U.S. patent application number 17/377863 was filed with the patent office on 2021-11-11 for antibodies specific for lox1 and use in treatment of cardiovascular disorders.
The applicant listed for this patent is MedImmune Limited. Invention is credited to Clare BALENDRAN, Daniel E. BERKOWITZ, Andrew BUCHANAN, Peter CARIUK, Fumin CHANG, Matthieu CHODORGE, Johanna HUSMARK, Deepesh PANDEY, Lewis H. ROMER.
Application Number | 20210347900 17/377863 |
Document ID | / |
Family ID | 1000005728512 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210347900 |
Kind Code |
A1 |
BUCHANAN; Andrew ; et
al. |
November 11, 2021 |
ANTIBODIES SPECIFIC FOR LOX1 AND USE IN TREATMENT OF CARDIOVASCULAR
DISORDERS
Abstract
This disclosure provides LOX1 (LOX1) binding proteins such as
anti-LOX1 antibodies, and compositions and methods for making these
binding proteins. In certain aspects the LOX1-binding proteins
provided herein, inhibit, or antagonize LOX1 activity. In addition,
the disclosure provides compositions and methods for diagnosing and
treating conditions associated with atherosclerosis, thrombosis,
coronary artery disease (CAD), ischemia (e.g., myocardial
ischemia), infarction (e.g., myocardial infarction), acute coronary
syndrome (ACS), stroke, reperfusion injury, restenosis, peripheral
vascular disease, hypertension, heart failure, inflammation (e.g.,
chronic inflammation), angiogenesis, preeclampsia, cancer and other
LOX1-mediated diseases and conditions.
Inventors: |
BUCHANAN; Andrew;
(Cambridge, GB) ; CHODORGE; Matthieu; (Cambridge,
GB) ; CARIUK; Peter; (Cambridge, GB) ;
HUSMARK; Johanna; (Macclesfield, GB) ; BALENDRAN;
Clare; (Moldnal, SE) ; PANDEY; Deepesh;
(Baltimore, MD) ; CHANG; Fumin; (Baltimore,
MD) ; BERKOWITZ; Daniel E.; (Baltimore, MD) ;
ROMER; Lewis H.; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune Limited |
Cambridge |
|
GB |
|
|
Family ID: |
1000005728512 |
Appl. No.: |
17/377863 |
Filed: |
July 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16179135 |
Nov 2, 2018 |
11078284 |
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17377863 |
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15516115 |
Mar 31, 2017 |
10117889 |
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PCT/EP2015/072644 |
Sep 30, 2015 |
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16179135 |
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62058254 |
Oct 1, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/04 20180101; C07K
2317/76 20130101; C12N 15/09 20130101; C07K 2317/94 20130101; C07K
2317/565 20130101; C07K 16/28 20130101; A61K 39/395 20130101; A61K
39/3955 20130101; C12N 5/0695 20130101; C12N 15/1138 20130101; C07K
2317/33 20130101; C07K 2318/00 20130101; C07K 2317/21 20130101;
G01N 33/00 20130101; C07K 2317/92 20130101; C12N 15/63 20130101;
C07K 14/47 20130101; C12N 2310/14 20130101; A61P 9/10 20180101;
C12N 15/11 20130101; C07K 2317/622 20130101; C12N 2800/10 20130101;
C07K 16/2851 20130101; C07K 16/30 20130101; A61P 9/12 20180101;
A61K 31/7068 20130101; A61K 2039/505 20130101; G01N 33/574
20130101; C12N 5/00 20130101; C12N 2310/531 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; C12N 15/63 20060101
C12N015/63; C12N 15/09 20060101 C12N015/09; C12N 15/11 20060101
C12N015/11; A61P 9/10 20060101 A61P009/10; A61P 9/12 20060101
A61P009/12; A61P 9/04 20060101 A61P009/04 |
Claims
1. A method of making a Lectin-like oxidized low density
lipoprotein receptor-1 (LOX1) binding protein comprising culturing
a host cell under suitable conditions for producing the LOX-1
binding protein, wherein the host cell comprises a nucleic acid
molecule or set of nucleic acid molecules encoding the LOX1-binding
protein, wherein the LOX1-binding protein comprises a set of
complementary determining regions (CDRs): heavy chain variable
region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain variable region
(VL)-CDR1, VL-CDR2 and VL-CDR3, wherein: (a) (i) VH-CDR1 has the
amino acid sequence of SEQ ID NO:1; (ii) VH-CDR2 has the amino acid
sequence of SEQ ID NO:2; (iii) VH-CDR3 has the amino acid sequence
of SEQ ID NO:3; (iv) VL-CDR1 has the amino acid sequence of SEQ ID
NO:30; (v) VL-CDR2 has the amino acid sequence of SEQ ID NO:31; and
(vi) VL-CDR3 has the amino acid sequence of SEQ ID NO:32; (b) (i)
VH-CDR1 has the amino acid sequence of SEQ ID NO:1; (ii) VH-CDR2
has the amino acid sequence of SEQ ID NO:5; (iii) VH-CDR3 has the
amino acid sequence of SEQ ID NO:14; (iv) VL-CDR1 has the amino
acid sequence of SEQ ID NO:30; (v) VL-CDR2 has the amino acid
sequence of SEQ ID NO:31; and (vi) VL-CDR3 has the amino acid
sequence of SEQ ID NO:32; (c) (i) VH-CDR1 has the amino acid
sequence of SEQ ID NO:38; (ii) VH-CDR2 has the amino acid sequence
of SEQ ID NO:39; (iii) VH-CDR3 has the amino acid sequence of SEQ
ID NO:44; (iv) VL-CDR1 has the amino acid sequence of SEQ ID NO:55;
(v) VL-CDR2 has the amino acid sequence of SEQ ID NO:60; and (vi)
VL-CDR3 has the amino acid sequence of SEQ ID NO:61; or (d) (i)
VH-CDR1 has the amino acid sequence of SEQ ID NO:38; (ii) VH-CDR2
has the amino acid sequence of SEQ ID NO:39; (iii) VH-CDR3 has the
amino acid sequence of SEQ ID NO:40; (iv) VL-CDR1 has the amino
acid sequence of SEQ ID NO:55; (v) VL-CDR2 has the amino acid
sequence of SEQ ID NO:56; and (vi) VL-CDR3 has the amino acid
sequence of SEQ ID NO:57.
