01 mg/kg
to about 50 mg/kg, or about 0.1 mg/kg to about 30 mg/kg, or about
0.1 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 5 mg/kg,
or about 0.1 mg/kg to about 2 mg/kg or about 0.1 mg/kg to about 1
mg/kg. In one example, the inhibitor of FXII is administered at a
dose ranging from about 0.01 mg/kg to about 1000 mg/kg, or about
0.1 mg/kg to about 1000 mg/kg, or about 1 mg/kg to about 1000
mg/kg, or about 1 mg/kg to about 500 mg/kg, or about 10 mg/kg to
about 200 mg/kg, or about 10 mg/kg to about 100 mg/kg, or about 50
mg/kg to about 500 mg/kg, or about 50 mg/kg to about 200 mg/kg, or
about 100 mg/kg to about 200 mg/kg. In one example, the inhibitor
of FXII is administered at a dose of about 10 mg/kg. In one
example, the inhibitor of FXII is administered at a dose of about
20 mg/kg.
[0104] In one example, the subject has atherosclerosis. In one
example, the subject has been diagnosed as suffering from
atherosclerosis. In one example, the subject is receiving treatment
for atherosclerosis. In one example, the subject is receiving
treatment for atherosclerosis associated condition (i.e.,
myocardial infarct). For example, the subject is receiving
treatment with a statin or warfarin or a .beta.-blocker or
anti-platelet drug.
[0105] In one example, the subject has previously suffered a
myocardial infarction. In one example the subject is receiving
treatment with a statin, warfarin and a .beta.-blocker.
[0106] In one example of any method or an inhibitor of FXII for use
described herein, the subject is at risk of developing
atherosclerosis. In this regard, the inhibitor of FXII is used in a
preventative or prophylactic manner or can be said to be used in a
primary preventative manner. An exemplary subject at risk of
developing atherosclerosis suffers from diabetes and/or obesity.
For example, the diabetes is type 2 diabetes.
[0107] Additional or alternative characteristics of a subject at
risk of suffering from atherosclerosis include one or more of the
following characteristics: [0108] has already suffered from angina,
and/or stroke and/or heart attack; [0109] has peripheral artery
disease; [0110] has a family history of heart disease; [0111] has
high plasma low density lipoprotein levels; [0112] has low plasma
high density lipoprotein levels; and/or [0113] has high blood
pressure.
[0114] In one example, a subject at risk of suffering from
atherosclerosis has a high plasma low density lipoprotein level.
For example, the plasma low density lipoprotein level is at least
160 mg/dL.
[0115] In one example, a subject at risk of suffering from
atherosclerosis has a low plasma high density lipoprotein level.
For example, the plasma high density lipoprotein level is less than
about 50 mg/dL.
[0116] In one example, a subject at risk of suffering from
atherosclerosis has high blood pressure.
[0117] For example, the blood pressure level is at least 140/90
mmHg.
[0118] In one example, the subject is additionally aged 55 years or
more, e.g., 65 years or more, or 75 years or more.
[0119] In one example, the subject has increased levels of an
inflammatory marker. For example, the subject has increased levels
of C-reactive protein, e.g., 3-5 mg/L.
[0120] In one example of any method described herein, the inhibitor
of FXII is administered to the subject before or after the onset of
atherosclerosis. For example, the inhibitor of FXII is administered
prophylactically or therapeutically. In one example, the inhibitor
is administered to the subject prophylactically. In one example,
the inhibitor is administered to the subject therapeutically.
[0121] In one example of any method or an inhibitor of FXII for use
described herein, the atherosclerosis in the subject may result in
a myocardial infarct or stroke. For example, atherosclerotic plaque
rupture may result in occlusive arterial thrombosis at the site of
rupture which clinically manifests as a myocardial infarct or
stroke. Accordingly, the present disclosure additionally provides a
method for reducing the risk of occlusive arterial thrombosis
and/or myocardial infarction and/or stroke by performing a method
described herein. In another example, the present disclosure
provides a method for preventing occlusive arterial thrombosis
and/or myocardial infarction and/or stroke by performing a method
described herein.
[0122] Methods for assessing each of the foregoing are known in the
art and/or described herein.
[0123] In one example, the inhibitor of FXII is administered in an
amount sufficient to have one or more of the following effects:
[0124] (i) reducing the likelihood of occlusive arterial thrombus
in a subject; [0125] (ii) reducing atherosclerotic plaque lesion
size in a subject; [0126] (iii) increasing plaque stabilization in
a subject; [0127] (iv) reducing inflammatory cell accumulation in
an atherosclerotic plaque lesion in a subject; and/or [0128] (v)
reducing pro-atherogenic cell populations in a subject.
[0129] In one example, the inhibitor of FXII is administered in an
amount sufficient to stabilize atherosclerotic plaques. For
example, lipid accumulation in an atherosclerotic plaque lesion in
a subject is reduced. In one example, collagen deposition in an
atherosclerotic plaque lesion in a subject is increased. In one
example, necrotic core area in an atherosclerotic plaque lesion in
a subject is reduced. In one example, smooth muscle cell number in
an atherosclerotic plaque lesion in a subject is increased. In
another example, the expression of vascular cell adhesion
molecule-1 (VCAM-1) in an atherosclerotic plaque lesion in a
subject is reduced. Accordingly, the present disclosure provides a
method for or an inhibitor of FXII for use in reducing lipid
accumulation and/or increasing collagen deposition and/or reducing
necrotic core area and/or increasing smooth muscle cell number
and/or reducing expression of vascular cell adhesion molecule-1 in
an atherosclerotic plaque lesion in a subject by performing a
method or an inhibitor of FXII for use described herein. For
example, the reduction and/or increase are relative to an
atherosclerotic plaque in a subject that is not treated with the
inhibitor of FXII. In one example, the reduction and/or increase is
relative to an atherosclerotic plaque in a subject prior to the
commencement of treatment with an inhibitor of FXII.
[0130] In one example, the inhibitor of FXII is administered in an
amount sufficient to reduce inflammatory cell accumulation in an
atherosclerotic plaque lesion in a subject. For example, macrophage
accumulation in an atherosclerotic plaque lesion in a subject is
reduced.
[0131] In one example, the inhibitor of FXII is administered in an
amount sufficient to reduce pro-atherogenic cell populations in the
atherosclerotic plaque. For example, circulating natural killer
and/or natural killer T cell populations in a plaque are
reduced.
[0132] In one example of any method described herein, the inhibitor
of FXII is administered before or after the development of
atherosclerosis. In one example, the inhibitor of FXII is
administered before the development of atherosclerosis. In one
example, the inhibitor of FXII is administered after the
development of atherosclerosis.
[0133] In one example, the inhibitor of FXII is administered after
the onset of symptoms of atherosclerosis. In one example, the
inhibitor of FXII is administered at a dose that alleviates or
reduces one or more of the symptoms of atherosclerosis.
[0134] Symptoms of atherosclerosis will be apparent to the skilled
person and include, for example: [0135] chest pain on exertion or
angina; [0136] chest pain at rest; [0137] pain in the arms,
shoulder, abdomen, or jaw; [0138] cardiac arrest; [0139] shortness
of breath; [0140] being generally unwell; [0141] numbness or
weakness in the subject's arm(s) or leg(s); [0142] difficulty
speaking or slurred speech; [0143] temporary loss of vision; [0144]
drooping muscles in the subject's face; and/or [0145] fatigue.
[0146] In one example of any method or an inhibitor of FXII for use
described herein, the subject is a mammal, for example a primate,
such as a human.
[0147] Methods of treatment or inhibitors of FXII for use described
herein can additionally comprise administering a further compound
to reduce, treat or prevent the effect of atherosclerosis.
[0148] The present disclosure also provides a composition
comprising an inhibitor of FXII for use in treating or preventing
atherosclerosis in a subject in need thereof.
[0149] The present disclosure also provides use of an inhibitor of
FXII in the manufacture of a medicament for treating or preventing
atherosclerosis in a subject.
[0150] The present disclosure also provides a kit comprising at
least one inhibitor of FXII packaged with instructions for use in
treating or preventing atherosclerosis in a subject. Optionally,
the kit additionally comprises a therapeutically active compound or
drug.
[0151] The present disclosure also provides a kit comprising at
least one inhibitor of FXII packaged with instructions to
administer the inhibitor of FXII to a subject who is suffering from
or at risk of suffering from atherosclerosis, optionally, in
combination with a therapeutically active compound or drug.
[0152] Exemplary effects of atherosclerosis and inhibitors of FXII
are described herein and are to be taken to apply mutatis mutandis
to the examples of the disclosure set out in the previous five
paragraphs.
[0153] An inventor has also produced an inhibitor of FXII, e.g., an
anti-FXII antibody or antigen binding fragment thereof suitable for
use in treating a human subject. This inhibitor is an affinity
matured human antibody that has been modified to make most, but not
all, residues in the framework regions the same as those in a
germline human antibody thereby reducing the potential for
immunogenicity. This antibody is also capable of inhibiting FXIIa
and has good manufacturability characteristics. Thus, the present
disclosure also provides an anti-FXII antibody or antigen binding
fragment thereof, wherein the anti-FXII antibody comprises a
V.sub.H comprising a sequence set forth in SEQ ID NO: 18 and a
V.sub.L comprising a sequence set forth in SEQ ID NO: 19.
[0154] In one example, the anti-FXII antibody comprises lambda
light chain constant regions.
[0155] In one example, the anti-FXII antibody comprises IgG4 or
stabilized IgG4 constant regions. For example, the stabilized IgG4
constant regions comprise a proline at position 241 of the hinge
region according to the system of Kabat (Kabat et al., Sequences of
Proteins of Immunological Interest Washington D.C. United States
Department of Health and Human Services, 1987 and/or 1991).
[0156] In one example, the anti-FXII antibody comprises a heavy
chain comprising a sequence set forth in SEQ ID NO: 20 and a light
chain comprising a sequence set forth in SEQ ID NO: 21.
[0157] In one example, the disclosure provides a composition
comprising the anti-FXII antibody or antigen binding fragment and a
carrier, e.g., a pharmaceutically acceptable carrier.
[0158] In one example, the composition additionally comprises one
or more variants of the protein or antibody. For example, that
comprises a variant missing an encoded C-terminal lysine residue, a
deamidated variant and/or a glycosylated variant and/or a variant
comprising a pyroglutamate, e.g., at the N-terminus of a protein
and/or a variant lacking a N-terminal residue, e.g., a N-terminal
glutamine in an antibody or V region and/or a variant comprising
all or part of a secretion signal. Deamidated variants of encoded
asparagine residues may result in isoaspartic, and aspartic acid
isoforms being generated or even a succinamide involving an
adjacent amino acid residue. Deamidated variants of encoded
glutamine residues may result in glutamic acid. Compositions
comprising a heterogeneous mixture of such sequences and variants
are intended to be included when reference is made to a particular
amino acid sequence.
[0159] The present disclosure also provides the anti-FXII antibody
or antigen binding fragment thereof for medical use.
[0160] The present disclosure also provides a method for treating
or preventing a disorder in a subject, the method comprising
administering the anti-FXII antibody or antigen binding fragment
thereof, wherein the disorder is selected from the group consisting
of venous, arterial or capillary thrombus formation, thrombus
formation in the heart, thrombus formation during and/or after
contacting blood of a human or animal subject with artificial
surfaces, thromboembolism, by preventing the formation and/or the
stabilization of thrombi and thereby three-dimensional intraluminal
thrombus growth, or by preventing and/or treating intraluminal
thrombi; interstitial lung disease, inflammation, a neurological
inflammatory disease, complement activation, fibrinolysis,
angiogenesis and diseases related to FXII/FXIIa-induced kinin
formation or FXII/FXIIa-mediated complement activation.
[0161] The present disclosure also provides a method of treating
intraluminal thrombi in a human or animal subject, the method
comprising administering an anti-FXII antibody or antigen binding
fragment thereof to the subject, wherein the intraluminal thrombi
are related to a disorder selected from the group consisting of
venous, arterial or capillary thrombus formation, thrombus
formation in the heart, thrombus formation during and/or after
contacting blood of a human or animal subject with artificial
surfaces, thromboembolism; interstitial lung disease, inflammation,
a neurological inflammatory disease, complement activation,
fibrinolysis, angiogenesis and diseases related to
FXII/FXIIa-induced kinin formation or FXII/FXIIa-mediated
complement activation. For example, the venous or arterial thrombus
formation is stroke, myocardial infarction, deep vein thrombosis,
portal vein thrombosis, renal vein thrombosis, jugular vein
thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome
or Paget-Schroetter disease.
[0162] In one example, the diseases related to FXII/FXIIa-induced
kinin formation are selected from the group hereditary angioedema,
bacterial infections of the lung, trypanosoma infections,
hypotensive shock, pancreatitis, chagas disease, articular gout,
arthritis, disseminated intravascular coagulation (DIC) and
sepsis.
[0163] In one example, the interstitial lung disease is
fibroproliferative and/or idiopathic pulmonary fibrosis.
[0164] In one example, the thrombus formation occurs during and/or
after contacting blood of a human or animal subject with artificial
surfaces during and/or after a medical procedure performed on said
human or animal subject and said antibody or antigen-binding
fragment thereof is administered before and/or during and/or after
said medical procedure, and further [0165] (i) the artificial
surface is exposed to at least 80% of the blood volume of the
subject and the artificial surface is at least 0.2 m.sup.2 or
[0166] (ii) the artificial surface is a container for collection of
blood outside the body of the subject or [0167] (iii) the
artificial surface is a stent, valve, intraluminal catheter, or a
system for internal assisted pumping of blood.
[0168] The present disclosure also provides a medical device coated
with the antibody or antigen-binding fragment thereof of the
invention, wherein the device is a cardiopulmonary bypass machine,
an extracorporeal membrane oxygenation system for oxygenation of
blood, a device for assisted pumping of blood, a blood dialysis
device, a device for the extracorporeal filtration of blood, a
repository for use in the collection of blood, an intraluminal
catheter, a stent, an artificial heart valve, and/or accessories
for any one of said devices including tubing, cannulae, centrifugal
pump, valve, port, and/or diverter.
[0169] The present disclosure also provides a method comprising
administering the anti-FXII antibody or antigen binding fragment
thereof to a patient receiving a medical procedure, wherein the
medical procedure comprises contact with at least one of: [0170]
(a) heart, [0171] (b) at least one blood vessel chosen from: the
aorta, the aortic arch, a carotid artery, a coronary artery,
brachiocephalic artery, vertebrobasilar circulation, intracranial
arteries, renal artery, a hepatic artery, a mesenteric artery,
and/or a blood vessel of the arterial system cranial to the heart,
[0172] (c) a venous blood vessel if the patient has a known septal
defect; and wherein the medical procedure comprises release of at
least one embolus in at least one of said blood vessels in the body
that could result in ischemia in at least one target organ and
administration of the antibody or antigen binding fragment thereof
before, during and/or after the medical procedure.
[0173] The present disclosure also provides a method for treating
or preventing a condition associated with increased vascular
permeability, in particular increased retinal vascular
permeability, including progressive retinopathy, sight-threatening
complication of retinopathy, macular edema, non-proliferative
retinopathy, proliferative retinopathy, retinal edema, diabetic
retinopathy, hypertensive retinopathy, and retinal trauma, wherein
the method comprises administering the anti-FXII antibody or
antigen binding fragment thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0174] FIG. 1 is a graphical representation showing the effect of
anti-FXII antibody treatment on atherosclerosis development in
aortic sinus lesions (A-E) and aortic arch lesions (F-H). Anti-FXII
antibody treatment attenuates total lesion size (A, F); lipid
accumulation (B, G), macrophage numbers (C, H), collagen
accumulation (D) and necrotic core area (E). Mean.+-.SEM; n=6; *:
P<0.05 compared to control, unpaired T-test.
[0175] FIG. 2 is a graphical representation showing flow cytometric
analysis of the lymphocyte profile in blood of mice with
atherosclerotic lesions treated with anti-FXII antibody.
Mean.+-.SEM; n=6; *: P<0.05 compared to control, unpaired
T-test.
[0176] FIG. 3 is a graphical representation showing flow cytometric
analysis of lymphocyte profile in lymph nodes of mice with
atherosclerotic lesions treated with anti-FXII. Mean.+-.SEM; n=6;
*: P<0.05 compared to control, unpaired T-test.
[0177] FIG. 4 is a graphical representation showing anti-FXII
treatment reduces local inflammation and results in plaque
stabilization of aortic sinus lesions (A, C, E, G) and aortic arch
lesions (B, D, F, H). Aortic sinus lesions were analysed for VCAM-1
expression (A, B); necrotic core area (C, D), .alpha.-smooth muscle
actin expression (E, F) and collagen accumulation (G, H).
Mean.+-.SEM; n=6; *: P<0.05 compared to control, unpaired
T-test.
[0178] FIG. 5 is a graphical representation showing anti-FXII
antibody treatment achieves plaque stabilization. Unstable
atherosclerotic plaques were generated in segment I using the
described tandem stenosis surgery (A) and analysed for
atherosclerotic plaque area (B); lipid content (C); macrophage
accumulation (D); VCAM-1 expression (E); necrotic core area (F);
.alpha.-smooth muscle actin expression (G) and collagen
accumulation (H). Mean.+-.SEM, anti-FXIIa mAb: n=17, IgG isotype
control: n=16, *: P<0.05 compared to control, unpaired
T-test.
Key to Sequence Listing
[0179] SEQ ID NO: 1 is an amino acid sequence of wild-type
Infestin-4 SEQ ID NO: 2 is an amino acid sequence of wild-type
SPINK-1 SEQ ID NO: 3 is an amino acid sequence of SPINK-1 mutant K1
SEQ ID NO: 4 is an amino acid sequence of SPINK-1 mutant K2 SEQ ID
NO: 5 is an amino acid sequence of SPINK-1 mutant K3 SEQ ID NO: 6
is an amino acid sequence from the V.sub.H of anti-FXII antibody
3F7 SEQ ID NO: 7 is an amino acid sequence from the V.sub.L of
anti-FXII antibody 3F7 SEQ ID NO: 8 is an amino acid sequence from
a V.sub.H CDR1 of an anti-FXII antibody SEQ ID NO: 9 is an amino
acid sequence from a V.sub.H CDR2 of an anti-FXII antibody SEQ ID
NO: 10 is an amino acid sequence from a V.sub.H CDR2 of an
anti-FXII antibody SEQ ID NO: 11 is an amino acid sequence from a
V.sub.H CDR3 of an anti-FXII antibody SEQ ID NO: 12 is an amino
acid sequence from a V.sub.H CDR3 of an anti-FXII antibody SEQ ID
NO: 13 is an amino acid sequence from a V.sub.L CDR1 of an
anti-FXII antibody SEQ ID NO: 14 is an amino acid sequence from a
V.sub.L CDR2 of an anti-FXII antibody SEQ ID NO: 15 is an amino
acid sequence from a V.sub.L CDR3 of an anti-FXII antibody SEQ ID
NO: 16 is an amino acid sequence from a V.sub.L CDR3 of an
anti-FXII antibody SEQ ID NO: 17 is an amino acid sequence from a
V.sub.L CDR1 of an anti-FXII antibody SEQ ID NO: 18 is an amino
acid sequence of the V.sub.H of anti-FXII antibody gVR115 SEQ ID
NO: 19 is an amino acid sequence of the V.sub.L of anti-FXII
antibody gVR115 SEQ ID NO: 20 is an amino acid sequence of the
heavy chain of anti-FXII antibody gVR115 SEQ ID NO: 21 is an amino
acid sequence of the light chain of anti-FXII antibody gVR115 SEQ
ID NO: 22 is an amino acid sequence from a human Factor XII SEQ ID
NO: 23 is an amino acid sequence of a mature form of human albumin
SEQ ID NO: 24 is an amino acid sequence of an Infestin-4 variant
SEQ ID NO: 25 is an amino acid sequence of an Infestin-4
variant
DESCRIPTION
General
[0180] Throughout this specification, unless specifically stated
otherwise or the context requires otherwise, reference to a single
step, composition of matter, group of steps or group of
compositions of matter shall be taken to encompass one and a
plurality (i.e. one or more) of those steps, compositions of
matter, groups of steps or groups of compositions of matter.
[0181] Those skilled in the art will appreciate that the present
disclosure is susceptible to variations and modifications other
than those specifically described. It is to be understood that the
disclosure includes all such variations and modifications. The
disclosure also includes all of the steps, features, compositions
and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations or any
two or more of said steps or features.
[0182] The present disclosure is not to be limited in scope by the
specific examples described herein, which are intended for the
purpose of exemplification only. Functionally-equivalent products,
compositions and methods are clearly within the scope of the
present disclosure.
[0183] Any example of the present disclosure herein shall be taken
to apply mutatis mutandis to any other example of the disclosure
unless specifically stated otherwise.
[0184] Unless specifically defined otherwise, all technical and
scientific terms used herein shall be taken to have the same
meaning as commonly understood by one of ordinary skill in the art
(for example, in cell culture, molecular genetics, immunology,
immunohistochemistry, protein chemistry, and biochemistry).
[0185] Unless otherwise indicated, the recombinant protein, cell
culture, and immunological techniques utilized in the present
disclosure are standard procedures, well known to those skilled in
the art. Such techniques are described and explained throughout the
literature in sources such as, J. Perbal, A Practical Guide to
Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al.
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989), T. A. Brown (editor), Essential Molecular
Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991),
D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical
Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel
et al. (editors), Current Protocols in Molecular Biology, Greene
Pub. Associates and Wiley-Interscience (1988, including all updates
until present), Ed Harlow and David Lane (editors) Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E.
Coligan et al. (editors) Current Protocols in Immunology, John
Wiley & Sons (including all updates until present).
[0186] The description and definitions of variable regions and
parts thereof, immunoglobulins, antibodies and fragments thereof
herein may be further clarified by the discussion in Kabat
Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J
Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol.
196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989 and/or
or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.
[0187] Any discussion of a protein or antibody herein will be
understood to include any variants of the protein or antibody
produced during manufacturing and/or storage. For example, during
manufacturing or storage an antibody can be deamidated (e.g., at an
asparagine or a glutamine residue) and/or have altered
glycosylation and/or have a glutamine residue converted to
pyroglutamine and/or have a N-terminal or C-terminal residue
removed or "clipped" and/or have part or all of a signal sequence
incompletely processed and, as a consequence, remain at the
terminus of the antibody. It is understood that a composition
comprising a particular amino acid sequence may be a heterogeneous
mixture of the stated or encoded sequence and/or variants of that
stated or encoded sequence.
[0188] The term "and/or", e.g., "X and/or Y" shall be understood to
mean either "X and Y" or "X or Y" and shall be taken to provide
explicit support for both meanings or for either meaning.
[0189] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0190] As used herein the term "derived from" shall be taken to
indicate that a specified integer may be obtained from a particular
source albeit not necessarily directly from that source.
Selected Definitions
[0191] Coagulation Factor XII, also known as Hageman factor or
FXII, is a plasma protein. It is the zymogen form of Factor XIIa,
an enzyme of the serine protease (or serine endopeptidase) class.
In humans, Factor XII is encoded by the F12 gene. For the purposes
of nomenclature only and not limitation exemplary sequences of
human Factor XII is set out in NCBI Reference Sequence:
NP_000496.2; in NCPI protein accession number NP_000496 and in SEQ
ID NO: 22. Additional sequences of Factor XII can be determined
using sequences provided herein and/or in publically available
databases and/or determined using standard techniques (e.g., as
described in Ausubel et al., (editors), Current Protocols in
Molecular Biology, Greene Pub. Associates and Wiley-Interscience
(1988, including all updates until present) or Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989)).
[0192] As used herein, the term "Factor XII inhibitor" or "FXII
inhibitor" or "inhibitor of FXII" refers to an inhibitor of either
or both of Factor XII (prior to activation, i.e., its zymogen) and
activated Factor XII (FXIIa) as well as to the activation of FXII.
Thus, "inhibitor(s) of FXII" can include inhibitors of either or
both of FXII and FXIIa (also termed .alpha.FXIIa) as well as the
activation of FXII, including the FXIIa cleavage products FXIIa
alpha and FXIIa beta (also termed FXIIf). FXII inhibitors encompass
functional variants and fragments of the wild-type inhibitor. A
functional variant or fragment is a molecule that retains at least
50% (e.g., about 50%, or about 60%, or about 70%, or about 80%, or
about 90%, or about 95%, or about 99%, or about 100%) of the
ability of the wild-type molecule to inhibit FXII, FXIIa or the
activation of FXII. In one example, the FXII inhibitors are
non-endogenous inhibitors; that is, they are not inhibitors that
occur naturally in the human or animal body.
[0193] The term "direct FXII inhibitor" or "direct inhibitor", as
used herein, refers to an inhibitor that acts via contact (e.g.,
binding) with FXII (or FXIIa), i.e., the FXII inhibitor binds to
FXII and/or FXIIa and inhibits its activity and/or activation. In
contrast, an indirect inhibitor may act without contacting FXII (or
FXIIa) protein. For example, antisense RNA can be used to decrease
expression of the FXII gene, or a small molecule can inhibit the
effects of FXIIa via interactions with downstream FXIIa reaction
partners like Factor XI; these do not interact directly with the
FXII protein. Thus, an indirect inhibitor, in contrast to a direct
inhibitor, acts upstream or downstream from the FXII protein. In
one example, the FXII inhibitors are specific to FXII or FXIIa, in
particular specific to human FXII or FXIIa.
[0194] As used herein, the term "binds" in reference to the
interaction of a protein or an antigen binding site thereof with an
antigen means that the interaction is dependent upon the presence
of a particular structure (e.g., an antigenic determinant or
epitope) on the antigen. For example, an antibody recognizes and
binds to a specific protein structure rather than to proteins
generally. If an antibody binds to epitope "A", the presence of a
molecule containing epitope "A" (or free, unlabeled "A"), in a
reaction containing labeled "A" and the protein, will reduce the
amount of labeled "A" bound to the antibody.
[0195] As used herein, the term "specifically binds" or "binds
specifically" shall be taken to mean that a protein or an antigen
binding site thereof reacts or associates more frequently, more
rapidly, with greater duration and/or with greater affinity with a
particular antigen or cell expressing same than it does with
alternative antigens or cells. For example, a protein or an antigen
binding site thereof binds to FXII (or FXIIa) with materially
greater affinity (e.g., 1.5 fold or 2 fold or 5 fold or 10 fold or
20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or
200 fold) than it does to other blood clotting factors or to
antigens commonly recognized by polyreactive natural antibodies
(i.e., by naturally occurring antibodies known to bind a variety of
antigens naturally found in humans). Generally, but not
necessarily, reference to binding means specific binding, and each
term shall be understood to provide explicit support for the other
term.
[0196] The term "amidolytic activity" refers to the ability of the
inhibitor of FXII to catalyse the hydrolysis of at least one
peptide bond in another polypeptide.
[0197] The term "identity" or "identical" as used herein refers to
the percentage number of amino acids that are identical or
constitute conservative substitutions. Homology may be determined
using sequence comparison programs such as GAP (Deveraux et al.,
1984, Nucleic Acids Research 12, 387-395), which is incorporated
herein by reference. In this way sequences of a similar or
substantially different length to those cited herein could be
compared by insertion of gaps into the alignment, such gaps being
determined, for example, by the comparison algorithm used by
GAP.
[0198] A "half-life enhancing polypeptide" or "HLEP" is a
polypeptide fusion partner that may increase the half-life of the
FXII inhibitor in vivo in a patient or in an animal. Examples
include albumin and immunoglobulins and their fragments, such as Fc
domains, or derivatives, which may be fused to a FXII inhibitor
directly or via a cleavable or non-cleavable linker. Ballance et
al. (WO 2001/79271) described fusion polypeptides comprising a
multitude of different therapeutic polypeptides fused to human
serum albumin.
[0199] As used herein, the terms "albumin" and "serum albumin"
encompass human albumin (HA) and variants thereof. For the purposes
of nomenclature only and not limitation exemplary sequences of the
full mature form of albumin is set out in SEQ ID NO: 23, as well as
albumin from other species and variants thereof. As used herein,
"albumin" refers to an albumin polypeptide or amino acid sequence,
or an albumin variant, having one or more functional activities
(e.g. biological activities) of albumin. In certain examples,
albumin is used to stabilize or prolong the therapeutic activity of
a FXII inhibitor. The albumin may be derived from any vertebrate,
especially any mammal, for example human, monkey, cow, sheep, or
pig. Non-mammalian albumin can also be used and includes, but is
not limited to, albumin from chicken and salmon. The albumin
portion of the albumin-linked polypeptide may be from a different
animal than the therapeutic polypeptide portion. See WO 2008/098720
for examples of albumin fusion proteins, incorporated herein by
reference in its entirety.
[0200] The term "recombinant" shall be understood to mean the
product of artificial genetic recombination. Accordingly, in the
context of a recombinant protein comprising an antibody variable
region, this term does not encompass an antibody
naturally-occurring within a subject's body that is the product of
natural recombination that occurs during B cell maturation.
However, if such an antibody is isolated, it is to be considered an
isolated protein comprising an antibody variable region. Similarly,
if nucleic acid encoding the protein is isolated and expressed
using recombinant means, the resulting protein is a recombinant
protein. A recombinant protein also encompasses a protein expressed
by artificial recombinant means when it is within a cell, tissue or
subject, e.g., in which it is expressed.
[0201] The term "protein" shall be taken to include a single
polypeptide chain, i.e., a series of contiguous amino acids linked
by peptide bonds or a series of polypeptide chains covalently or
non-covalently linked to one another (i.e., a polypeptide complex).
For example, the series of polypeptide chains can be covalently
linked using a suitable chemical or a disulfide bond. Examples of
non-covalent bonds include hydrogen bonds, ionic bonds, Van der
Waals forces, and hydrophobic interactions.
[0202] The term "polypeptide" or "polypeptide chain" will be
understood from the foregoing paragraph to mean a series of
contiguous amino acids linked by peptide bonds.
[0203] The skilled artisan will be aware that an "antibody" is
generally considered to be a protein that comprises a variable
region made up of a plurality of polypeptide chains, e.g., a
polypeptide comprising a light chain variable region (V.sub.L) and
a polypeptide comprising a heavy chain variable region (V.sub.H).
An antibody also generally comprises constant domains, some of
which can be arranged into a constant region, which includes a
constant fragment or fragment crystallizable (Fc), in the case of a
heavy chain. A V.sub.H and a V.sub.L interact to form a Fv
comprising an antigen binding region that is capable of
specifically binding to one or a few closely related antigens.
Generally, a light chain from mammals is either a .kappa. light
chain or a .lamda. light chain and a heavy chain from mammals is
.alpha., .delta., , .gamma., or .mu.. Antibodies can be of any type
(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2) or
subclass. The term "antibody" also encompasses humanized
antibodies, primatized antibodies, human antibodies, synhumanized
antibodies and chimeric antibodies.
[0204] An "anti-FXII antibody" includes antibodies that bind to
and/or inhibit either or both of the zymogen of FXII and the
activated protein (FXIIa), including the FXIIa alpha and FXIIa beta
cleavage fragments. In some examples, the antibody binds
specifically to FXIIa or the alpha or beta chain fragments of
FXIIa.
[0205] The terms "full-length antibody," "intact antibody" or
"whole antibody" are used interchangeably to refer to an antibody
in its substantially intact form, as opposed to an antigen binding
fragment of an antibody. Specifically, whole antibodies include
those with heavy and light chains including an Fc region. The
constant domains may be wild-type sequence constant domains (e.g.,
human wild-type sequence constant domains) or amino acid sequence
variants thereof.
[0206] As used herein, "variable region" refers to the portions of
the light and/or heavy chains of an antibody as defined herein that
is capable of specifically binding to an antigen and includes amino
acid sequences of complementarity determining regions (CDRs); i.e.,
CDR1, CDR2, and CDR3, and framework regions (FRs). Exemplary
variable regions comprise three or four FRs (e.g., FR1, FR2, FR3
and optionally FR4) together with three CDRs. In the case of a
protein derived from an IgNAR, the protein may lack a CDR2. V.sub.H
refers to the variable region of the heavy chain. V.sub.L refers to
the variable region of the light chain.
[0207] As used herein, the term "complementarity determining
regions" (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the
amino acid residues of an antibody variable domain the presence of
which are necessary for antigen binding. Each variable domain
typically has three CDR regions identified as CDR1, CDR2 and CDR3.
The amino acid positions assigned to CDRs and FRs can be defined
according to Kabat Sequences of Proteins of Immunological Interest,
National Institutes of Health, Bethesda, Md., 1987 and 1991 or
other numbering systems in the performance of this disclosure,
e.g., the canonical numbering system of Chothia and Lesk J. Mol
Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989;
and/or AI-Lazikani et al., J Mol Biol 273: 927-948, 1997; the IMGT
numbering system of Lefranc et al., Devel. And Compar. Immunol.,
27: 55-77, 2003; or the AHO numbering system of Honnegher and
Plukthun J. Mol. Biol., 309: 657-670, 2001.
[0208] "Framework regions" (FRs) are those variable domain residues
other than the CDR residues. As used herein, the term "variable
region fragment" or "Fv" shall be taken to mean any protein,
whether comprised of multiple polypeptides or a single polypeptide,
comprising a V.sub.L and a V.sub.H, wherein the V.sub.L and a
V.sub.H are associated to form a complex having an antigen binding
site, i.e., capable of specifically binding to an antigen. The
V.sub.H and the V.sub.L which form the antigen binding site can be
in a single polypeptide chain or in different polypeptide chains.