2. The method of claim 1, wherein the LOX1-binding protein
comprises a heavy chain variable region (VH) and a light chain
variable region (VL) selected from the group consisting of: (a) a
VH having the amino acid sequence of SEQ ID NO:4 and a VL having
the amino acid sequence of SEQ ID NO:33; (b) a VH having the amino
acid sequence of SEQ ID NO:29 and a VL having the amino acid
sequence of SEQ ID NO:33; (c) a VH having the amino acid sequence
of SEQ ID NO:41 and a VL having the amino acid sequence of SEQ ID
NO:58 and (d) a VH having the amino acid sequence of SEQ ID NO:54
and a VL having the amino acid sequence of SEQ ID NO:70.
3. The method of claim 1, wherein the LOX1-binding protein
comprises a heavy chain variable region (VH) comprising SEQ ID NO:4
and a light chain variable region (VL) comprising SEQ ID NO:33.
4. The method of claim 1, wherein the LOX1-binding protein reduces,
inhibits or antagonizes LOX1 activity.
5. The method of claim 1, wherein the LOX1-binding protein is an
antibody.
6. The method of claim 5, wherein the antibody is a monoclonal
antibody, a recombinant antibody, a human antibody, a humanized
antibody, a chimeric antibody, a bi-specific antibody, a
multi-specific antibody, or a LOX1-binding antibody fragment.
7. The method of claim 6, wherein the LOX1-binding antibody
fragment is selected from the group consisting of: a Fab fragment,
a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody, and
a single chain antibody molecule.
8. The method of claim 1, wherein the LOX1-binding protein further
comprises a heavy chain immunoglobulin constant domain selected
from the group consisting of: (a) a human IgA constant domain (b) a
human IgD constant domain; (c) a human IgE constant domain; (d) a
human IgG1 constant domain; (e) a human IgG2 constant domain; (f) a
human IgG3 constant domain; (g) a human IgG4 constant domain; and
(h) a human IgM constant domain.
9. The method of claim 1, wherein the LOX1-binding protein further
comprises a light chain immunoglobulin constant domain selected
from the group consisting of: (a) a human Ig kappa constant domain;
and (b) a human Ig lambda constant domain.
10. The method of claim 1, wherein the LOX1-binding protein further
comprises a human IgG1 heavy chain constant domain and a human
lambda light chain constant domain.
11. The method of claim 10, wherein the IgG1 heavy chain constant
domain contains a mutation at positions 234, 235 and 331, wherein
the position numbering is according to the EU index as in
Kabat.
12. The method of claim 11, wherein the IgG1 heavy chain constant
domain contains the mutations L234F, L235E and P331S, wherein the
position numbering is according to the EU index as in Kabat.
13. The method of claim 1, wherein the nucleic acid molecule is
operably linked to a control sequence.
14. The method of claim 1, wherein the host cell comprises a vector
comprising the nucleic acid molecule according to claim 1.
15. The method of claim 1, wherein the host cell is a mammalian
host cell.
16. The method of claim 15, wherein the host cell is a NSO murine
myeloma cell, a PER.C6.RTM. human cell, or a Chinese hamster ovary
(CHO) cell.
17. The method of claim 1 further comprising isolating the
LOX1-binding protein secreted from the host cell.
18. An isolated LOX1-binding protein that binds the same epitope as
a LOX1-binding protein produced according to claim 1.
19. A method of making a Lectin-like oxidized low density
lipoprotein receptor-1 (LOX1) binding protein comprising culturing
a host cell under suitable conditions for producing the LOX-1
binding protein, wherein the host cell comprises a nucleic acid
molecule or set of nucleic acid molecules encoding the LOX1-binding
protein, wherein the LOX1-binding protein comprises a set of
complementary determining regions (CDRs): heavy chain variable
region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain variable region
(VL)-CDR1, VL-CDR2 and VL-CDR3, wherein the set of CDRs has a total
of 18 or fewer amino acid substitutions, deletions, and/or
insertions from a reference set of CDRs in which: (i) VH-CDR1 has
the amino acid sequence of SEQ ID NO:1; (ii) VH-CDR2 has the amino
acid sequence of SEQ ID NO:5; (iii) VH-CDR3 has the amino acid
sequence of SEQ ID NO:14; (iv) VL-CDR1 has the amino acid sequence
of SEQ ID NO:30; (v) VL-CDR2 has the amino acid sequence of SEQ ID
NO:31; and (vi) VL-CDR3 has the amino acid sequence of SEQ ID
NO:32.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 16/179,135, filed Nov. 2, 2018, which is a
continuation of U.S. patent application Ser. No. 15/516,115, filed
Mar. 31, 2017, which issued as U.S. Pat. No. 10,117,889. U.S.
patent application Ser. No. 15/516,115 is a U.S. National Stage
application of International Patent Application No.