Furthermore, an Fv of the disclosure (as well as any protein of the
disclosure) may have multiple antigen binding sites which may or
may not bind the same antigen. This term shall be understood to
encompass fragments directly derived from an antibody as well as
proteins corresponding to such a fragment produced using
recombinant means. In some examples, the V.sub.H is not linked to a
heavy chain constant domain (C.sub.H)1 and/or the V.sub.L is not
linked to a light chain constant domain (C.sub.L). Exemplary Fv
containing polypeptides or proteins include a Fab fragment, a Fab'
fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a
tetrabody or higher order complex, or any of the foregoing linked
to a constant region or domain thereof, e.g., C.sub.H2 or C.sub.H3
domain, e.g., a minibody or an antibody. A "Fab fragment" consists
of a monovalent antigen-binding fragment of an antibody, and can be
produced by digestion of a whole antibody with the enzyme papain,
to yield a fragment consisting of an intact light chain and a
portion of a heavy chain or can be produced using recombinant
means. A "Fab' fragment" of an antibody can be obtained by treating
a whole antibody with pepsin, followed by reduction, to yield a
molecule consisting of an intact light chain and a portion of a
heavy chain comprising a V.sub.H and a single constant domain. Two
Fab' fragments are obtained per antibody treated in this manner. A
Fab' fragment can also be produced by recombinant means. A "F(ab')2
fragment" of an antibody consists of a dimer of two Fab' fragments
held together by two disulfide bonds, and is obtained by treating a
whole antibody molecule with the enzyme pepsin, without subsequent
reduction. A "Fab.sub.2" fragment is a recombinant fragment
comprising two Fab fragments linked using, for example a leucine
zipper or a C.sub.H3 domain. A "single chain Fv" or "scFv" is a
recombinant molecule containing the Fv of an antibody in which the
variable region of the light chain and the variable region of the
heavy chain are covalently linked by a suitable, flexible
polypeptide linker. As will be apparent from the foregoing
discussion, this term encompasses an antibody or an antigen binding
fragment thereof comprising a V.sub.H and a V.sub.L.
[0209] As used herein, the terms "preventing", "prevent" or
"prevention" include administering a compound of the disclosure to
thereby stop or hinder the development of at least one symptom of a
condition.
[0210] As used herein, the terms "treating", "treat" or "treatment"
include administering a protein described herein to thereby reduce
or eliminate at least one symptom of a specified disease or
condition or to slow progression of the disease or condition.
[0211] As used herein, the term "subject" shall be taken to mean
any animal including humans, for example a mammal. Exemplary
subjects include but are not limited to humans and non-human
primates. For example, the subject is a human.
Treating or Preventing Atherosclerotic Lesions
[0212] The disclosure herein provides, for example, a method for
treating or preventing atherosclerosis in a subject comprising
administering to the subject an inhibitor of Factor XII.
[0213] The disclosure also provides, a method for preventing
atherosclerotic plaque rupture in a subject comprising
administering to the subject an inhibitor of Factor XII.
[0214] In one example, the subject suffers from atherosclerosis, or
atherosclerotic lesions. The atherosclerotic lesion can be stable
or unstable. In one example, the atherosclerotic lesion is
unstable. For example, a subject suffering from atherosclerosis has
suffered a clinically acceptable symptom of atherosclerosis, such
as: [0215] chest pain on exertion or angina; [0216] chest pain at
rest; [0217] pain in the arms, shoulder, abdomen, or jaw; [0218]
cardiac arrest; [0219] shortness of breath; [0220] being generally
unwell; [0221] numbness or weakness in the subject's arm(s) or
leg(s); [0222] difficulty speaking or slurred speech; [0223]
temporary loss of vision; [0224] drooping muscles in the subject's
face; and/or [0225] fatigue.
[0226] In one example, the subject has suffered or suffers from a
condition associated with atherosclerosis. For example, the subject
has suffered a myocardial infarct. In one example, the subject has
suffered a stroke.
[0227] The methods of the present disclosure can be readily applied
to any form of atherosclerosis in the arterial system. For example,
the subject can present with sign(s) and/or symptoms of
atherosclerosis of the heart, or brain, or legs, or pelvis, or
arms, or kidneys. Thus, the methods of the present disclosure will
be taken to apply to treating or preventing atherosclerosis in the
arterial system.
[0228] In one example, the subject is at risk of developing
atherosclerosis, or atherosclerotic lesions, but the onset of
atherosclerosis has not yet occurred. A subject is at risk if he or
she has a higher risk of developing atherosclerosis than a control
population. The control population may include one or more subjects
selected at random from the general population (e.g., matched by
age, gender, race and/or ethnicity) who have not suffered from or
have a family history of angina, stroke and/or heart attack. A
subject can be considered at risk for atherosclerosis if a "risk
factor" associated with atherosclerosis is found to be associated
with that subject. A risk factor can include any activity, trait,
event or property associated with a given disorder, for example,
through statistical or epidemiological studies on a population of
subjects. A subject can thus be classified as being at risk for
atherosclerosis even if studies identifying the underlying risk
factors did not include the subject specifically. For example, a
subject who has high plasma low density lipoprotein levels is at
risk of developing atherosclerosis because the frequency of
atherosclerosis is increased in a population of subjects who have
high plasma low density lipoprotein levels as compared to a
population of subjects who do not.
[0229] In one example, a subject at risk of atherosclerosis include
those patients who are hyper-cholesterolemic, diabetic, obese
and/or hypertensive. Subjects especially at a high risk are those
who have already suffered from angina, and/or stroke and/or heart
attack.
[0230] As discussed above, methods of the disclosure achieve one or
more of the following effects: [0231] reducing the likelihood of
occlusive arterial thrombus in a subject; [0232] reducing
atherosclerotic plaque lesion size in a subject; [0233] increasing
plaque stabilization in a subject; [0234] reducing inflammatory
cell accumulation in an atherosclerotic plaque lesion in a subject;
and/or [0235] reducing pro-atherogenic cell populations in a
subject.
[0236] Methods for assessing plaque lesion size are known in the
art and include, for example, angiographic analysis, intravascular
ultrasound or optical coherence tomography (OCT).
[0237] Methods for assessing plaque stabilization are known in the
art and include, for example, intravascular ultrasound analysis or
MRI of carotid arteries.
[0238] In one example, a method of the disclosure reduces any
symptom of atherosclerosis known in the art or described
herein.
[0239] As will be apparent to the skilled person a "reduction" in a
symptom or effect of atherosclerosis in a subject will be
comparative to another subject who has also suffered from
atherosclerosis but who has not received treatment with a method
described herein or to the subject prior to treatment. This does
not necessarily require a side-by-side comparison of two subjects.
Rather population data can be relied upon. For example a population
of subjects suffering from atherosclerosis who have not received
treatment with a method described herein (optionally, a population
of similar subjects to the treated subject, e.g., age, weight,
diabetic status, cholesterol levels) are assessed and the mean
values are compared to results of a subject or population of
subjects treated with a method described herein.
Inhibitors of Factor XII
[0240] In one example, the inhibitor of FXII is a direct FXII
inhibitor, such as a specific FXII inhibitor. For example, the
specific FXII inhibitor inhibits plasmatic serine proteases or
other endogenous proteins other than FXII and/or FXIIa less than or
equal to about 25% if used in a molar ratio of 1:1. For example,
the specific inhibitor of FXII/FXIIa inhibitor inhibits plasmatic
serine proteases other than FXII and/or FXIIa less than or equal to
about 25% when said inhibitor is used in a molar ratio of 1:1 of
the respective plasmatic serine protease to said inhibitor. In one
example, the FXII inhibitor inhibits plasmatic serine proteases
other than FXII and/or FXIIa less than or equal to about 20%, or
less than or equal to about 15%, or less than or equal to about
10%, or less than or equal to about 5%, or less than or equal to
about 1% if used in a molar ratio of 1:1. For example, a specific
FXII antibody inhibits the plasmatic serine protease FXIa by about
5%, wherein the molar ratio of FXIa to said antibody is 1:1 whereas
the same FXII antibody inhibits FXIIa by at least about 80%, or at
least about 90%.
[0241] In one example, one other plasmatic serine protease is
inhibited by more than about 50% if used in a molar ratio of 1:1 of
the respective plasmatic serine protease to the inhibitor.
[0242] In another example of the disclosure, two other plasmatic
serine proteases are inhibited by more than about 50% if used in a
molar ratio of 1:1 of the respective plasmatic serine protease to
the inhibitor.
Serine Protease Inhibitors
[0243] In one example, the inhibitor of FXII is a serine protease
inhibitor. For example, the inhibitor of FXII comprises a sequence
corresponding to Infestin-4 or variants thereof. In one example,
the inhibitor of FXII comprises a sequence corresponding to SPINK-1
or variants thereof.
Infestin-4
[0244] In one example, the disclosure provides an inhibitor of FXII
comprising infestin domain 4 (referred to as "Infestin-4").
Infestins are a class of serine protease inhibitors derived from
the midgut of the hematophagous insect, Triatoma infestans, a major
vector for the parasite Trypanosoma cruzi, known to cause Chagas
disease. (Campos TIN et al. 32 Insect Biochem. Mol. Bio. 991-997,
2002; Campos ITN et al. 577 FEBS Lett. 512-516, 2004; WO
2008/098720.) This insect uses these inhibitors to prevent
coagulation of ingested blood. The infestin gene encodes 4 domains
that result in proteins that can inhibit different factors in the
coagulation pathway. In particular, domain 4 encodes a protein
(Infestin-4) that is a strong inhibitor of FXIIa. Infestin-4 has
been administered in mice without resulting in bleeding
complications. (WO 2008/098720; Hagedorn et al., Circulation 2010;
121:1510-17.)
[0245] In one embodiment, the inhibitor of FXII comprises
Infestin-4. The term "Infestin-4," as used herein, encompasses
variants or fragments of the wild-type peptide that retain the
ability to inhibit FXII. For the purposes of nomenclature only and
not limitation an exemplary sequence of Infestin-4 is set out in
SEQ ID NO: 1.
[0246] In one example, the Infestin-4 is chosen for its ability to
inhibit FXIIa. In one example, the Infestin-4 comprises a variant
of Infestin-4, wherein the variant comprises Infestin domain 4, and
optionally, Infestin domains 1, 2, and/or 3. In one example, the
Infestin-4 is a (His).sub.6-tagged Infestin-4 construct.
[0247] In another example, the Infestin-4 is a fusion protein
comprising a fusion partner, such as a half-life enhancing
polypeptide (e.g., albumin, an Fc domain of an IgG, or PEG), linked
or bound to infestin-4. In one example, a linker connects the
fusion partner to Infestin-4. In various embodiments, the
Infestin-4 is the rHA-Infestin-4 protein described in Hagedorn et
al., Circulation 2010; 117:1153-60. In one example, a composition
comprises albumin bound to the rHA-Infestin-4 protein described in
Hagedorn et al., Circulation 2010; 117:1153-60, by a flexible
linker. In another example, other Infestin-4 inhibitors of FXII are
used, examples of which are described in WO 2008/098720 and
Hagedorn et al., Circulation 2010; 117:1153-60, both of which are
incorporated by reference in their entirety.
[0248] In one example, the inhibitor of FXII is a variant of
Infestin-4. As used here, the term "variant" of Infestin-4 refers
to a polypeptide with one or more amino acid mutation, wherein
"mutation" is defined as a substitution, a deletion, or an
addition, to the wild type Infestin-4 sequence (SEQ ID NO: 1). The
term "variant" of Infestin-4 also includes functional fragments of
the wild type or a mutated Infestin-4 sequence.
[0249] In one example, the one or more mutations to the wild type
Infestin-4 sequence do not substantially alter the functional
ability of the polypeptide to inhibit FXII. For example, the one or
more mutations do not completely or substantially remove the
ability of the polypeptide to inhibit FXII. For example, the
variant retains at least about 20%, or about 30%, or about 40%, or
about 50%, or about 60%, or about 70%, or about 80%, or about 90%,
or about 95%, or about 98%, or about 99%, or more of the inhibitory
ability of wild type Infestin-4.
[0250] In one example, the inhibitor of FXII comprises an
Infestin-4 variant comprising residues 2-13 from the amino terminal
of the wild type Infestin-4 sequence as set forth in SEQ ID NO: 1.
For example, the Infestin-4 variant comprises the amino acid
sequence set forth in SEQ ID NO: 24.
[0251] In one example, the inhibitor of FXII comprises an
Infestin-4 variant comprising residues 2-13 of SEQ ID NO: 1 and
also comprising at least one amino acid mutations, as compared to
the wild type Infestin-4 sequence (SEQ ID NO: 1), outside residues
2-13 of SEQ ID NO: 1. For example, the Infestin-4 variant comprises
at least two amino acid mutations, or at least three amino acid
mutations, or at least four amino acid mutations, or at least five
amino acid mutations. For example, the inhibitor of FXII comprises
a polypeptide sequence comprising SEQ ID NO: 1 modified to contain
between 1 and 5 amino acid mutations outside of N-terminal amino
acid positions 2-13 of SEQ ID NO: 1.
[0252] In one example, the inhibitor of FXII is an Infestin-4
variant which retains six conserved cysteine residues from the wild
type Infestin-4 sequence. In one example, the six conserved
cysteine residues are amino acids at positions 6, 8, 16, 27, 31,
and 48 of the wild type Infestin-4 sequence (SEQ ID NO: 1). In one
example, the Infestin-4 variant comprises the final conserved
cysteine at position 48. In another example, the exact positions of
the cysteine residues, and relative positions to each other, may
change from positions 6, 8, 16, 27, 31, and 48 of the wild type
Infestin-4 sequence due to insertions or deletions in the
Infestin-4 variant sequence.
[0253] In one example, the Infestin-4 variant is at least about 70%
identical to the wild type Infestin-4 sequence. For example, the
Infestin-4 has an identity of at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 98%, or at least about 99% to the wild type Infestin-4
sequence. For example, the inhibitor of FXII comprises a
polypeptide sequence comprising a sequence at least 70% identical
to SEQ ID NO: 1 and retaining six conserved cysteine residues from
SEQ ID NO: 1.
[0254] In one example, the inhibitor of FXII is an Infestin-4
variant retains six conserved cysteine residues from the wild type
Infestin-4 sequence and/or has a sequence of at least about 70%
identical to the wild type Infestin-4 sequence.
[0255] In one example, the inhibitor of FXII is an Infestin-4
variant comprising SEQ ID NO: 1 modified to contain 1-5 amino acid
mutations outside of N-terminal amino acid positions 2-13 of SEQ ID
NO: 1; and a sequence at least 70% identical to SEQ ID NO: 1 and
retaining six conserved cysteine residues from SEQ ID NO: 1.
[0256] In one example, an Infestin-4 variant comprises the
conserved N-terminal region amino acids 2-13 of the wild type
Infestin-4 sequence, and at least one, and optionally up to five,
amino acid mutations outside these conserved N-terminal amino
acids, resulting in differences from the wild type Infestin-4
sequence. As used here, the term "outside the N-terminal amino
acids" of an Infestin variant refers to any amino acid along the
polypeptide chain of the variant other than the contiguous stretch
of amino acids that comprises the sequence of SEQ ID NO: 24, which
are amino acids 2-13 from SEQ ID NO: 1.
[0257] In one example, an Infestin-4 variant comprises all six
conserved cysteine residues from SEQ ID NO: 1 and/or a sequence at
least about 70% identical to the wild type Infestin-4 sequence (SEQ
ID NO: 1). For example, the Infestin-4 variant may comprise a
sequence with about 70%, or about 75%, or about 85%, or about 90%,
or about 91%, or about 92%, or about 93%, or about 94%, or about
95%, or about 96%, or about 97%, or about 98% or about 99% identity
to the wild type Infestin-4 sequence (SEQ ID NO: 1).
[0258] In one example, the Infestin-4 variant retains amino acids
2-13 from SEQ ID NO: 1 as well as all six conserved cysteine
residues, and is at least about 70% identical to the wild type
Infestin-4 sequence (SEQ ID NO: 1). For example, the Infestin-4
variant may comprise a sequence with about 70%, or about 75%, or
about 85%, or about 90%, or about 91%, or about 92%, or about 93%,
or about 94%, or about 95%, or about 96%, or about 97%, or about
98% or about 99% identity to the wild type Infestin-4 sequence (SEQ
ID NO: 1).
[0259] In one embodiment, the FXII inhibitor comprises variant of
the wild type Infestin-4 polypeptide sequence, wherein the
Infestin-4 variant comprises the N-terminal amino acids 2-13 of SEQ
ID NO: 1; at least one, and optionally up to five, amino acid
mutations outside the N-terminal amino acids; six conserved
cysteine residues; and/or at least 70% identity to the wild type
Infestin-4 sequence (SEQ ID NO: 1). For example, the Infestin-4
variant comprises a sequence with at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, or at least
99% identity to the wild type Infestin-4 sequence (SEQ ID NO:
1).
[0260] In one example, an Infestin-4 variant comprises the sequence
set forth in SEQ ID NO: 25. In one example, SEQ ID NO: 24 is added
at or near the N-terminus of a fragment or full length wild type
Infestin-4 sequence (SEQ ID NO: 1) and derives from the human
protein SPINK-1.
[0261] In one example, an Infestin-4 comprises a fusion construct
between wild-type Infestin-4 or a variant Infestin-4 and a fusion
partner. For example, the fusion partner comprises a PEG or
half-life enhancing polypeptide. In one example, the Infestin the
half-life enhancing polypeptide comprises human albumin (referred
to as "HA"). In some embodiments, the HA is a recombinant protein
(referred to as "rHA"). In certain embodiments, the Infestin-4 and
HA proteins are joined directly, or via a linker peptide. For
example, the inhibitor of FXII is a fusion protein comprising human
albumin linked to an Infestin-4 via a linker peptide.
[0262] In one example, the inhibitor of FXII is a variant of
Infestin-4 that retains the ability to inhibit FXII. For example,
the variant of Infestin-4 has the same ability as Infestin-4 to
inhibit FXII. In one example, the variant of Infestin-4 inhibits
FXII activity and/or activation of FXII.
[0263] In one example, the inhibitor of FXII of the present
disclosure competes with Infestin-4 for binding to human Factor
XIIa-beta.
[0264] Methods for assessing functional inhibitory activity of FXII
are known in the art and include, for example, in vitro and/or in
vivo characterization, including direct assays to test inhibition
of FXII enzyme activity, prolonged coagulation time (e.g.,
activated partial thromboplastin time, aPTT), clinical clotting
tests that address the intrinsic pathway of coagulation, or in vivo
methods that evaluate coagulation.
Spink-1
[0265] In one example, the inhibitor of FXII comprises a human
protein with high similarity to Infestin-4. For example, the
inhibitor of FXII is SPINK-1. SPINK-1 is a Kazal-type serine
protease inhibitor expressed in the pancreas (also known as
pancreatic secretory trypsin inhibitor, or PSTI). The Kazal-type
serine protease inhibitor family is one of numerous families of
known serine protease inhibitors. Many similar proteins from
different species have been described (Laskowski M and Kato I, 49
Ann. Rev. Biochem. 593-626, 1980). For the purposes of nomenclature
only and not limitation an exemplary sequence of SPINK-1 is set out
in SEQ ID NO: 2.
[0266] In one example, the inhibitor of FXII comprises the
wild-type sequence of SPINK-2 as set out in SEQ ID NO: 2.
[0267] The term "SPINK-1" also encompasses functional variants and
fragments of SPINK-1 that substantially retain the ability to
inhibit FXII. In one example, the inhibitor of FXII is a SPINK-1
variant. For example, variants of the wild-type sequence (i.e., SEQ
ID NO: 2) may be generated in order to increase the identity of the
SPINK-1 sequence to Infestin-4. As used herein, the term "variant"
includes fragments of a SPINK-1 or mutated SPINK-1 sequence.
[0268] In one example, SPINK-1 (SEQ ID NO: 2) is mutated to replace
the N-terminal amino acids at positions 2-13 with the N-terminal
amino acids 2-13 of SEQ ID NO: 1.
[0269] In one example, the inhibitor of FXII is a SPINK-1 variant
comprising an N-terminal portion of a wild type Infestin-4
sequence. For example, the SPINK-1 variant comprises amino acids
2-13 of SEQ ID NO: 1.
[0270] In one example, the inhibitor of FXII is a SPINK-1 variant
comprising at least one additional amino acid mutation outside the
N-terminal amino acids that increase the identity of the SPINK-1
variant to the wild type Infestin-4 sequence. For example, the
SPINK-1 variant comprises at least one, or at least two, or at
least three, or at least four, or at least five additional amino
acid mutations outside the N-terminal amino acids that increase the
identity of the SPINK-1 variant to the wild type Infestin-4
sequence.
[0271] A mutation may comprise a substitution, a deletion, and/or
an addition. A mutation that is "outside the N-terminal amino
acids" refers to one or more mutations in any amino acids along the
polypeptide chain of the variant other than the contiguous stretch
of amino acids that comprises the sequence SEQ ID NO: 24, i.e.,
amino acids 2-13 of SEQ ID NO: 1.
[0272] In one example, the inhibitor of FXII comprises a
polypeptide sequence comprising SEQ ID NO: 2 mutated to replace the
N-terminal amino acids at positions 2-13 with the N-terminal amino
acids at positions 2-13 of SEQ ID NO: 1; and optionally further
modified to contain 1-5 additional amino acid mutations that
increase the identity of the polypeptide sequence to sequence of
SEQ ID NO: 1.
[0273] In one example, the inhibitor of FXII comprises a SPINK-1
variant comprising a SPINK-1 sequence that has been mutated to
increase the identity of the variant to the wild type Infestin-4
sequence (i.e., SEQ ID NO: 1).
[0274] In one example, the SPINK-1 variant comprises an N-terminal
portion of a wild type Infestin-4 sequence. For example, the
SPINK-1 variant comprises the amino acids 2-13 of SEQ ID NO: 1.
[0275] In one example, the inhibitor of FXII comprises a SPINK-1
variant comprising six conserved cysteine residues of SEQ ID NO:
2.
[0276] In one example, the six conserved cysteine residues of
SPINK-1 may be the amino acids at positions 9, 16, 24, 35, 38, and
56 of the wild type SPINK-1 sequence (e.g., SEQ ID NO: 2).
[0277] In one example, the variant comprises the final cysteine of
the wild type SPINK-1 sequence (i.e., the cysteine at position 56
of SEQ ID NO: 2). In one example, the six conserved cysteine
residues are not mutated but the exact positions of the cysteine
residues, and relative positions to each other, may change from
positions 9, 16, 24, 35, 38, and 56 of the wild type SPINK-1
sequence due to insertions and/or deletions elsewhere in the
SPINK-1 variant sequence. Nevertheless, in these examples, a
SPINK-1 variant comprises all six cysteine residues.
[0278] In one example, the inhibitor of FXII comprises a SPINK-1
variant comprising a sequence at least 70% identical to SEQ ID NO:
2. For example, the SPINK-1 variant comprises a sequence at least
about 75%, or at least about 80%, or at least about 85%, or at
least about 90%, or at least about 95%, or at least about 98%, or
at least about 99% identical to SEQ ID NO: 2.
[0279] In one example, the inhibitor of FXII comprises a SPINK-1
variant comprising a sequence at least 70% identical to SEQ ID NO:
2 and retaining six conserved cysteine residues from SEQ ID NO:
2.
[0280] In one example, the six conserved cysteine residues of
SPINK-1 are not mutated. In one example, the SPINK-1 sequence is
mutated to comprise an N-terminal portion of a wild type Infestin-4
sequence. For example, the SPINK-1 sequence is mutated to comprise
amino acids 2-13 of SEQ ID NO: 1.
[0281] In one example, the SPINK-1 sequence is mutated to comprise
an N-terminal portion of a wild type Infestin-4 sequence and/or to
have a sequence at least 70% identical to the wild type SPINK-1
sequence. For example, the SPINK-1 sequence is mutated to comprise
a sequence at least about 70%, or at least about 75%, or at least
about 80%, or at least about 85%, or at least about 90%, or at
least about 95%, or at least about 98%, or at least 99% identical
to the wild type SPINK-1 sequence.
[0282] In one example, the SPINK-1 sequence is mutated to comprise
an N-terminal portion of a wild type Infestin-4 sequence and/or to
have a sequence at least 70% identical to the wild type SPINK-1
sequence and/or to include at least one mutation in the SPINK-1
sequence outside the N-terminal amino acids.
[0283] In one example, the inhibitor of FXII comprises a SPINK-1
variant comprising SEQ ID NO: 2 mutated to replace the N-terminal
amino acids at positions 2-13 with the N-terminal amino acids at
positions 2-13 of SEQ ID NO: 1; and optionally further modified to
contain 1-5 additional amino acid mutations that increase the
identity of the polypeptide sequence to sequence of SEQ ID NO:
1.
[0284] In one example, the inhibitor of FXII comprises a SPINK-1
variant comprising SEQ ID NO: 2 mutated to replace the N-terminal
amino acids at positions 2-13 with the N-terminal amino acids at
positions 2-13 of SEQ ID NO: 1; and optionally further modified to
contain 1-5 additional amino acid mutations that increase the
identity of the polypeptide sequence to sequence of SEQ ID NO: 1
and a sequence at least 70% identical to SEQ ID NO: 2 and retaining
six conserved cysteine residues from SEQ ID NO: 2.
[0285] In one example, the inhibitor of FXII comprises a SPINK-1
variant that substantially retains its ability to inhibit FXII. For
example, the SPINK-1 variant retains at least about 20%, or about
30%, or about 40%, or about 50%, or about 60%, or about 70%, or
about 80%, or about 90%, or about 95%, or about 98%, or about 99%
of the inhibitory activity of wild type SPINK-1.
[0286] In one example, the inhibitor of FXII of the present
disclosure competes with SPINK-1 for binding to human Factor
XIIa-beta.
[0287] In one example, the inhibitor of FXII is a SPINK-1 variant
selected from the group consisting of K1 (SEQ ID NO: 3), K2 (SEQ ID
NO: 4), and K3 (SEQ ID NO: 5).
[0288] In one example, the SPINK-1 variant is K1, as set forth in
SEQ ID NO: 3.
[0289] In one example, the SPINK-1 variant is K2, as set forth in
SEQ ID NO: 4.
[0290] In one example, the SPINK-1 variant is K3, as set forth in
SEQ ID NO: 5.
[0291] In one example, further amino acid substitutions can be made
outside of the N-terminus relative to K1 in order to increase
identity to Infestin-4.
[0292] In one example, further amino acid substitutions can be made
outside of the N-terminus relative to K3 in order to increase
identity to Infestin-4. For example, five amino acid substitutions
outside of the N-terminus relative to K3 increase identity to
Infestin-4.
[0293] In one example, a SPINK-1 variant shares at least about 70%
identity with the wild type SPINK-1 sequence. For example, the
SPINK-1 variant shares at least about 75%, or at least about 80%,
at least about 85%, or at least about 90%, or at least about 91%,
or at least about 92%, or at least about 93%, or at least about
94%, or at least about 95%, or at least about 96%, or at least
about 97%, or at least about 98% or at least about 99% identity
with the wild type SPINK-1 sequence.
[0294] Factor XII Antibodies
[0295] An exemplary inhibitor of Factor XII (FXII) comprises an
antigen variable region, e.g., is an antibody or an antigen binding
fragment thereof that binds to FXII and neutralizes FXII
signalling. For example, the anti-FXII antibody or antigen binding
fragment thereof binds to and inhibits activation of and/or
activity of FXII and/or FXIIa.
[0296] In one example, the antibody variable region specifically
binds to FXII.
[0297] For example, the inhibitor of FXII binds to FXII and/or
FXIIa and inhibits the activity of FXII and/or FXIIa.
[0298] In another example, the inhibitor of FXII binds to FXII
and/or FXIIa and inhibits the activation of FXII to FXIIa.
[0299] Suitable antibodies and proteins comprising variable regions
thereof are known in the art.
[0300] For example, anti-Factor XII antibodies and fragments
thereof are described in WO 2006/066878, and in Ravon et al., Blood
86: 4134-43 (1995). Additional anti-Factor XII antibodies are
described in WO 2013/014092.
[0301] In one example, the anti-FXII antibody or antigen binding
fragment thereof is an antibody that binds to FXII and/or
FXIIa.
[0302] In one example, the activity of FXII and/or FXIIa is
inhibited by at least about 50%. For example, the activity of FXII
and/or FXIIa is inhibited by about 60%, or about 70%, or about 80%,
or about 85%, or about 90%, or about 95%, or about 99%, or about
100%.
[0303] In one example, the inhibitor of FXII of the present
disclosure inhibits FXIIa by at least about 80%. For example, the
inhibitor of FXII of the present disclosure inhibits Factor
XIIa-alpha by at least about 80% when used at a molar ratio of
1:0.5 of FXIIa to inhibitor. For example, the inhibitor of FXII
inhibits Factor XIIa by at least about 85%, or at least about 90%,
or at least about 95%, or at least about 96%, or at least about
97%, or at least about 98%, or at least about 99%, or about 100%
when used at a molar ratio of 1:0.5 of Factor XIIa to
inhibitor.
[0304] Methods for detecting inhibition of Factor XIIa activity are
known in the art and include, for example, an in vitro FXIIa
amidolytic activity assay as disclosed in WO 2013/014092).
[0305] In one example, the anti-FXII antibody is capable of
inhibiting the amidolytic activity of human Factor XIIa.
[0306] In one example, the inhibitor of FXII of the present
disclosure has a binding affinity or specificity for human FXIIa
that is similar to the binding affinity or specificity of antibody
3F7 or gVR115.
[0307] In one example, the inhibitor of FXII of the present
disclosure competitively inhibits the binding of antibody 3F7 to
FXII or gVR115.
[0308] In one example, the anti-FXII antibody has a binding
affinity to human Factor XIIa-beta at least 2 fold higher than to
inactivated human FXII. For example, the binding affinity to human
Factor XIIa-beta is at least about 3 fold, or at least about 4
fold, or at least about 5 fold higher than to inactivated human
FXII.
[0309] In one example, the inhibitor of FXII of the present
disclosure binds human Factor XIIa-beta with an equilibrium
dissociation constant (K.sub.D) of about 1.times.10.sup.-8M or
less, such as 9.5.times.10.sup.-9 M or less, such as
9.times.10.sup.-9 M or less, such as 8.5.times.10.sup.-9 M or less,
such as 8.times.10.sup.-9 M or less, such as 7.5.times.10.sup.-9 M
or less, such as 7.times.10.sup.-9M or less, such as
6.5.times.10.sup.-9 M or less, such as 6.times.10.sup.-9 M or less,
such as 5.5.times.10.sup.-9 M or less, such as 5.times.10.sup.-9
M.
[0310] In one example, the anti-FXII antibody or fragment thereof
comprises a V.sub.H comprising a sequence set forth in SEQ ID NO: 6
and a V.sub.L comprising a sequence set forth in SEQ ID NO: 7.
[0311] In one example, the anti-FXII antibody or fragment thereof
comprises a V.sub.H comprising a sequence which is at least 85%
identical to the sequence set forth in SEQ ID NO: 6. For example,
the V.sub.H sequence is at least about 90%, or at least about 95%,
or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 6. Exemplary positions for
mutations to CDRs are described herein. The skilled person will be
readily capable of identifying positions for mutations to framework
regions. In one example, the position of any change relative to the
recited sequence is in a framework region.
[0312] In one example, the anti-FXII antibody or fragment thereof
comprises a V.sub.L comprising a sequence which is at least 85%
identical to the sequence set forth in SEQ ID NO: 7. For example,
the V.sub.L sequence is at least about 90%, or at least about 95%,
or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 7. Exemplary positions for
mutations to CDRs are described herein. The skilled person will be
readily capable of identifying positions for mutations to framework
regions. In one example, the position of any change relative to the
recited sequence is in a framework region.
[0313] In one example, the inhibitor of FXII is a protein
comprising the complementary determining regions (CDRs) of the
V.sub.H (SEQ ID NO: 6) and the V.sub.L (SEQ ID NO: 7) of the
anti-FXII antibody.
[0314] For example, the protein comprises:
(i) a V.sub.H comprising: [0315] (a) a sequence set forth in SEQ ID
NO: 6; or [0316] (b) a CDR1 comprising a sequence set forth in SEQ
ID NO: 8; [0317] (c) a CDR2 comprising a sequence set forth in SEQ
ID NO: 9 or SEQ ID NO: 10; [0318] (d) a CDR3 comprising a sequence
set forth in SEQ ID NO: 11 or SEQ ID NO: 12; and/or (ii) a V.sub.L
comprising: [0319] (a) a sequence set forth in SEQ ID NO: 7; or
[0320] (b) a CDR1 comprising a sequence set forth in SEQ ID NO: 13
or SEQ ID NO: 17; [0321] (c) a CDR2 comprising a sequence set forth
in SEQ ID NO: 14; [0322] (d) a CDR3 comprising a sequence set forth
in SEQ ID NO: 15 or SEQ ID NO: 16.
[0323] In another example, the protein comprises:
(i) a V.sub.H comprising: [0324] (a) a CDR1 comprising a sequence
set forth in SEQ ID NO: 8; [0325] (b) a CDR2 comprising a sequence
set forth in SEQ ID NO: 10; and [0326] (c) a CDR3 comprising a
sequence set forth in SEQ ID NO: 12; and/or (ii) a V.sub.L
comprising: [0327] (a) a CDR1 comprising a sequence as set forth in
SEQ ID NO: 13; [0328] (b) a CDR2 comprising a sequence as set forth
in SEQ ID NO: 14; and [0329] (c) a CDR3 comprising a sequence as
set forth in SEQ ID NO: 16.