PCT/EP2015/072644, filed on Sep. 30, 2015, which claims the benefit
of U.S. Provisional Patent Application No. 62/058,254, filed Oct.
1, 2014. Each of the aforementioned patent applications is
incorporated by reference herein in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing
in ASCII text file (Name: LOX1-100-US-CNT-SequenceListing.txt;
Size: 50,980 bytes; and Date of Creation: Nov. 2, 2018) filed with
the application is incorporated herein by reference in its
entirety.
BACKGROUND
[0003] This disclosure provides Lectin-like oxidized low density
lipoprotein receptor-1 (LOX1) binding proteins and methods for the
use of such binding proteins, e.g., for the treatment, prevention
and/or amelioration of a disease or condition associated with LOX1
including, e.g., vascular dysfunction, atherosclerosis (plaque
progression, rupture and/or thrombosis) coronary artery disease
(CAD), ischemia (e.g., myocardial ischemia), infarction (e.g.,
myocardial infarction), stroke and acute coronary syndrome
(ACS).
[0004] Atherosclerosis is a complex disease that results from the
accumulation of lipids, macrophages and fibrous elements as lesions
in the arterial wall. The lesions develop into complex plaques that
narrow the artery lumen and are a focus of chronic inflammation.
The plaques are vulnerable to rupture triggering thrombosis that
results in adverse cardiovascular events including stroke and
myocardial infarction. Atherosclerosis is the primary cause of
coronary artery disease, stroke and peripheral vascular disease and
therefore represents the most common cause of morbidity worldwide
(World Health Organization 2011).
[0005] Lectin-like oxidized low density lipoprotein receptor-1
(LOX1) is a disulphide linked type II transmembrane protein. It was
first identified as a major receptor of oxidized low density
lipoprotein (oxLDL) (Kume et al., 70th Scientific Sessions of the
American Heart Association Ser. 96, 1997). The receptor consists of
a short N terminal cytoplasmic domain, transmembrane domain, neck
domain and a C-type lectin domain (CTLD), with the structure of the
CTLD has been solved (Ohki et al., Structure 13:905-917 (2005)). In
addition, LOX1 can be proteolytically cleaved in the neck domain
releasing soluble LOX1 (sLOX1). LOX1 is a class E scavenger
receptor and binds multiple ligands including oxLDL, C-reactive
protein (CRP), phosphatidylserine, advanced glycation end products
(AGEs), small dense lipoproteins (sdLDL), oxidized HDL,
N4-oxononanoyl lysine (ONL), heat shock proteins (hsp), Chlamydia
pneumoniae, platelets, leukocytes and apoptotic cells. Many of
these ligands, particularly oxLDL, are associated with
atherosclerosis. Multiple signal transduction pathways are
associated with LOX1 activation including RhoA/Racl, nitrogen
monoxide, p38MAPK, protein kinase B and C, ERK1/2, and NF.kappa.B.
See, e.g., Taye et. al., Eur J Clin Invest. 43(7):740-5 (2013).
[0006] Preclinical evidence implicates LOX1 in the promotion of
vascular dysfunction, plaque progression, rupture and thrombosis,
atherosclerosis and inflammatory conditions. See, e.g.,
Ulrich-Merzenich et al., Expert Opin Ther Targets. 17(8):905-19
(2013). For example, whereas LOX1 knockout mice have reduced aortic
atherosclerosis and decreased vessel wall collagen deposition
(Mehta et al., Circ. Res. 100:1634-1642 (2007)), LOX1
overexpression increased atherosclerotic plaque formation (Inoue et
al., Circ. Res. 97:176-84 (2005); and White et al., Cardiovascular
pathology 20:369-73 (2011)) with LOX1 expression observed on the
vulnerable plaque shoulders and associated with macrophage
accumulation, apoptosis, and MMP-9 expression (Li et al., Cir.
Cardio. Imaging 3:464-72 (2010)). Neutralizing LOX1 antibodies
restored acetylcholine induced coronary arteriolar dilation (Xu et
al., Arterioscler. Thromb. Vasc. Biol. 27(4) 871-877 (2007)) and
reduced intimal thickening after balloon injury in rats (Hinagata
et al., Cardiovasc. Res. 69:263-71 (2006)). LOX1 expression in
humans is not constitutive but dynamically inducible by
proinflammatory stimuli. In the atherosclerotic plaque LOX1 is
expressed on endothelial cells, smooth muscle cells and
macrophages. Interestingly serum sLOX1 has been proposed to be
diagnostic of plaque instability and rupture in acute coronary
syndrome (ACS) patients (Nakamura et al., J. Pharm. Biomed. Anal.
51:158-163 (2010)); to be predictive of ACS recurrence or death
(Kume et al., 70th Scientific Sessions of the American Heart
Association Ser. 96, 1997); and is associated with increasing
number of complex lesions (Zhao et al., Clin. Cardiol. 34:172-177
(2011)).
[0007] Atherosclerosis related mortality continues to rise due to
the increasing prevalence of hypertension, diabetes, dyslipidemia
and life-style characteristics (such as smoking and obesity) which
are risk factors for atherosclerosis. Intervention with standard of
care treatments including: platelet inhibitors, anti-hypertensives,
HMG CoA reductases inhibitors (statins), thrombolytic agents,
percutaneous arterial dilation, stenting or coronary artery bypass
surgery have had significant clinical benefit. However, despite the
use of preventative strategies and treatment there are still large
numbers of patients who suffer from secondary major adverse
cardiovascular events (MACE). Therefore there is a need for new
therapeutics that can be used alone or in combination with the
standard of care.
BRIEF SUMMARY
[0008] The disclosure provides LOX1-binding proteins and their
methods of use. In particular aspects, the LOX1-binding proteins
disclosed herein reduce, inhibit or block LOX1-binding to one or
more of its ligands. In some aspects, the LOX1-binding proteins
reduce, inhibit or block LOX1-binding with oxidized low density
lipoprotein (oxLDL), C-reactive protein (CRP) and/or advanced
glycation end products (AGEs). In some aspects, the disclosure
provides methods of using LOX1-binding proteins for the treatment,
prevention and/or amelioration of a disease or condition associated
with LOX1 expression and/or reduced HDL-mediated signaling. The
disclosure also provides methods of using LOX1-binding proteins for
the treatment, prevention and/or amelioration of acute coronary
syndrome (ACS), myocardial infarction (MI) or coronary artery
disease (CAD) or a condition associated with ACS, MI or CAD. In
some aspects, the disclosure provides methods of using LOX1-binding
proteins for the treatment, prevention and/or amelioration of a
disease or condition selected from the group including, but not
limited to: atherosclerosis, thrombosis, coronary artery disease
(CAD), ischemia (e.g., myocardial ischemia), infarction (e.g.,
myocardial infarction), acute coronary syndrome (ACS), stroke,
reperfusion injury, restenosis, peripheral vascular disease,
hypertension, heart failure, inflammation, angiogenesis,
preeclampsia and cancer.
[0009] In some aspects, the LOX1-binding protein comprises a set of
complementary determining regions (CDRs): heavy chain variable
region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain variable region
(VL)-CDR1, VL-CDR2 and VL-CDR3, wherein the set of CDRs is
identical to, or has a total of 18 or fewer (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) amino acid
substitutions, deletions, and/or insertions from a reference set of
CDRs in which: (a) VH-CDR1 has the amino acid sequence of SEQ ID
NO:1; (b) VH-CDR2 has the amino acid sequence of SEQ ID NO:5; (c)
VH-CDR3 has the amino acid sequence of SEQ ID NO:14; (d) VL-CDR1
has the amino acid sequence of SEQ ID NO:30; (e) VL-CDR2 has the
amino acid sequence of SEQ ID NO:31; and (0 VL-CDR3 has the amino
acid sequence of SEQ ID NO:32.
[0010] In some aspects, the LOX1-binding protein comprises a set of
six complementary determining regions (CDRs): heavy chain variable
region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain variable region
(VL)-CDR1, VL-CDR2 and VL-CDR3, wherein the set of CDRs is
identical to, or has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions, and/or
insertions from a reference set of CDRs in which: (a) VH-CDR1 has
the amino acid sequence of SEQ ID NO:1; (b) VH-CDR2 has the amino
acid sequence of SEQ ID NO:2; (c) VH-CDR3 has the amino acid
sequence of SEQ ID NO:3; (d) VL-CDR1 has the amino acid sequence of
SEQ ID NO:30; (e) VL-CDR2 has the amino acid sequence of SEQ ID
NO:31; and (0 VL-CDR3 has the amino acid sequence of SEQ ID
NO:32.
[0011] In some aspects, the LOX1-binding protein comprises a set of
complementary determining regions (CDRs): heavy chain variable
region (VH)-CDR1, VH-CDR2, VH-CDR3, light chain variable region
(VL)-CDR1, VL-CDR2 and VL-CDR3 wherein the set of CDRs is identical
to, or has a total of one, two, three, four, five, six, seven,
eight or fewer amino acid substitutions, deletions, and/or
insertions from a reference set of CDRs in which: (a) VH-CDR1 has
the amino acid sequence of SEQ ID NO:38; (b) VH-CDR2 has the amino
acid sequence of SEQ ID NO:39; (c) VH-CDR3 has the amino acid
sequence of SEQ ID NO:40; (d) VL-CDR1 has the amino acid sequence
of SEQ ID NO:55; (e) VL-CDR2 has the amino acid sequence of SEQ ID
NO:56; and (0 VL-CDR3 has the amino acid sequence of SEQ ID
NO:57.
[0012] In further aspects, the LOX1-binding protein comprises a set
of six CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and
VL-CDR3 wherein: (a) VH-CDR1 has the amino acid sequence of SEQ ID
NO:1; (b) VH-CDR2 has the amino acid sequence of SEQ ID NO:2; (c)
VH-CDR3 has the amino acid sequence of SEQ ID NO:3; (d) VL-CDR1 has
the amino acid sequence of SEQ ID NO:30; (e) VL-CDR2 has the amino
acid sequence of SEQ ID NO:31; and (0 VL-CDR3 has the amino acid
sequence of SEQ ID NO:32.
[0013] In further aspects, the LOX1-binding protein comprises a set
of six CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and
VL-CDR3 wherein: (a) VH-CDR1 has the amino acid sequence of SEQ ID
NO:38; (b) VH-CDR2 has the amino acid sequence of SEQ ID NO:39; (c)
VH-CDR3 has the amino acid sequence of SEQ ID NO:44; (d) VL-CDR1
has the amino acid sequence of SEQ ID NO:55; (e) VL-CDR2 has the
amino acid sequence of SEQ ID NO:60; and (f) VL-CDR3 has the amino
acid sequence of SEQ ID NO:61.