[0330] In one example, the protein comprises:
(i) a V.sub.H comprising: [0331] (a) a CDR1 comprising a sequence
set forth in SEQ ID NO: 8; [0332] (b) a CDR2 comprising a sequence
set forth in SEQ ID NO: 9; and [0333] (c) a CDR3 comprising a
sequence set forth in SEQ ID NO: 11; and/or (ii) a V.sub.L
comprising: [0334] (a) a CDR1 comprising a sequence as set forth in
SEQ ID NO: 13; [0335] (b) a CDR2 comprising a sequence as set forth
in SEQ ID NO: 14; and [0336] (c) a CDR3 comprising a sequence as
set forth in SEQ ID NO: 15.
[0337] In another example, the protein comprises:
(i) a V.sub.H comprising: [0338] (a) a CDR1 comprising a sequence
set forth in SEQ ID NO: 8; [0339] (b) a CDR2 comprising a sequence
set forth in SEQ ID NO: 10; and [0340] (c) a CDR3 comprising a
sequence set forth in SEQ ID NO: 11; and/or (ii) a V.sub.L
comprising: [0341] (a) a CDR1 comprising a sequence as set forth in
SEQ ID NO: 13; [0342] (b) a CDR2 comprising a sequence as set forth
in SEQ ID NO: 14; and [0343] (c) a CDR3 comprising a sequence as
set forth in SEQ ID NO: 15.
[0344] In another example, the protein comprises:
(i) a V.sub.H comprising: [0345] (a) a CDR1 comprising a sequence
set forth in SEQ ID NO: 8; [0346] (b) a CDR2 comprising a sequence
set forth in SEQ ID NO: 9; and [0347] (c) a CDR3 comprising a
sequence set forth in SEQ ID NO: 11; and/or (ii) a V.sub.L
comprising: [0348] (a) a CDR1 comprising a sequence as set forth in
SEQ ID NO: 17; [0349] (b) a CDR2 comprising a sequence as set forth
in SEQ ID NO: 14; and [0350] (c) a CDR3 comprising a sequence as
set forth in SEQ ID NO: 15.
[0351] In one example, the protein comprises a V.sub.H comprising a
CDR1 as set forth in SEQ ID NO: 8.
[0352] In one example, the protein comprises a V.sub.H comprising a
CDR1 at least 80% identical to the sequence set forth in SEQ ID NO:
8. For example, the protein comprises a V.sub.H comprising a CDR1
at least about 85%, or at least about 90%, or at least about 95%,
or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 8.
[0353] In one example, the protein comprises a V.sub.H comprising a
CDR2 as set forth in SEQ ID NO: 9.
[0354] In one example, the protein comprises a V.sub.H comprising a
CDR2 at least 60% identical to the sequence set forth in SEQ ID NO:
9. For example, the protein comprises a V.sub.H comprising a CDR2
at least about 70%, or at least about 80%, or at least about 85%,
or at least about 90%, or at least about 95%, or at least about
98%, or at least about 99% identical to the sequence set forth in
SEQ ID NO: 9.
[0355] In one example, the protein comprises a V.sub.H comprising a
CDR2 as set forth in SEQ ID NO: 10.
[0356] In one example, the amino acid sequence of V.sub.H CDR2
comprises Arginine (R), Asparagine (N) or Aspartic Acid (D) at
position 3 and/or Proline (P), Valine (V), Isoleucine (I) or
Methionine (M) at position 4 and/or Serine (S), Proline (P) or
Alanine (A) at position 5 and/or Glycine (G), Leucine (L), Valine
(V) or Threonine (T) at position 6 and/or any amino acid at
position 7 and/or Threonine (T), Glycine (G) or Serine (S) at
position 8.
[0357] In one example, the amino acid sequence of V.sub.H CDR2
comprises Asparagine (N) at position 3 and Valine (V) at position 4
and Proline (P) at position 5 and Leucine (L) at position 6 and
Tyrosine (Y) at position 7 and Glycine (G) at position 8.
[0358] In one example, the amino acid sequence of V.sub.H CDR2
comprises Asparagine (N) at position 3 and Valine (V) at position 4
and Proline (P) at position 5 and Valine (V) at position 6 and
Glutamine (Q) at position 7 and Glycine (G) at position 8.
[0359] In one example, the amino acid sequence of V.sub.H CDR2
comprises Aspartic acid (D) at position 3 and Isoleucine (I) at
position 4 and Proline (P) at position 5 and Threonine (T) at
position 6 and Lysine (K) at position 7 and Glycine (G) at position
8.
[0360] In one example, the amino acid sequence of V.sub.H CDR2
comprises Aspartic acid (D) at position 3 and Methionine (M) at
position 4 and Proline (P) at position 5 and Threonine (T) at
position 6 and Lysine (K) at position 7 and Glycine (G) at position
8.
[0361] In one example, the protein comprises a V.sub.H comprising a
CDR3 as set forth in SEQ ID NO: 11.
[0362] In one example, the protein comprises a V.sub.H comprising a
CDR3 at least 80% identical to the sequence set forth in SEQ ID NO:
11. For example, the protein comprises a V.sub.H comprising a CDR3
at least about 85%, or at least about 90%, or at least about 95%,
or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 11.
[0363] In one example, the protein comprises a V.sub.H comprising a
CDR3 as set forth in SEQ ID NO: 12.
[0364] In one example, the amino acid sequence of V.sub.H CDR3
comprises Isoleucine (I), Methionine (M) or Valine (V) at position
9 and/or Serine (S) or Lysine (K) at position 10 and/or Proline
(P), Lysine (K), Threonine (T) or Histidine (H) at position 11
and/or Histidine (H), Asparagine (N), Glycine (G) or Glutamine (Q)
at position 12.
[0365] In one example, the protein comprises a V.sub.L comprising a
CDR1 as set forth in SEQ ID NO: 13.
[0366] In one example, the protein comprises a V.sub.L comprising a
CDR1 at least 50% identical to the sequence set forth in SEQ ID NO:
13. For example, the protein comprises a V.sub.L comprising a CDR1
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 13.
[0367] In one example, the protein comprises a V.sub.L comprising a
CDR1 as set forth in SEQ ID NO: 17.
[0368] In one example, the protein comprises a V.sub.L comprising a
CDR1 at least 50% identical to the sequence set forth in SEQ ID NO:
17. For example, the protein comprises a V.sub.L comprising a CDR1
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 17.
[0369] In one example, the protein comprises a V.sub.L comprising a
CDR2 as set forth in SEQ ID NO: 14.
[0370] In one example, the protein comprises a V.sub.L comprising a
CDR2 at least 50% identical to the sequence set forth in SEQ ID NO:
14. For example, the protein comprises a V.sub.L comprising a CDR2
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 14.
[0371] In one example, the protein comprises a V.sub.L comprising a
CDR3 as set forth in SEQ ID NO: 15.
[0372] In one example, the protein comprises a V.sub.L comprising a
CDR3 at least 50% identical to the sequence set forth in SEQ ID NO:
15. For example, the protein comprises a V.sub.L comprising a CDR3
at least about 60%, or at least about 70%, or at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 98%, or at least about 99% identical to the
sequence set forth in SEQ ID NO: 15.
[0373] In one example, the protein comprises a V.sub.L comprising a
CDR3 as set forth in SEQ ID NO: 16.
[0374] In one example, the amino acid sequence of V.sub.L CDR3
comprises Alanine (A) or Serine (S) at position 2, and/or Aspartic
acid (D), Tyrosine (Y), Glutamic acid (E), Threonine (T),
Tryptophan (W) or Serine (S) at position 4 and/or Alanine (A),
Asparagine (N), Isoleucine (I), Leucine (L), Valine (V), Proline
(P), Glutamine (Q) or Glutamic acid (E) at position 5 and/or Serine
(S), Aspartic acid (D), Proline (P), Glutamic acid (E), Glutamine
(Q) or Arginine (R) at position 6 and/or Leucine (L) or Valine (V)
at position 7 and/or Glycine (G), Leucine (L) or Lysine (K) at
position 9 and/or Valine (V), Alanine (A), Aspartic acid (D),
Threonine (T), Methionine (M) or Glycine (G) at position 10.
[0375] In one example, the inhibitor of FXII is an affinity
matured, chimeric, CDR grafted, or humanized antibody, or antigen
binding fragment thereof. In one example, the anti-FXII antibody is
an affinity matured form of antibody 3F7. For example, the
anti-FXII antibody is selected from VR115, VR112, VR24, VR110 or
VR119 (SEQ ID NOs for HCDR 1-3 and LCDR1-3 of these antibodies are
shown below in Table 1).
TABLE-US-00001 TABLE 1 mAb HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 3F7
8 9 11 13 14 15 VR119 8 10 11 13 14 15 VR112 8 10 11 13 14 15 VR115
8 10 11 13 14 15 VR24 8 9 11 17 14 15 VR110 8 10 11 13 14 15
[0376] SEQ ID NO: 10 is a consensus sequence. VR119 comprises SEQ
ID NO: 10 wherein the X at position 3 is N, the X at position 4 is
V, the X at position 5 is P; the X at position 6 is L, the X at
position 7 is Y; and the X at position 8 is G. VR112 comprises SEQ
ID NO: 10 wherein the X at position 3 is N, the X at position 4 is
V, the X at position 5 is P, the X at position 6 is V, the X at
position 7 is Q, and the X at position 8 is G. VR115 comprises SEQ
ID NO: 10 wherein the X at position 3 is D, the X at position 4 is
I, the X at position 5 is P, the X at position 6 is T, the X at
position 7 is K, and the X at position 8 is G. VR110 comprises SEQ
ID NO: 10 wherein the X at position 3 is D, the X at position 4 is
M, the X at position 5 is P, the X at position 6 is T, the X at
position 7 is K, and the X at position 8 is G. VR24 comprises a
LCDR1 set forth in SEQ ID NO: 17.
[0377] In one example, an antibody described herein comprises IgG4
or stabilized IgG4 constant regions. The term "stabilized IgG4
constant regions" will be understood to mean IgG4 constant regions
that have been modified to reduce Fab arm exchange or the
propensity to undergo Fab arm exchange or formation of a
half-antibody or a propensity to form a half antibody. "Fab arm
exchange" refers to a type of protein modification for human IgG4,
in which an IgG4 heavy chain and attached light chain
(half-molecule) is swapped for a heavy-light chain pair from
another IgG4 molecule. Thus, IgG4 molecules may acquire two
distinct Fab arms recognizing two distinct antigens (resulting in
bispecific molecules). Fab arm exchange occurs naturally in vivo
and can be induced in vitro by purified blood cells or reducing
agents such as reduced glutathione. A "half antibody" forms when an
IgG4 antibody dissociates to form two molecules each containing a
single heavy chain and a single light chain.
[0378] In one example, a stabilized IgG4 constant region comprises
a proline at position 241 of the hinge region according to the
system of Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest Washington D.C. United States Department of
Health and Human Services, 1987 and/or 1991). This position
corresponds to position 228 of the hinge region according to the EU
numbering system (Kabat et al., Sequences of Proteins of
Immunological Interest Washington D.C. United States Department of
Health and Human Services, 2001 and Edelman et al., Proc. Natd.
Acad. Sci USA, 63, 78-85, 1969). In human gG4, this residue is
generally a serine. Following substitution of the serine for
proline, the IgG4 hinge region comprises a sequence CPPC. In this
regard, the skilled person will be aware that the "hinge region" is
a proline-rich portion of an antibody heavy chain constant region
that links the Fc and Fab regions that confers mobility on the two
Fab arms of an antibody. The hinge region includes cysteine
residues which are involved in inter-heavy chain disulfide bonds.
It is generally defined as stretching from Glu226 to Pro243 of
human IgG1 according to the numbering system of Kabat. Hinge
regions of other IgG isotypes may be aligned with the IgG1 sequence
by placing the first and last cysteine residues forming inter-heavy
chain disulphide (S--S) bonds in the same positions (see for
example WO 2010/080538).
[0379] Additional examples of stabilized IgG4 antibodies are
antibodies in which arginine at position 409 in a heavy chain
constant region of human IgG4 (according to the EU numbering
system) is substituted with lysine, threonine, methionine, or
leucine (e.g., as described in WO 2006/033386). The Fc region of
the constant region may additionally or alternatively comprise a
residue selected from the group consisting of: alanine, valine,
glycine, isoleucine and leucine at the position corresponding to
405 (according to the EU numbering system). Optionally, the hinge
region comprises a proline at position 241 (i.e., a CPPC sequence)
(as described above).
[0380] In one example, the inhibitor of FXII of the present
disclosure is antibody 3F7 or a chimeric, CDR grafted or humanized
version thereof or an antigen binding fragment thereof.
[0381] In one example, antibody 3F7 comprises a V.sub.H comprising
a sequence set forth in SEQ ID NO: 6 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 7. In one example, antibody 3F7
comprises the complementary determining regions (CDRs) of the
V.sub.H (SEQ ID NO: 6) and the V.sub.L (SEQ ID NO: 7).
[0382] For example, antibody 3F7 comprises a V.sub.H comprising a
CDR1 set forth in SEQ ID NO: 8, a CDR2 set forth in SEQ ID NO: 9, a
CDR3 set forth in SEQ ID NO: 11 and a V.sub.L comprising a CDR1 set
forth in SEQ ID NO: 13, CDR2 set forth in SEQ ID NO: 14 and a CDR3
set forth in SEQ ID NO: 15.
[0383] In one example, the inhibitor of XII of the present
disclosure is antibody VR115.
[0384] For example, antibody VR115 comprising a V.sub.H comprising
a CDR1 set forth in SEQ ID NO: 8, a CDR2 set forth in SEQ ID NO: 10
wherein the X at position 3 is D, the X at position 4 is I, the X
at position 5 is P, the X at position 6 is T, the X at position 7
is K, and the X at position 8 is G, and a CDR3 set forth in SEQ ID
NO: 11 and a V.sub.L comprising a CDR1 set forth in SEQ ID NO: 13,
CDR2 set forth in SEQ ID NO: 14 and a CDR3 set forth in SEQ ID NO:
15.
[0385] In one example, the antibody is a germlined antibody. A
"germlined" antibody is an antibody where some or all somatic
mutations that introduced changes into the framework residues are
reversed to the original sequence present in the genome, e.g., a
human genome. In this regard, not all changes need to be reversed
in a germlined antibody.
[0386] For example, the antibody is a germlined VR115 antibody
(gVR115).
[0387] For example, gVR115 comprises a V.sub.H comprising a
sequence set forth in SEQ ID NO: 18 and a V.sub.L comprising a
sequence set forth in SEQ ID NO: 19.
[0388] In one example, gVR115 comprises lambda light chain constant
regions.
[0389] In one example, gVR115 comprises IgG4 or stabilized IgG4
constant regions. For example, the stabilized IgG4 constant regions
comprise a proline at position 241 of the hinge region according to
the system of Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest Washington D.C. United States Department of
Health and Human Services, 1987 and/or 1991).
[0390] In one example, gVR115 comprises a heavy chain comprising a
sequence set forth in SEQ ID NO: 20 and a light chain comprising a
sequence set forth in SEQ ID NO: 21.
[0391] In another example, an antibody or protein comprising a
variable region thereof is produced using a standard method, e.g.,
as is known in the art.
Factor XII Inhibitor Fusion Partners
[0392] In one example, the inhibitor of FXII is linked to a fusion
partner. For example, the fusion partner comprises polyethylene
glycol (PEG). In one example, the fusion partner comprises a
half-life enhancing polypeptide (HLEPs).
Polyethylene Glycol (PEG)
[0393] In one example, the inhibitor of FXII is linked to a fusion
partner. For example, the fusion partner comprises polyethylene
glycol (PEG).
[0394] In one example, the fusion partner comprises mono- or poly-
(e.g., 2-4) polyethylene glycol (PEG) moieties. For example, the
mono- poly- (e.g., 2-4) polyethylene glycol (PEG) moieties extend
in vivo half-lives of the FXII inhibitor.
[0395] Pegylation may be carried out by any of the pegylation
reactions available. Exemplary methods for preparing pegylated
protein products can generally include (a) reacting a polypeptide
with polyethylene glycol (such as a reactive ester or aldehyde
derivative of PEG) under conditions whereby the protein becomes
attached to one or more PEG groups; and (b) obtaining the reaction
product(s).
[0396] The skilled person will be aware of different PEG attachment
methods which include, but are not limited to those described in
e.g., EP 0 401 384; Malik et al., Exp. Hematol., 20:1028-1035
(1992); Francis, Focus on Growth Factors, 3(2):4-10 (1992); EP 0
154 316; EP 0 401 384; WO 92/16221; WO 95/34326; U.S. Pat. No.