[0014] In further aspects, the LOX1-binding protein comprises a set
of six CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and
VL-CDR3 wherein: (a) VH-CDR1 has the amino acid sequence of SEQ ID
NO:38; (b) VH-CDR2 has the amino acid sequence of SEQ ID NO:39; (c)
VH-CDR3 has the amino acid sequence of SEQ ID NO:40; (d) VL-CDR1
has the amino acid sequence of SEQ ID NO:55; (e) VL-CDR2 has the
amino acid sequence of SEQ ID NO:56; and (f) VL-CDR3 has the amino
acid sequence of SEQ ID NO:57.
[0015] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) having at least 90, 95, 97, 98 or 99%
sequence identity to SEQ ID NO:4 and/or a light chain variable
region (VL) having at least 90, 95, 97, 98 or 99% sequence identity
to SEQ ID NO:33.
[0016] In some aspects, the LOX1-binding protein comprises a VH
having at least 90, 95, 97, 98 or 99% sequence identity to SEQ ID
NO:41 and/or a VL having at least 90, 95, 97, 98 or 99% sequence
identity to SEQ ID NO:58.
[0017] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) having at least 90, 95, 97, 98 or 99%
sequence identity to SEQ ID NOs:4, 19-29, 41, or 48-54; and a light
chain variable region (VL) having at least 90, 95, 97, 98 or 99%
sequence identity to SEQ ID NOs:33, 36, 37, 58 or 65-70.
[0018] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) having at least 90, 95, 97, 98 or 99%
sequence identity and a light chain variable region (VL) having at
least 90, 95, 97, 98 or 99% sequence identity to a VH and a VL
selected from: (a) a VH having the amino acid sequence of SEQ ID
NO:4 and a VL having the amino acid sequence of SEQ ID NO:33; (b) a
VH having the amino acid sequence of SEQ ID NO:29 and a VL having
the amino acid sequence of SEQ ID NO:33; (c) a VH having the amino
acid sequence of SEQ ID NO:41 and a VL having the amino acid
sequence of SEQ ID NO:58; and (d) a VH having the amino acid
sequence of SEQ ID NO:54 and a VL having the amino acid sequence of
SEQ ID NO:70.
[0019] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the VH sequence is identical to, or has a total of 15 or
fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15)
amino acid substitutions, deletions, and/or insertions from a
reference VH sequence of SEQ ID NO:4, 19-28, or 29, and wherein the
VL sequence is identical to, or that has a total of 6 or fewer
(e.g., 1, 2, 3, 4, 5, or 6) amino acid substitutions, additions
and/or deletions from a reference VL sequence of SEQ ID NO:33, 36
or 37.
[0020] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) selected from the group consisting of: a
VH comprising SEQ ID NO:4, 19-29, 41, or 48-54; and a light chain
variable region (VL) selected from the group consisting of a VL
comprising SEQ ID NO:33, 36, 37, 58 or 65-70.
[0021] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) and a light chain variable region (VL)
selected from the group consisting of: (a) a VH having the amino
acid sequence of SEQ ID NO:4 and a VL having the amino acid
sequence of SEQ ID NO:33; (b) a VH having the amino acid sequence
of SEQ ID NO:29 and a VL having the amino acid sequence of SEQ ID
NO:33; (c) a VH having the amino acid sequence of SEQ ID NO:41 and
a VL having the amino acid sequence of SEQ ID NO:58; and (d) a VH
having the amino acid sequence of SEQ ID NO:54 and a VL having the
amino acid sequence of SEQ ID NO:70.
[0022] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) comprising the amino acid sequence of
SEQ ID NO:4 and a light chain variable region (VL) comprising the
amino acid sequence of SEQ ID NO:33.
[0023] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) selected from a VH containing a VH-CDR1
having the amino acid sequence of SEQ ID NO:1, a VH-CDR2 having the
amino acid sequence of SEQ ID NO:2, 5-12, or 13, and a VH-CDR3
having the amino acid sequence of SEQ ID NO:3, 14-17 or 18; and a
light chain variable region (VL) selected from a VL containing a
VL-CDR1 having the amino acid sequence of SEQ ID NO:30, a VL-CDR2
having the amino acid sequence of SEQ ID NO:31, and a VL-CDR3
having the amino acid sequence of SEQ ID NO:32, 34 or 35.
[0024] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
wherein the VH sequence is identical to, or has a total of one,
two, three, four, five, six, seven, eight or fewer amino acid
substitutions, deletions, and/or insertions from a reference VH
sequence of SEQ ID NO:41, 48-53 or 54, and wherein the VL sequence
is identical to, or that has a total of one, two, three, four,
five, six, seven, eight or fewer amino acid substitutions,
additions and/or deletions from a reference VL sequence of SEQ ID
NO:58, 65-69 or 70.
[0025] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) selected from a VH containing a VH-CDR1
having the amino acid sequence of SEQ ID NO:38, a VH-CDR2 having
the amino acid sequence of SEQ ID NO:39, 42, or 43, and a VH-CDR3
having the amino acid sequence of SEQ ID NO:40, 44-46 or 47; and a
light chain variable region (VL) selected from a VL containing a
VL-CDR1 having the amino acid sequence of SEQ ID NO:55 or 59, a
VL-CDR2 having the amino acid sequence of SEQ ID NO:56 or 60, and a
VL-CDR3 having the amino acid sequence of SEQ ID NO:57, 61-63, or
64.