5,252,714.
Half-Life Enhancing Polypeptides (HLEPs)
[0397] In one example, the inhibitor of FXII is linked to a fusion
partner. For example, the fusion partner comprises a half-life
enhancing polypeptide (HLEPs).
[0398] A variety of half-life enhancing polypeptides are known to
the skilled artisan, and include, but are not limited to, those
described herein.
Albumin Proteins and Variants Thereof
[0399] In one example, the half-life enhancing polypeptide is
selected from the group consisting of albumin, afamin,
alpha-fetoprotein, vitamin D binding protein, human albumin, an
immunoglobulin, and an Fc of an IgG. For example, the half-life
enhancing polypeptide is albumin or a variant thereof.
[0400] In one example, the half-life enhancing polypeptide is
linked to the inhibitor of FXII via a linker. For example, the
inhibitor of FXII is a fusion protein comprising human albumin
linked to an inhibitor of FXII via a linker peptide.
[0401] In one example, an albumin variant is at least 10, or at
least 20, or at least 40, or at least 50, or at least 60, or at
least 70 amino acids long from a human albumin (HA) sequence (e.g.,
SEQ ID NO: 23).
[0402] In one example, an albumin variant is at least about 15, or
at least 20, or at least about 25, or at least about 30, or at
least about 50 or more contiguous amino acids from a human albumin
(HA) sequence (e.g., SEQ ID NO: 23).
[0403] In one example, an albumin variant includes part or all of
specific domains of HA. An albumin variant may include an amino
acid substitution, deletion, or addition, either conservative or
non-conservative substitution, wherein such changes do not
substantially alter the active site, or active domain, which
confers the therapeutic activities of the half-life enhancing
polypeptides. These variants may share identity of about 70%, or
about 75%, or about 80%, or about 85%, or about 90%, or about 91%,
or about 92%, or about 93%, or about 94%, or about 95%, or about
96%, or about 97%, or about 98% or about 99% from a human albumin
(HA) sequence.
[0404] In one example, an albumin variant is a fragment. In one
example, the albumin variant comprises at least one domain of
albumin and/or fragments of those domains. For example, the albumin
variant comprises at least one of domain 1 (amino acids 1-194 of
SEQ ID NO: 23), or domain 2 (amino acids 195-387 of SEQ ID NO: 23),
or domain 3 (amino acids 388-585 of SEQ ID NO 23). In one example,
the albumin variant comprises at least domains 1 and 2 (1-387 of
SEQ ID NO: 23), or domains 2 and 3 (195-585 of SEQ ID NO: 23), or
domains 1 and 3 (amino acids 1-194 and amino acids 388-585 of SEQ
ID NO: 23).
[0405] Each domain is itself made up of two homologous subdomains
namely residues 1-105, 120-194, 195-291, 316-387, 388-491 and
512-585, of SEQ ID NO: 23, with flexible inter-subdomain linker
regions comprising residues Lys106 to Glu119, Glu292 to Val315 and
Glu492 to Ala511.
[0406] In one example, the albumin variant comprises at least one
whole subdomain of albumin. For example, the albumin variant
comprises residues 1-105, or residues 120-194, or residues 195-291,
or residues 316-387, or residues 388-491, or residues 512-585 of
SEQ ID NO: 23.
[0407] In one example, other proteins that are structurally or
evolutionarily related to albumin ("albumin family proteins") may
be used as HLEPs, including, but not limited to alpha-fetoprotein
(WO 2005/024044; Beattie and Dugaiczyk, 20 Gene 415-422, 1982),
afamin (Lichenstein et al. 269 (27) J. Biol. Chem. 18149-18154,
1994), and vitamin D binding protein (Cooke and David, 76 J. Clin.
Invest. 2420-2424, 1985). The genes encoding these proteins
represent a multigene cluster with structural and functional
similarities mapping to the same chromosomal region in humans,
mice, and rats. The structural similarity of the albumin family
members suggests that they can be used as HLEPs. For example,
alpha-fetoprotein has been claimed to extend the half-life of an
attached therapeutic polypeptide in vivo (WO 2005/024044).
[0408] In one example, the half-life enhancing polypeptide is
selected from the group consisting of alpha-fetoprotein and vitamin
D binding protein. For example, the half-life enhancing polypeptide
is alpha-fetoprotein. In one example, the half-life enhancing
polypeptide is vitamin D binding protein.
[0409] In one example, the albumin family proteins or variants
thereof are capable of stabilizing or prolonging therapeutic
activity.
[0410] In one example, the albumin family proteins or variants
thereof are used as HLEPs linked to a FXII or FXIIa inhibitor.
[0411] In one example, the albumin family proteins or variants
thereof are derived from any vertebrate. For example, the
vertebrate is a mammal. In one example, the mammal is not a human,
monkey, cow, sheep, or pig. In one example, the vertebrate is
non-mammal. For example, the non-mammal is a hen or salmon.
[0412] In one example, the albumin variant comprises at least 10
amino acids in length. For example, the albumin variant comprises
about 15, or about 20, or about 25, or about 30, or about 50
contiguous amino acids of the respective protein sequence from
which they are derived. In one example, the albumin variant
comprises part or all of specific domains of the respective
proteins.
[0413] As discussed herein, albumin family member fusion proteins
may include naturally occurring polymorphic variants.
Immunoglobulin
[0414] In one example, the half-life enhancing polypeptide is an
immunoglobulin (Ig).
[0415] As discussed above, the term "immunoglobulin" encompasses
functional fragments and variants thereof, such as an Fc region or
one or more Ig constant domains. In one example, the Ig comprises
an Fc region or portions of the immunoglobulin constant domain(s).
The constant region may be that of an IgM, IgG, IgD, IgA, or IgE
immunoglobulin. In one example, the therapeutic polypeptide portion
is connected to the Ig via the hinge region of the antibody or a
peptide linker, which may be cleavable.
[0416] Methods for the fusion of therapeutic proteins to
immunoglobulin constant regions to extend the therapeutic protein's
half-life in vivo are known in the art and are described in e.g.,
US 2004/0087778, WO 2005/001025, WO 2005/063808, WO 2003/076567, WO
2005/000892, WO 2004/101740, U.S. Pat. No. 6,403,077.
[0417] In one example, the half-life enhancing polypeptide is an
immunoglobulin region. For example, the immunoglobulin region is an
Fc domain, or an Fc fragment of immunoglobulins, and/or variants
thereof.
[0418] In one example, an inhibitor of FXII is fused to Fc domains
or portions of immunoglobulin constant regions as HLEPs.
[0419] In one example, fusion proteins are prepared as recombinant
molecules expressed in prokaryotic or eukaryotic host cells. For
example, the fusion proteins are prepared in bacteria, or yeast, or
plant, or animal (including insect) or human cell lines or in
transgenic animals.
[0420] Methods of the expression of fusion proteins in prokaryotic
or eukaryotic cells are known in the art and are described in e.g.,
WO 2008/098720.
Linkers
[0421] In one example, the half-life enhancing polypeptide is
linked to the inhibitor of FXII via a linker. For example, the
linker is a linker peptide.
[0422] In one example, an intervening peptidic linker may be
introduced between a therapeutic polypeptide and a HLEP.
[0423] In one example, the linker is a cleavable linker. For
example, the linker is a cleavable linker if the HLEP has the
potential to interfere with the therapeutic polypeptide's specific
activity, for example, interference by steric hindrance.
[0424] In one example, the linker is cleavable by enzymes involved
in coagulation. For example, the linker is cleavable by coagulation
proteases of the intrinsic, extrinsic, or common coagulation
pathway. Coagulation proteases of the intrinsic pathway include
proteases in the contact activation pathway, e.g., FXIIa, FXIa, or
FIXa. In one embodiment, the linker is cleaved by FXIIa. Proteases
of the extrinsic pathway include proteases in the tissue factor
pathway, for example, FXIIa. Proteases of the common pathway
include proteases involved in the conversion of fibrinogen to
fibrin, for example, FXa, FIIa, and FXIIIa.
Screening Assays
[0425] Compounds that inhibit FXII signaling can be identified
using techniques known in the art, e.g., as described below.
Similarly, amounts of FXII inhibitors suitable for use in a method
described herein can be determined or estimated using techniques
known in the art, e.g., as described below.
FXIIa Amidolytic Activity
[0426] For inhibitors that bind FXII an in vitro assay to determine
the level of inhibitor of FXIIa amidolytic activity can be
used.
[0427] In one example, the amidolytic activity can be measured by
assay of the cleavage of FXII in the presence of an inhibitor of
FXII and a buffer. For example, FXII is incubated in the presence
of absence of an inhibitor of FXII or control. Following incubation
and addition of a detection substrate, the amidolytic activity is
spectrophotometrically determined as a change in optical density
(i.e., colour change).
[0428] Compounds that are found to effectively inhibit amidolytic
activity are identified as inhibitors of FXII.
In Vivo Animal Models
[0429] In one example, an inhibitor of FXII is tested for
therapeutic or prophylactic effects in an animal model of
atherosclerosis.
[0430] Animal models for assessing the effect of an inhibitor of
FXII on atherosclerotic lesions are known in the art and/or
exemplified herein. Exemplary animal models include, for example,
ApoE knockout (-/-) mouse model and a tandem stenosis mouse
model.
ApoE Knockout Animal Model
[0431] In one example, the animal model is an ApoE knockout mouse
model.
[0432] For example, an inhibitor of FXII or control is administered
to a model of atherosclerosis, for example, an ApoE-/- mouse model
and the therapeutic efficacy of the inhibitor of FXII assessed.
[0433] In one example, the ApoE-/- (ApoE-KO) mice are treated with
an inhibitor of FXII or control subcutaneously on alternate days.
In one example, the ApoE-/- mice are fed a high-fat diet (HFD)
containing 21% fat and 0.15% cholesterol.
[0434] In one example, the therapeutic efficacy of the inhibitor of
FXII is assessed by histological and/or morphological analysis of
atherosclerotic lesions.
[0435] Attenuation of atherosclerotic lesions in the animal model
in the presence of an inhibitor of FXII compared to in the absence
of an inhibitor of FXII indicates that the inhibitor of FXII is
useful for treating atherosclerosis and stabilization of
atherosclerotic plaques.
Tandem Stenosis Animal Model
[0436] In one example, the animal model is a tandem stenosis mouse
model.
[0437] For example, an inhibitor of FXII or control is administered
to a model of atherosclerosis, for example, a tandem stenosis mouse
model and the therapeutic efficacy of the inhibitor of FXII
assessed.
[0438] In one example, the mice are ApoE-/- knockout mice. In one
example, the mice are fed a high-fat diet (HFD) containing 21% fat
and 0.15% cholesterol.
[0439] In one example, the tandem stenosis is inserted into the
right common carotid artery near the carotid artery
bifurcation.
[0440] Attenuation of atherosclerotic lesions in the animal model
in the presence of an inhibitor of FXII compared to in the absence
of an inhibitor of FXII indicates that the inhibitor of FXII is
useful for treating atherosclerosis and stabilization of
atherosclerotic plaques.
Atherosclerotic Lesion Morphological Analysis
[0441] In one example, an inhibitor of FXII that attenuates
development of atherosclerotic lesions is identified by performing
histological analysis.
[0442] Suitable methods of morphological analysis are known in the
art and include, for example, analysis of total lesion size, lipid
area, necrotic core area, collagen accumulation, macrophage
accumulation and expression of a number of markers including e.g.,
VCAM-1 and .alpha.-smooth muscle actin.
[0443] In one example, cryo-sections of atherosclerotic lesions
from animal models are histologically stained with either Oil-red O
(ORO) standard to detect lipids and total lesion area, Mayer's
hematoxylin/eosin (H&E) to determine necrotic core areas
(acellular areas) in atherosclerotic lesions, Picro-sirius Red to
detect collagen. Quantification of histological samples for each
segment is performed on sequential 6 .mu.m sections obtained at 120
.mu.m intervals. The percentage of the various plaque components is
quantified as the positive area for each specific parameter divided
by the total intimal plaque area. The necrotic core is defined as
the total plaque area devoid of cellular tissue. Relative cap
thickness is defined as the ratio of the cap thickness at the
shoulder and mid-plaque region divided by maximal intimal
thickness. Imaging to ORO positive and acellular regions within
atherosclerotic lesions is carried out using light microscopy and
cross sectional area of lipid deposition quantified using image
analysis software (e.g., Optimas 6.2 Video Pro-32). For each mouse,
lesion size is measured in at least 4 cross-sectional areas at 30
mm intervals.
Flow Cytometry
[0444] In one example, flow cytometric analysis is performed on for
analysis of cell populations in the spleen, lymph node and
blood.
[0445] In one example, B lymphocytes and non-B-lymphocyte
populations in spleen, lymph node and blood are analysed with
fluorochrome conjugated antibodies on a BD FACS Canto-II. For
example, for analysis of B cells, PE-conjugated anti-CD19,
APC-conjugated anti-CD5, and APC-Cy7-conjugated anti-CD11b Abs are
used. For example, for analysis of non-B lymphocyte populations,
Pacific Blue-conjugated anti-CD4, PerCP-conjugated anti-CD8a,
FITC-conjugated anti-TCR-b, and PE-Cy7-conjugated anti-NK1.1 Abs
are used.
[0446] Attenuation of atherosclerotic lesions in the animal model
in the presence of an inhibitor of FXII compared to in the absence
of an inhibitor of FXII indicates that the inhibitor of FXII is
useful for treating atherosclerosis and stabilization of
atherosclerotic plaques.
Pharmaceutical Compositions and Methods of Treatment
[0447] In some examples of the disclosure, the FXII inhibitor may
have a purity of greater than 80%, or greater than 95%, 96%, 97%,
98%, or 99%. In one example, the FXII inhibitor may have a
pharmaceutically pure state that is greater than 99.9% pure with
respect to contaminating macromolecules, such as other proteins and
nucleic acids, and may be free of infectious and pyrogenic
agents.
[0448] A compound that inhibits FXII (syn. active ingredient) is
useful for parenteral, topical, oral, or local administration,
aerosol administration, or transdermal administration, for
prophylactic or for therapeutic treatment. In one example, the
inhibitor of FXII is administered parenterally, such as
subcutaneously or intravenously. For example, the inhibitor of FXII
is administered subcutaneously.
[0449] Formulation of a FXII inhibitor to be administered will vary
according to the route of administration and formulation (e.g.,
solution, emulsion, capsule) selected. An appropriate
pharmaceutical composition comprising a FXII inhibitor to be
administered can be prepared in a physiologically acceptable
carrier. For solutions or emulsions, suitable carriers include, for
example, aqueous or alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles can include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils. A
variety of appropriate aqueous carriers are known to the skilled
artisan, including water, buffered water, buffered saline, polyols
(e.g., glycerol, propylene glycol, liquid polyethylene glycol),
dextrose solution and glycine. Intravenous vehicles can include
various additives, preservatives, or fluid, nutrient or electrolyte
replenishers (See, generally, Remington's Pharmaceutical Science,
16th Edition, Mack, Ed. 1980). The compositions can optionally
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents and toxicity adjusting agents, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride and sodium lactate. Additional pharmaceutical
additives include, e.g., mannitol, starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, dried skim milk, and
ethanol.
[0450] The FXII inhibitor can be stored in the liquid stage or can
be lyophilized for storage and reconstituted in a suitable carrier
prior to use according to art-known lyophilization and
reconstitution techniques.
[0451] Alternatively, or in addition, the carrier or excipient
comprises a compound that enhances the activity of a FXII inhibitor
and/or reduces inhibition of the FXII inhibitor, e.g., a protease
inhibitor and/or a DNase inhibitor and/or an RNase inhibitor to
thereby enhance the stability of the inhibitor.
[0452] The optimum concentration of the active ingredient(s) in the
chosen medium can be determined empirically, according to
procedures known to the skilled artisan, and will depend on the
ultimate pharmaceutical formulation desired.
[0453] The dosage ranges for the administration of the FXII
inhibitor of the disclosure are those large enough to produce the
desired effect. For example, the composition comprises a
therapeutically or prophylactically effective amount of the FXII
inhibitor.
[0454] As used herein, the term "effective amount" shall be taken
to mean a sufficient quantity of the FXII inhibitor to
inhibit/reduce/prevent signaling of FXII and/or FXIIa in a subject.
The skilled artisan will be aware that such an amount will vary
depending on, for example, the FXII inhibitor and/or the particular
subject and/or the type and/or the severity of atherosclerosis
being treated. Accordingly, this term is not to be construed to
limit the disclosure to a specific quantity, e.g., weight or number
of FXII inhibitors.