[0026] In some aspects, the LOX1-binding protein comprises a set of
CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR3 and VL-CDR3 as
described in Table 1, FIG. 4 or FIG. 5 (e.g. a set of CDRs from
Lox514, LX5140011, LX5140014, LX5140016, LX5140038, LX5140094,
LX5140108, LX5140110, LX5140092, LX5140092_D, LX5140093,
LX5140093_D, Lox696, LX6960067_ngl1, LX6960071_ngl1,
LX6960073_ngl1, LX6960086_ngl1, LX6960094_ngl1, LX6960101_ngl1,
LX6960102_ngl1, LX6960116_ngl1, LX6960073_gl, or
LX6960073_G82bs_gl).
[0027] In some additional aspects, the LOX1-binding protein
comprises a heavy chain variable region (VH) and light chain
variable region (VL) as described in Table 1, FIG. 4 or FIG. 5
(e.g. a VH and VL from Lox514, LX5140011, LX5140014, LX5140016,
LX5140038, LX5140094, LX5140108, LX5140110, LX5140092, LX5140092_D,
LX5140093, LX5140093_D, Lox696, LX6960067_ngl1, LX6960071_ngl1,
LX6960073_ngl1, LX6960086_ngl1, LX6960094_ngl1, LX6960101_ngl1,
LX6960102_ngl1, LX6960116_ngl1, LX6960073_gl, or
LX6960073_G82bs_gl).
[0028] In some aspects, the isolated LOX1-binding protein comprises
a set of complementary determining regions (CDRs): heavy chain
variable region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain
variable region (VL)-CDR1, VL-CDR2 and VL-CDR3 wherein: (a) VH-CDR1
comprises the amino acid sequence: E L S M H (SEQ ID NO: 1); (b)
VH-CDR2 comprises the amino acid sequence: G F D P E D HX1 HX2 HX3
HX4 HX5 HX6 Q K F Q G, wherein HX1 is selected from the group
consisting of G, W, Y and F, HX2 is selected from the group
consisting of E, T, Q, K, A, and S, HX3 is selected from the group
consisting of T, Y, I, and N, HX4 is selected from the group
consisting of I, A, R and H, HX5 is selected from the group
consisting of Y, V, T, L and Q, and HX6 is selected from the group
consisting of A, D, G, S and H (SEQ ID NO: 71); (c) VH-CDR3
comprises the amino acid sequence: HX7 HX8 G HX9 HX10 HX11 HX12
GVRGWDYYYGMD V, wherein HX7 is selected from the group consisting
of P, S and V, HX8 is selected from the group consisting of N, W,
D, and T, HX9 is selected from the group consisting of Q, R and T,
HX10 is selected from the group consisting of Q and H, HX11 is
selected from the group consisting of G and Q, and HX12 is selected
from the group consisting of K and G (SEQ ID NO: 72); (d) VL-CDR1
comprises the amino acid sequence: T G S S S N I G A G Y D V H (SEQ
ID NO: 30); (e) VL-CDR2 comprises the amino acid sequence: G N S N
R P S (SEQ ID NO: 31); and (f) VL-CDR3 comprises the amino acid
sequence: Q S Y D S LX1 LX2 LX3 LX4 LX5 LX6, wherein LX1 is
selected from the group consisting of M and S, LX2 is selected from
the group consisting of L, Y and H, LX3 is selected from the group
consisting of S and R, LX4 is selected from the group consisting of
A and G or is omitted (no amino acid), LX5 is selected from the
group consisting of W and F, and LX6 is selected from the group
consisting of V, G and A (SEQ ID NO: 73).
[0029] In some aspects, the LOX1-binding protein binds the same
epitope as a LOX-1 binding protein (e.g. a LOX-1 antibody or
fragment thereof) comprising a heavy chain variable region (VH) and
a light chain variable region (VL) described in Table 1, FIG. 4 or
FIG. 5, including a LOX-1 binding protein (e.g. a LOX-1 antibody or
fragment thereof) comprising a VH sequence of SEQ ID NO:4 and a VL
sequence of SEQ ID NO:33.
[0030] In other aspects, the disclosure provides LOX1-binding
proteins (e.g., antibodies such as, full length LOX1-antibodies,
LOX1-binding antibody fragments, and variants and derivatives
thereof), that compete or cross-compete for binding to LOX1 with a
LOX-1 binding protein (e.g. a LOX-1 antibody or fragment thereof)
comprising a heavy chain variable region (VH) and a light chain
variable region (VL) described in Table 1, FIG. 4 or FIG. 5,
including a LOX-1 binding protein (e.g. a LOX-1 antibody or
fragment thereof) comprising a VH sequence of SEQ ID NO:4 and a VL
sequence of SEQ ID NO:33.
[0031] In some aspects, the LOX1-binding protein binds the same
epitope as an antibody comprising a VH sequence of SEQ ID NO:4 and
a VL sequence of SEQ ID NO:33.
[0032] In other aspects, the disclosure provides LOX1-binding
proteins (e.g., antibodies such as, full length LOX1-antibodies,
LOX1-binding antibody fragments, and variants and derivatives
thereof), that compete or cross-compete for binding to LOX1 with an
antibody comprising a VH sequence of SEQ ID NO:4 and a VL sequence
of SEQ ID NO:33.