[0455] The dosage should not be so large as to cause adverse side
effects, such as hyper viscosity syndromes, pulmonary edema,
congestive heart failure, and the like. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the
patient and can be determined by one of skill in the art. The
dosage can be adjusted by the individual physician in the event of
any complication.
[0456] Dosage can vary from about 0.1 mg/kg to about 300 mg/kg,
e.g., from about 0.2 mg/kg to about 200 mg/kg, such as, from about
0.5 mg/kg to about 20 mg/kg, for example about 10 mg/kg in one or
more dose administrations daily, for one or several days.
[0457] In some examples, the FXII inhibitor is administered at an
initial (or loading) dose which is higher than subsequent
(maintenance doses). For example, the FXII inhibitor is
administered at an initial dose of between about 10 mg/kg to about
30 mg/kg. The FXII inhibitor is then administered at a maintenance
dose of between about 0.0001 mg/kg to about 10 mg/kg. The
maintenance doses may be administered every 7-100 days, such as,
every 14 or 28 or 56 or 84 days.
[0458] In some examples, a dose escalation regime is used, in which
a FXII inhibitor is initially administered at a lower dose than
used in subsequent doses. This dosage regime is useful in the case
of subject's initially suffering adverse events.
[0459] In the case of a subject that is not adequately responding
to treatment, multiple doses in a week may be administered.
Alternatively, or in addition, increasing doses may be
administered.
[0460] A subject may be retreated with the FXII inhibitor, by being
given more than one exposure or set of doses, such as at least
about two exposures of the compound, for example, from about 2 to
60 exposures, and more particularly about 2 to 40 exposures, most
particularly, about 2 to 20 exposures.
[0461] In another example, any retreatment may be given at defined
intervals. For example, subsequent exposures may be administered at
various intervals, such as, for example, about 24-28 weeks or 48-56
weeks or longer. For example, such exposures are administered at
intervals each of about 24-26 weeks or about 38-42 weeks, or about
50-54 weeks.
[0462] A method of the present disclosure may also include
co-administration of the at least one FXII inhibitor according to
the disclosure together with the administration of another
therapeutically effective agent for the prevention or treatment of
atherosclerosis or atherosclerotic plaque rupture.
[0463] In one example, the FXII inhibitor of the disclosure is used
in combination with at least one additional known compound which is
currently being used or is in development for preventing or
treating atherosclerosis. Examples of such known compounds include
but are not limited to statins (e.g., Lovastatin, Pravastatin,
Rosuvastatin, Simvastatin, Atorvastatin, and Fluvastatin) and blood
thinning drugs (e.g., Asprin, Warfarin and Heparin).
[0464] As will be apparent from the foregoing, the present
disclosure provides methods of concomitant therapeutic treatment of
a subject, comprising administering to a subject in need thereof an
effective amount of a first compound and a second compound, wherein
said first agent is a FXII inhibitor, and the second agent is for
the prevention or treatment of atherosclerosis.
[0465] As used herein, the term "concomitant" as in the phrase
"concomitant therapeutic treatment" includes administering a first
agent in the presence of a second agent. A concomitant therapeutic
treatment method includes methods in which the first, second, third
or additional agents are co-administered. A concomitant therapeutic
treatment method also includes methods in which the first or
additional agents are administered in the presence of a second or
additional agent, wherein the second or additional agent, for
example, may have been previously administered. A concomitant
therapeutic treatment method may be executed step-wise by different
actors. For example, one actor may administer to a subject a first
agent and as a second actor may administer to the subject a second
agent and the administering steps may be executed at the same time,
or nearly the same time, or at distant times, so long as the first
agent (and/or additional agents) are after administration in the
presence of the second agent (and/or additional agents). The actor
and the subject may be the same entity (e.g. a human).
[0466] In one example, the disclosure also provides a method for
treating or preventing a atherosclerosis in a subject, the method
comprising administering to the subject a first pharmaceutical
composition comprising a FXII inhibitor of the disclosure and a
second pharmaceutical composition comprising one or more additional
compounds.
[0467] In one example, a method of the disclosure comprises
administering a FXII inhibitor to a subject suffering from
atherosclerosis and receiving another treatment (e.g., for diabetes
and/or cholesterol).
Kits and Other Compositions of Matter
[0468] Another example of the disclosure provides kits containing a
FXII inhibitor useful for the treatment of atherosclerosis as
described above.
[0469] In one example, the kit comprises (a) a container comprising
a FXII inhibitor as described herein, optionally in a
pharmaceutically acceptable carrier or diluent; and (b) a package
insert with instructions for treating atherosclerosis in a
subject.
[0470] In accordance with this example of the disclosure, the
package insert is on or associated with the container. Suitable
containers include, for example, bottles, vials, syringes, etc. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds or contains a composition that is
effective for treating atherosclerosis and may have a sterile
access port (for example, the container may be an intravenous
solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). At least one active agent in the composition is
the FXII inhibitor. The label or package insert indicates that the
composition is used for treating a subject eligible for treatment,
e.g., one having or predisposed to atherosclerosis, with specific
guidance regarding dosing amounts and intervals of FXII inhibitor
and any other medicament being provided. The kit may further
comprise an additional container comprising a pharmaceutically
acceptable diluent buffer, such as bacteriostatic water for
injection (BWFI), phosphate-buffered saline, Ringer's solution,
and/or dextrose solution.
[0471] The kit may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0472] The kit optionally further comprises a container comprises a
second medicament, wherein the FXII inhibitor is a first
medicament, and which article further comprises instructions on the
package insert for treating the subject with the second medicament,
in an effective amount or another treatment for atherosclerosis.
The second medicament may be any of those set forth above.
[0473] The present disclosure includes the following non-limiting
examples.
EXAMPLES
Example 1: Factor XII Inhibitor Treatment Attenuates Progression
and Development of Atherosclerotic Lesions
[0474] Male ApoE-/- (ApoE-KO) mice with C57BL/6J background (6
weeks old) received anti-factor XIIa monoclonal antibody 3F7 (1
mg/kg) or IgG isotype control (MuBM4-MuG1K) subcutaneously on
alternate days whilst being fed a high-fat diet (HFD) containing
21% fat and 0.15% cholesterol (SF00-219, Specialty Feeds, Western
Australia) for 8 weeks.
[0475] Animals were sacrificed and a catheter was placed in the
left ventricle for perfusion with 10 ml PBS, pH 7.4 under
physiological pressure. After perfusion, the entire aortic arch
with the brachiocephalic artery and the right and left carotid
artery was embedded in optimal cutting temperature (OCT) compound
(Sakura Finetechnical), frozen over liquid nitrogen and stored at
-80.degree. C. until sectioning.
[0476] Frozen carotid artery, aorta arch and aortic sinus sections
of 6 .mu.m thick transversal cryo sections were prepared using a
cryostat (Zeiss MICROM HM 550). Sections were histologically
stained with either Oil-red O (ORO) standard to detect lipids and
total lesion area, Mayer's hematoxylin/eosin (H&E) to determine
necrotic core areas (acellular areas) in atherosclerotic lesions,
Picro-sirius Red to detect collagen. Quantification of histological
samples for each segment was performed on sequential 6 .mu.m
sections obtained at 120 .mu.m intervals. The percentage of the
various plaque components was quantified as the positive area for
each specific parameter divided by the total intimal plaque area.
The necrotic core was defined as the total plaque area devoid of
cellular tissue. Relative cap thickness was defined as the ratio of
the cap thickness at the shoulder and mid-plaque region divided by
maximal intimal thickness. Imaging to ORO positive and acellular
regions within atherosclerotic lesions were carried out using light
microscopy and cross sectional area of lipid deposition quantified
using image analysis software (Optimas 6.2 Video Pro-32, Bedford
Park, South Australia, Australia). For each mouse, lesion size was
measured in 4 cross-sectional areas at 30 mm intervals. Oil-Red O
staining of atherosclerotic lesions in the aorta root revealed a
highly significant reduction in total lesion size (FIG. 1A) and
lipid accumulation (FIG. 1B) by 60% and 70%, respectively (all
P<0.05).
[0477] As macrophages are a main component of atherosclerotic
lesions, CD68 staining was performed, revealing that macrophage
accumulation was also markedly reduced by 45% (P<0.05; FIG. 1C).
Further evaluation of atherosclerotic lesions demonstrated a
significant increase in collagen deposition in the lesion by 50%
(P<0.05; FIG. 1D). H&E staining revealed a significant
reduction in necrotic core area in the lesion by 52% (P<0.05;
FIG. 1E). Atherosclerosis in the aortic arch was also studied,
revealing a reduction in total lesion, lipid and macrophage
accumulation by 50%, 47% and 60%, respectively (all P<0.05; FIG.
1F-H).
[0478] Flow cytometric analysis was performed for analysis of cell
populations in the spleen, lymph node and blood. B lymphocytes and
non-B-lymphocyte populations in spleen, LN and blood were analysed
with fluorochrome conjugated antibodies (from BD Pharmingen, San
Diego, Calif. unless otherwise stated) on a BD FACS Canto-II (BD
Biosciences). For B cells, PE-conjugated anti-CD19, APC-conjugated
anti-CD5, and APC-Cy7-conjugated anti-CD11 b Abs were used. For
non-B lymphocyte populations, Pacific Blue-conjugated anti-CD4,
PerCP-conjugated anti-CD8a, FITC-conjugated anti-TCR-b, and
PE-Cy7-conjugated anti-NK1.1 Abs were used.
[0479] Flow cytometric analysis of lymphocytic cell populations
revealed that chronic treatment with a FXII inhibitor reduced NK
and NKT cell populations in blood (FIG. 2) by 35% and 42%,
respectively and in lymph node (FIG. 3) by 38% and 47%,
respectively, without affecting other lymphocytes (P<0.05).
Example 2: Factor XII Inhibitor Treatment Reduces Arterial
Inflammation and Increases Smooth Muscle Cell Number in
Atherosclerotic Plaques
[0480] To determine whether FXII inhibitor treatment reduced
inflammation as a driving force of plaque development, the effect
of chronic FXII inhibitor (3F7) treatment on the expression of
vascular cell adhesion molecule-1 (VCAM-1; clone sc-1504, Santa
Cruz) was investigated in atherosclerotic lesions of the aortic
root and the aortic arch. A reduction of VCAM-1 expression was
observed in the atherosclerotic lesions of the aortic root (43%)
and the aortic arch (33%) (all P<0.05; FIG. 4A, B).
[0481] H&E staining also revealed a significant reduction in
the size of the acellular area (necrotic core) in the
atherosclerotic lesions of the aortic root and the aortic arch 33%
and 50%, respectively, in 3F7 treated mice compared to the istotype
IgG monoclonal antibody treated control group (all P<0.05; FIG.
4C, D).
[0482] In addition, we assessed the effect of FXII inhibitor
treatment on the number of smooth muscle cells in atherosclerotic
plaques by immunohistochemistry using .alpha.-smooth muscle actin
antibody (clone 1A4; 1:100 dilution; Sigma Aldrich). FXII inhibitor
treatment increased the number of smooth muscle cells in
atherosclerotic plaques in the aortic root as well as in the aortic
arch (all P<0.05; FIG. 4E, F). Interestingly, the collagen
content in lesions neither in the aortic root nor in the aortic
arch was changed (FIG. 4G, H).
[0483] In all immunohistochemical analysis detection was achieved
by Vectastain ABC kit and the DAB substrate. Rat IgG2B control
antibodies were used to validate the staining specificity of the
applied rat antibody. Other isotype control antibodies (Goat IgG,
rabbit IgG) were used for the validation of each immunostaining
according to the primary antibodies. Expression of antigens was
quantified using Optimus 6.2 VideoPro-32 and the stained segments
were expressed as a percentage of the total plaque area.
Example 3: 3F7 Treatment Achieves Plaque Stabilization
[0484] To specifically address the question whether 3F7 has the
potential to stabilize plaques, a recently developed unique mouse
model of plaque instability/rupture was used.
[0485] In this model male ApoE-/- (ApoE-KO) mice were fed a high
fat diet for 6 weeks to develop established atherosclerosis. At 12
weeks of age, 6 weeks after commencement of the high fat diet,
ApoE-KO mice were anaesthetized by a ketamine (100 mg/kg) and
xylazine (10 mg/kg) mixture through intraperitoneal injection. An
incision was made in the neck and the right common carotid artery
was dissected from circumferential connective tissues. A tandem
stenosis with 150 .mu.m (or 450 .mu.m) outer diameters was
introduced with the distal point 1 mm from the carotid artery
bifurcation and the proximal point 3 mm from the distal stenosis.
The stenosis diameter was obtained by placing a 6-0 blue braided
polyester fibre suture around the carotid artery together with a
150- or 450-.mu.m needle that was tied to it and later removed.
Immediately after tandem stenosis surgery, mice were treated with
the same regimen of either FXII inhibitor or IgG control while they
were fed a high fat diet for a further 7 weeks.
[0486] Animals and tissues were processed as described above in
Examples 1 and 2.
[0487] As shown in FIG. 5A, segment 1 represents the area of plaque
instability. A reduction of total lesion size of 32% was observed
in mice treated with the FXII inhibitor (FIG. 5B). Lipid and
macrophage accumulation were also markedly reduced, by 52% and 53%
respectively (all P<0.05; FIGS. 5C, D). VCAM-1 expression as a
major measure of plaque inflammation was reduced by 30% in segment
1 of mice treated with 3F7 (FIG. 5E). Necrotic core size, again
considered a marker of plaque instability, was reduced by 3F7
treatment by 32% (FIG. 5F). Most impressive was a significant
increase in expression of smooth muscle cells expression in
atherosclerotic lesion in segment 1 of nearly 2.5 fold (P<0.05;
FIG. 5G). Interestingly, FXIIa inhibition by 3F7 did not result in
a change of the collagen inhibition arguing for very specific
effects on various plaque stabilizing mechanisms (FIG. 5H).
Example 4: Lipid Profile of Treated Animals
[0488] Cholesterol profiles (total cholesterol, high-density
lipoprotein cholesterol, very-low-density lipoprotein/LDL
cholesterol, and triglycerides) in plasma from ApoE-/- (ApoE-KO)
mice either treated with 3F7 or control antibody were measured as
described below.
Cholesterol
[0489] Cholesterol levels were measured by a standard commercial
enzymatic assay using a Beckman Coulter LX20PRO Analyzer, with
reagents and calibrators supplied by Beckman Coulter Diagnostics
Australia.
[0490] CHOL reagent is used to measure cholesterol concentration by
a timed-endpoint method. In the reaction, cholesterol esterase (CE)
hydrolyzes cholesterol esters to free cholesterol and fatty acids.
Free cholesterol is oxidized to cholestene-3-one and hydrogen
peroxide by cholesterol oxidase (CO). Peroxidase catalyzes the
reaction of hydrogen peroxide with 4-aminoantipyrine (4-AAP) and
phenol to produce a colored quinoneimine product.
[0491] The SYNCHRON LX.RTM. System(s) automatically proportions the
appropriate sample and reagent volumes into the cuvette. The ratio
used is one part sample to 100 parts reagent. The system monitors
the change in absorbance at 520 nanometers. This change in
absorbance is directly proportional to the concentration of CHOL in
the sample and is used by the System to calculate and express CHOL
concentration.
HDL Cholesterol
[0492] HDL cholesterol was measured by a standard commercial
enzymatic assay using a Beckman Coulter LX20PRO Analyser, with
reagents and calibrators supplied by Beckman Coulter Diagnostics
Australia.
[0493] This direct HDL Cholesterol method is a homogeneous assay
without the need for any offline pretreatment or centrifugation
steps. The method uses a detergent which solubilizes only the HDL
lipoprotein particles and releases HDL cholesterol to react with
cholesterol esterase and cholesterol oxidase in the presence of
chromogens, to produce a color product. The same detergent also
inhibits the reaction of the cholesterol enzymes with LDL, VLDL,
and chylomicrons lipoproteins by adsorbing to their surfaces. A
polyanion contained in the reagent enhances the selectivity for HDL
cholesterol assay by complexing LDL, VLDL, and chylomicrons
lipoproteins.
[0494] HDLD reagent was used to measure the cholesterol
concentration by a timed-endpoint method. The SYNCHRON LX.RTM.
System(s) automatically proportions the appropriate HDL cholesterol
sample and reagent volumes into a cuvette. The ratio used is one
part sample to 93 parts reagent. The System monitors the change in
absorbance at 560 nanometers. This change in absorbance is directly
proportional to the concentration of cholesterol in the sample and
is used by the System to calculate and express the HDL-cholesterol
concentration.
Triglycerides
[0495] Triglycerides were measured by a standard commercial
enzymatic assay using a Beckman Coulter LX20PRO Analyser, with
reagents and calibrators supplied by Beckman Coulter Diagnostics
Australia.
[0496] Triglycerides GPO reagent was used to measure the
triglycerides concentration by a timed endpoint method.