[0033] In some aspects, the LOX1-binding proteins disclosed herein
reduce, inhibit or antagonize LOX1 activity. In some aspects, the
LOX1-binding protein has at least one property selected from the
group consisting of: (a) reduces or inhibits binding of oxLDL,
C-reactive protein (CRP) and/or advanced glycation end products
(AGEs) to LOX1 as determined by any suitable assay including an
assay disclosed herein (see, e.g., Example 10, assays 1, 2 and/or 3
or Example 11); (b) decreases or inhibits RhoA/Racl, nitrogen
monoxide (NO), p38MAPK, protein kinase B and C, ERK1/2, and/or
NF.kappa.B signaling in an endothelial cell expressing cell surface
LOX1 as determined by any suitable assay including an assay
disclosed herein (see, e.g., Example 11); (c) decreases or inhibits
caspase-8, caspase-9, and/or BAX activity in an endothelial cell
expressing cell surface LOX1 as determined by any suitable assay
including an assay disclosed herein; (d) binds to LOX1 having the
single nucleotide polymorphism K167N as determined by any suitable
assay including an assay disclosed herein (see, e.g., Example 10,
assays 1, 2 and/or 3 or Example 11); (e) reduces or inhibits oxLDL
internalization as determined by any suitable assay including an
assay disclosed herein (see, e.g., Example 10, assay 4 or Example
11); (f) reduces or inhibits oxLDL-induced LOX1 signaling as
determined by any suitable assay including an assay disclosed
herein (see, e.g., Example 10, assay 5 or Example 11); (g) binds to
LOX1 with a dissociation constant (1(D) of about 150 pM to about
600 pM (e.g. about 400 pM) as determined by BIACORE or KinExA; (h)
binds to LOX1 with a Kon rate of about 1.times.10.sup.5 M.sup.-1
s.sup.-1 to about 6.times.10.sup.6 M.sup.-1 s.sup.-1 (e.g. about
5.times.10.sup.5 M.sup.-1 s.sup.-1) as determined by BIACORE; and
(i) binds to LOX1 with a Koff rate of about 1.times.10.sup.-4
s.sup.-1 to about 3.times.10.sup.-4 s.sup.-1 (e.g. about
2.3.times.10.sup.-4 s.sup.-1) as determined by BIACORE.
[0034] In additional aspects, the LOX1-binding protein is an
antibody. In some aspects, the antibody is a monoclonal antibody, a
recombinant antibody, a human antibody, a humanized antibody, a
chimeric antibody, a bi-specific antibody, a multi-specific
antibody, or an LOX1-binding antibody fragment. In some aspects the
antibody is a LOX1 binding antibody fragment selected from the
group consisting of: a Fab fragment, a Fab' fragment, a F(ab')2
fragment, a Fv fragment, a diabody, and a single chain antibody
molecule.
[0035] In some aspects, the LOX1-binding protein is an antibody
that comprises an IgG1 heavy chain immunoglobulin constant region.
In some aspects, the IgG1 constant region comprises a mutation that
decreases effector function. In further aspects, the IgG1 constant
region comprises the triple mutation L234F/L235E/P331S, that
results in an effector null IgG1.
[0036] The disclosure provides an isolated nucleic acid or a set of
nucleic acids encoding a LOX1-binding protein (e.g. a LOX-1
antibody or fragment thereof). Also provided is a vector or set of
vectors containing the nucleic acids or set of nucleic acids, and
host cells transformed with the isolated nucleic acids or vectors.
In some aspects, the host cell is a mammalian host cell such as, a
NSO murine myeloma cell, a PER.C6C.RTM. human cell, or a Chinese
hamster ovary (CHO) cell. Host cells and hybridomas producing
LOX1-binding proteins (e.g. a LOX-1 antibody or fragment thereof)
are also provided.
[0037] The disclosure also provides a method for making an
LOX1-binding protein disclosed herein. In some aspects, the method
comprises culturing a host cell or hybridoma capable of expressing
the LOX1-binding protein (e.g. a LOX-1 antibody or fragment
thereof) under suitable conditions and optionally provides a method
for isolating the LOX1-binding protein secreted from the host cell
or hybridoma. And the disclosure additionally provides the
LOX1-binding protein (e.g. a LOX-1 antibody or fragment thereof)
isolated using these methods.
[0038] Also provided are pharmaceutical compositions comprising a
LOX1-binding protein (e.g. a LOX-1 antibody or fragment thereof)
and a pharmaceutically acceptable carrier. Methods for treating,
preventing and/or ameliorating a condition associated with LOX1,
elevated LOX1 activity and/or elevated LOX1 expression levels,
including, for example, atherosclerosis, thrombosis, coronary
artery disease (CAD), ischemia (e.g., myocardial ischemia),
infarction (e.g., myocardial infarction), acute coronary syndrome
(ACS), stroke, reperfusion injury, restenosis, peripheral vascular
disease, hypertension, heart failure, inflammation (e.g., chronic
inflammation), angiogenesis, preeclampsia and/or cancer in a
subject are also provided herein. In some aspects, the methods
comprise administering to a subject in need thereof, a
pharmaceutical composition comprising an effective amount of a
LOX1-binding protein.
[0039] In some aspects, the LOX1-binding protein is administered
alone. In other aspects, the LOX1-binding protein is administered
as a combination therapy. In some aspects, the LOX1-binding protein
is administered as a combination therapy to the standard of care
treatment/therapy.
[0040] Also provided is a method of reducing LOX1 activity in a
subject comprising administering an effective amount of a
LOX1-binding protein to a subject in need thereof.
[0041] Additionally provided are methods of treating, preventing,
and/or ameliorating atherosclerosis. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having atherosclerosis. In other
aspects, the subject to which the LOX1-binding protein is
administered is at risk of developing atherosclerosis. In some
aspects, the subject has a proatherogenic condition. In further
aspects, the proatherogenic condition is systemic lupus
erythematosus (SLE), diabetes, hypertension, hyperglycemia, heart
failure, vascular injury, organ transplantation, dyslipidemia
(e.g., hyperlipidemia), inflammation (e.g., chronic inflammation
and endotoxin induced inflammation) and/or bacterial infection.