Triglycerides in the sample were hydrolyzed to glycerol and free
fatty acids by the action of lipase. A sequence of three coupled
enzymatic steps using glycerol kinase (GK), glycerophosphate
oxidase (GPO), and horseradish peroxidase (HPO) causes the
oxidative coupling of 3,5-dichloro-2-hydroxybenze-nesulfonic acid
(DHBS) with 4-aminoantipyrine to form a red quinoneimine dye.
[0497] The SYNCHRON LX.RTM. System(s) automatically proportions the
appropriate sample and reagent volumes into the cuvette. The ratio
used is one part sample to 100 parts reagent. The system monitors
the change in absorbance at 520 nanometers. This change in
absorbance is directly proportional to the concentration of TG in
the sample and is used by the System to calculate and express the
TG concentration.
Results
[0498] Results of the assays are shown in Table 2. These results
show that an inhibitor of FXII (3F7) provides a beneficial effect
in the context of atherosclerosis without changing cholesterol
levels.
TABLE-US-00002 TABLE 2 Body Spleen Total VLDL/LDL Trigly- HDL
Experimental weight weight Cholestrol Cholestrol ceride Cholestrol
details (gr) (mgr) (mmol/L) (mmol/L) (mmol/L) (mmol/L) F-XII Ab.
Treated 36.5 147 21.9 19.2 0.87 2.3 T/S-ApoE-/- (.+-.1.3)
(.+-.7.76) (.+-.1.3) (.+-.1.2) (.+-.0.04) (.+-.0.09) IgG ctrl.
Treated 38.4 138 24.3 21.4 0.88 2.4 T/S-ApoE-/- (.+-.1.4)
(.+-.7.61) (.+-.1.05) (.+-.0.9) (.+-.0.04) (.+-.0.13) F-XII Ab.
Treated 32.7 173 22.8 19.7 0.9 2.7 HFD-ApoE-/- (.+-.1.5) (.+-.13.3)
(.+-.1.7) (.+-.1.5) (.+-.0.09) (.+-.0.18) IgG ctrl. Treated 34.5
149 24.9 21.4 1.1 2.9 HFD-ApoE-/- (.+-.1.7) (.+-.8.79) (.+-.1.8)
(.+-.1.6) (.+-.0.12) (.+-.0.24)
Sequence CWU 1
1
25148PRTArtificial Sequenceamino acid sequence of wild-type
Infestin-4 1Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn Tyr Val Pro
Val Cys1 5 10 15Gly Ser Asp Gly Lys Thr Tyr Gly Asn Pro Cys Met Leu
Asn Cys Ala 20 25 30Ala Gln Thr Lys Val Pro Gly Leu Lys Leu Val His
Glu Gly Arg Cys 35 40 45256PRTArtificial Sequenceamino acid
sequence of wild-type SPINK-1 2Asp Ser Leu Gly Arg Glu Ala Lys Cys
Tyr Asn Glu Leu Asn Gly Cys1 5 10 15Thr Lys Ile Tyr Asp Pro Val Cys
Gly Thr Asp Gly Asn Thr Tyr Pro 20 25 30Asn Glu Cys Val Leu Cys Phe
Glu Asn Arg Lys Arg Gln Thr Ser Ile 35 40 45Leu Ile Gln Lys Ser Gly
Pro Cys 50 55353PRTArtificial SequenceSPINK-1 mutant K1 3Asp Ser
Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn1 5 10 15Tyr
Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Pro Asn Glu Cys 20 25
30Val Leu Cys Phe Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln
35 40 45Lys Ser Gly Pro Cys 50453PRTArtificial SequenceSPINK-1
mutant K2 4Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe
Arg Asn1 5 10 15Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Gly
Asn Glu Cys 20 25 30Met Leu Cys Ala Glu Asn Arg Lys Arg Gln Thr Ser
Ile Leu Ile Gln 35 40 45Lys Glu Gly Pro Cys 50554PRTArtificial
SequenceSPINK-1 mutant K3 5Asp Ser Leu Gly Arg Glu Val Arg Asn Pro
Cys Ala Cys Phe Arg Asn1 5 10 15Tyr Val Pro Val Cys Gly Thr Asp Gly
Asn Thr Tyr Gly Asn Glu Cys 20 25 30Met Leu Asn Cys Ala Glu Asn Arg
Lys Arg Gln Thr Ser Ile Leu Ile 35 40 45Gln Lys Glu Gly Pro Cys
506129PRTArtificial Sequenceamino acid sequence from a VH of an
anti-FXII antibody 6Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Lys Tyr 20 25 30Ile Met Gln Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Arg Pro Ser Gly Gly Thr
Thr Val Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Leu Pro
Arg Ser Gly Tyr Leu Ile Ser Pro His Tyr Tyr 100 105 110Tyr Tyr Ala
Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser7110PRTArtificial Sequenceamino acid sequence from a VL of an
anti-FXII antibody 7Gln Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly
Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
Asn Ile Gly Arg Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Val Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Val Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Ala Ser Leu 85 90 95Arg Gly Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 11085PRTArtificial
Sequenceamino acid sequence from a VH CDR1 of an anti-FXII antibody
8Lys Tyr Ile Met Gln1 5917PRTArtificial Sequenceamino acid sequence
from a VH CDR2 of an anti-FXII antibody 9Gly Ile Arg Pro Ser Gly
Gly Thr Thr Val Tyr Ala Asp Ser Val Lys1 5 10
15Gly1017PRTArtificial Sequenceamino acid sequence from a VH CDR2
of an anti-FXII antibodyX(3)..(3)R or N or DX(4)..(4)P or V or I or
MX(5)..(5)S or P or AX(6)..(6)G or L or V or
Tmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidX(8)..(8)G or S 10Gly Ile Xaa Xaa Xaa Xaa Xaa Xaa Thr Val Tyr
Ala Asp Ser Val Lys1 5 10 15Gly1120PRTArtificial Sequenceamino acid
sequence from a VH CDR3 of an anti-FXII antibody 11Ala Leu Pro Arg
Ser Gly Tyr Leu Ile Ser Pro His Tyr Tyr Tyr Tyr1 5 10 15Ala Leu Asp
Val 201220PRTArtificial Sequenceamino acid sequence from a VH CDR3
of an anti-FXII antibodyX(9)..(9)I or M or VX(10)..(10)S or
KX(11)..(11)P or K or T or HX(12)..(12)H or N or G or Q 12Ala Leu
Pro Arg Ser Gly Tyr Leu Xaa Xaa Xaa Xaa Tyr Tyr Tyr Tyr1 5 10 15Ala
Leu Asp Val 201313PRTArtificial Sequenceamino acid sequence from a
VL CDR1 of an anti-FXII antibody 13Ser Gly Ser Ser Ser Asn Ile Gly
Arg Asn Tyr Val Tyr1 5 10147PRTArtificial Sequenceamino acid
sequence from a VL CDR2 of an anti-FXII antibody 14Ser Asn Asn Gln
Arg Pro Ser1 51510PRTArtificial Sequenceamino acid sequence from a
VL CDR3 of an anti-FXII antibody 15Ala Ala Trp Asp Ala Ser Leu Arg
Gly Val1 5 101610PRTArtificial Sequenceamino acid sequence from a
VL CDR3 of an anti-FXII antibodyX(2)..(2)A or SX(4)..(4)D or Y or E
or T or W or SX(5)..(5)A or N or I or L or V or P or Q or
EX(6)..(6)S or D or P or E or Q or RX(7)..(7)L or VX(9)..(9)G or L
or KX(10)..(10)V or A or D or T or M or G 16Ala Xaa Trp Xaa Xaa Xaa
Xaa Arg Xaa Xaa1 5 101713PRTArtificial Sequenceamino acid sequence
from a VL CDR1 of an anti-FXII antibody 17Ser Gly Ser Ser Glu Met
Thr Val His His Tyr Val Tyr1 5 1018129PRTartificial sequenceVH of
anti-FXII antibody gVR115 18Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Lys Tyr 20 25 30Ile Met Gln Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Asp Ile Pro Thr
Lys Gly Thr Val Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala
Leu Pro Arg Ser Gly Tyr Leu Ile Ser Pro His Tyr Tyr 100 105 110Tyr
Tyr Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser19110PRTartificial sequenceVL of anti-FXII antibody gVR115
19Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg
Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Ala Ser Leu 85 90 95Arg Gly Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 11020456PRTartificial sequenceheavy
chain of anti-FXII antibody gVR1 20Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30Ile Met Gln Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Asp Ile
Pro Thr Lys Gly Thr Val Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ala Leu Pro Arg Ser Gly Tyr Leu Ile Ser Pro His Tyr Tyr 100 105
110Tyr Tyr Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
115 120 125Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser 130 135 140Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp145 150 155 160Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr 165 170 175Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr 180 185 190Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 195 200 205Thr Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 210 215 220Lys
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala225 230
235 240Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 245 250 255Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val 260 265 270Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val 275 280 285Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 290 295 300Phe Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln305 310 315 320Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 325 330 335Leu Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 340 345
350Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
355 360 365Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 370 375 380Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr385 390 395 400Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 405 410 415Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe 420 425 430Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440 445Ser Leu Ser
Leu Ser Leu Gly Lys 450 45521215PRTartificial sequencelight chain
of anti-FXII antibody gVR115 21Gln Ser Val Leu Thr Gln Pro Pro Ser
Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Arg Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp
Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Ala Ser Leu 85 90 95Arg Gly
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105
110Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr Pro 130 135 140Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro Val Lys Ala145 150 155 160Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln Ser Asn Asn Lys Tyr Ala 165 170 175Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205Val Ala Pro
Thr Glu Cys Ser 210 21522615PRTHomo sapiens 22Met Arg Ala Leu Leu
Leu Leu Gly Phe Leu Leu Val Ser Leu Glu Ser1 5 10 15Thr Leu Ser Ile
Pro Pro Trp Glu Ala Pro Lys Glu His Lys Tyr Lys 20 25 30Ala Glu Glu
His Thr Val Val Leu Thr Val Thr Gly Glu Pro Cys His 35 40 45Phe Pro
Phe Gln Tyr His Arg Gln Leu Tyr His Lys Cys Thr His Lys 50 55 60Gly
Arg Pro Gly Pro Gln Pro Trp Cys Ala Thr Thr Pro Asn Phe Asp65 70 75
80Gln Asp Gln Arg Trp Gly Tyr Cys Leu Glu Pro Lys Lys Val Lys Asp
85 90 95His Cys Ser Lys His Ser Pro Cys Gln Lys Gly Gly Thr Cys Val
Asn 100 105 110Met Pro Ser Gly Pro His Cys Leu Cys Pro Gln His Leu
Thr Gly Asn 115 120 125His Cys Gln Lys Glu Lys Cys Phe Glu Pro Gln
Leu Leu Arg Phe Phe 130 135 140His Lys Asn Glu Ile Trp Tyr Arg Thr
Glu Gln Ala Ala Val Ala Arg145 150 155 160Cys Gln Cys Lys Gly Pro
Asp Ala His Cys Gln Arg Leu Ala Ser Gln 165 170 175Ala Cys Arg Thr
Asn Pro Cys Leu His Gly Gly Arg Cys Leu Glu Val 180 185 190Glu Gly
His Arg Leu Cys His Cys Pro Val Gly Tyr Thr Gly Ala Phe 195 200
205Cys Asp Val Asp Thr Lys Ala Ser Cys Tyr Asp Gly Arg Gly Leu Ser
210 215 220Tyr Arg Gly Leu Ala Arg Thr Thr Leu Ser Gly Ala Pro Cys
Gln Pro225 230 235 240Trp Ala Ser Glu Ala Thr Tyr Arg Asn Val Thr
Ala Glu Gln Ala Arg 245 250 255Asn Trp Gly Leu Gly Gly His Ala Phe
Cys Arg Asn Pro Asp Asn Asp 260 265 270Ile Arg Pro Trp Cys Phe Val
Leu Asn Arg Asp Arg Leu Ser Trp Glu 275 280 285Tyr Cys Asp Leu Ala
Gln Cys Gln Thr Pro Thr Gln Ala Ala Pro Pro 290 295 300Thr Pro Val
Ser Pro Arg Leu His Val Pro Leu Met Pro Ala Gln Pro305 310 315
320Ala Pro Pro Lys Pro Gln Pro Thr Thr Arg Thr Pro Pro Gln Ser Gln
325 330 335Thr Pro Gly Ala Leu Pro Ala Lys Arg Glu Gln Pro Pro Ser
Leu Thr 340 345 350Arg Asn Gly Pro Leu Ser Cys Gly Gln Arg Leu Arg
Lys Ser Leu Ser 355 360 365Ser Met Thr Arg Val Val Gly Gly Leu Val
Ala Leu Arg Gly Ala His 370 375 380Pro Tyr Ile Ala Ala Leu Tyr Trp
Gly His Ser Phe Cys Ala Gly Ser385 390 395 400Leu Ile Ala Pro Cys
Trp Val Leu Thr Ala Ala His Cys Leu Gln Asp 405 410 415Arg Pro Ala
Pro Glu Asp Leu Thr Val Val Leu Gly Gln Glu Arg Arg 420 425 430Asn
His Ser Cys Glu Pro Cys Gln Thr Leu Ala Val Arg Ser Tyr Arg 435 440
445Leu His Glu Ala Phe Ser Pro Val Ser Tyr Gln His Asp Leu Ala Leu
450 455 460Leu Arg Leu Gln Glu Asp Ala Asp Gly Ser Cys Ala Leu Leu
Ser Pro465 470 475 480Tyr Val Gln Pro Val Cys Leu Pro Ser Gly Ala
Ala Arg Pro Ser Glu 485 490 495Thr Thr Leu Cys Gln Val Ala Gly Trp
Gly His Gln Phe Glu Gly Ala 500 505 510Glu Glu Tyr Ala Ser Phe Leu
Gln Glu Ala Gln Val Pro Phe Leu Ser 515 520 525Leu Glu Arg Cys Ser
Ala Pro Asp Val His Gly Ser Ser Ile Leu Pro 530 535 540Gly Met Leu
Cys Ala Gly Phe Leu Glu Gly Gly Thr Asp Ala Cys Gln545 550 555
560Gly Asp Ser Gly Gly Pro Leu Val Cys Glu Asp Gln Ala Ala Glu Arg
565 570 575Arg Leu Thr Leu Gln Gly Ile Ile Ser Trp Gly Ser Gly Cys
Gly Asp 580 585 590Arg Asn Lys Pro Gly Val Tyr Thr Asp Val Ala Tyr
Tyr Leu Ala Trp 595 600 605Ile Arg Glu His Thr Val Ser 610
61523609PRTHomo sapiens 23Met Lys Trp Val Thr Phe Ile Ser Leu Leu
Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg Asp
Ala His Lys Ser Glu Val Ala
20 25 30His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val
Leu 35 40 45Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp
His Val 50 55 60Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys
Val Ala Asp65 70 75 80Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His
Thr Leu Phe Gly Asp 85 90 95Lys Leu Cys Thr Val Ala Thr Leu Arg Glu
Thr Tyr Gly Glu Met Ala 100 105 110Asp Cys Cys Ala Lys Gln Glu Pro
Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125His Lys Asp Asp Asn Pro
Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140Asp Val Met Cys
Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys145 150 155 160Lys
Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170
175Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys
180 185 190Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu
Asp Glu 195 200 205Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln
Arg Leu Lys Cys 210 215 220Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala
Phe Lys Ala Trp Ala Val225 230 235 240Ala Arg Leu Ser Gln Arg Phe
Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255Lys Leu Val Thr Asp
Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270Asp Leu Leu
Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285Cys
Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295
300Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn
Asp305 310 315 320Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp
Phe Val Glu Ser 325 330 335Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala
Lys Asp Val Phe Leu Gly 340 345 350Met Phe Leu Tyr Glu Tyr Ala Arg
Arg His Pro Asp Tyr Ser Val Val 355 360 365Leu Leu Leu Arg Leu Ala
Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380Cys Ala Ala Ala
Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu385 390 395 400Phe
Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410
415Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu
420 425 430Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr
Leu Val 435 440 445Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys
Cys Cys Lys His 450 455 460Pro Glu Ala Lys Arg Met Pro Cys Ala Glu
Asp Tyr Leu Ser Val Val465 470 475 480Leu Asn Gln Leu Cys Val Leu
His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495Val Thr Lys Cys Cys
Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510Ser Ala Leu
Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525Glu
Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535
540Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His
Lys545 550 555 560Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met
Asp Asp Phe Ala 565 570 575Ala Phe Val Glu Lys Cys Cys Lys Ala Asp
Asp Lys Glu Thr Cys Phe 580 585 590Ala Glu Glu Gly Lys Lys Leu Val
Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605Leu2412PRTArtificial
Sequenceamino acid sequence of an Infestin-4 variant 24Val Arg Asn
Pro Cys Ala Cys Phe Arg Asn Tyr Val1 5 102512PRTArtificial
Sequenceamino acid sequence of an Infestin-4 variant 25Asp Ser Leu
Gly Arg Glu Val Arg Asn Pro Cys Ala1 5 10
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