Also provided are methods of decreasing atherosclerosis. In some
instances, the disclosure provides a method of decreasing
atherosclerosis in a subject that comprises administering a
LOX1-binding protein to a subject having atherosclerosis.
[0042] Also provided are methods of treating, preventing, and/or
ameliorating thrombosis. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof) to a subject having thrombosis. In other
aspects, the subject to which the LOX1-binding protein is
administered is at risk of developing thrombosis. In some aspects
the thrombosis is an arterial thrombosis. In further aspects, the
thrombosis is an arterial thrombosis.
[0043] The disclosure also provides methods of treating,
preventing, and/or ameliorating coronary artery disease (CAD) or a
condition associated with CAD. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having CAD. In other aspects, the
subject to which the LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof) is administered is at risk of
developing CAD. In some aspects, the subject has a proatherogenic
condition. In further aspects, the proatherogenic condition is
systemic lupus erythematosus (SLE), diabetes, hypertension,
hyperglycemia, heart failure, vascular injury, organ
transplantation, dyslipidemia (e.g., hyperlipidemia), inflammation
(e.g., chronic inflammation and endotoxin induced inflammation)
and/or bacterial infection.
[0044] The disclosure also provides methods of treating,
preventing, and/or ameliorating ischemia or a condition associated
with ischemia. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject having ischemia. In other aspects, the subject
to which the LOX1-binding protein (e.g., an anti-LOX1 antibody or
fragment thereof) is administered is at risk of developing
ischemia. In some aspects, the subject has myocardial ischemia. In
other aspects, the subject is at risk of developing myocardial
ischemia. In additional aspects, the subject has systemic lupus
erythematosus (SLE), diabetes, hypertension, hyperglycemia, heart
failure, vascular injury, organ transplantation, dyslipidemia
(e.g., hyperlipidemia), inflammation (e.g., chronic inflammation
and endotoxin induced inflammation) and/or a bacterial
infection.
[0045] Also provided are methods of treating, preventing, and/or
ameliorating an infarction or a condition associated with an
infarction. In some instances, the method comprises administering a
LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment
thereof in a pharmaceutical composition described herein) to a
subject having an infarction. In other aspects, the subject to
which the LOX1-binding protein (e.g., an anti-LOX1 antibody or
fragment thereof) is administered is at risk of developing an
infarction. In some aspects, the subject has a myocardial
infarction. In other aspects, the subject is at risk of developing
a myocardial infarction. In additional aspects, the subject has
ischemia, systemic lupus erythematosus (SLE), diabetes,
hypertension, hyperglycemia, heart failure, vascular injury, organ
transplantation, dyslipidemia (e.g., hyperlipidemia), inflammation
(e.g., chronic inflammation and endotoxin induced inflammation)
and/or a bacterial infection.
[0046] The disclosure also provides methods of treating,
preventing, and/or ameliorating acute coronary syndrome (ACS) or a
condition associated with ACS. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having ACS. In other aspects, the
subject to which the LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof) is administered is at risk of
developing ACS. In some aspects, the subject has elevated soluble
LOX1 (sLOX1) serum levels or elevated LOX1 activity. In additional
aspects, the subject has atherosclerosis.
[0047] The disclosure also provides methods of treating,
preventing, and/or ameliorating a stroke or a condition associated
with a stroke. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject that has had a stroke. In other aspects, the
subject to which the LOX1-binding protein is administered is at
risk of having a stroke. In some aspects, the subject has elevated
sLOX serum levels and/or elevated LOX1 activity. In additional
aspects, the subject has atherosclerosis.
[0048] Also provided are methods of treating, preventing, and/or
ameliorating reperfusion injury or a condition associated with
reperfusion injury. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject having reperfusion injury. In other aspects,
the subject to which the LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof) is administered is at risk of
developing reperfusion injury. In some aspects, the subject is
about to have surgery. In other aspects, the subject has had
surgery. In some aspects the surgery is transplantation or coronary
bypass surgery. In additional aspects the patient has, or is at
risk of developing, myocardial ischemia-reperfusion injury. In
further aspects the method decreases myocardial injury, reduces
serum creatine kinase-MB isoenzyme (CK-MB) and serum
malondialdehyde (MDA) levels, reduces cardiomyocyte size, reduces
leukocyte infiltration at the site of the injury, and/or reduces
cardiac dysfunction (e.g., reduces left ventricular pressure (LVP)
and increases left ventricular end-diastolic pressure (LVEDP). In
further aspects, the method increases heart stroke volume,
fractional shortening, and/or injection fraction.
[0049] The disclosure also provides methods of treating,
preventing, and/or ameliorating restenosis or a condition
associated with restenosis. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject having restenosis. In other aspects, the
subject to which the LOX1-binding protein is administered is at
risk of developing restenosis. In some aspects, the subject is
about to have surgery. In other aspects, the subject has had
surgery. In some aspects the surgery is an endovascular procedure
is vascular surgery, cardiac surgery or angioplasty. In additional
aspects the restenosis, the procedure is transplantation or
coronary bypass surgery. In additional aspects the treated,
prevented, and/or ameliorated restenosis is in-stent restenosis or
post-angioplasty restenosis.
[0050] In additional aspects, the disclosure provides methods of
treating, preventing, and/or ameliorating peripheral vascular
disease (PVD) or a condition associated with PVD. In some
instances, the method comprises administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof in a
pharmaceutical composition described herein) to a subject having
PVD. In other aspects, the subject to which the LOX1-binding
protein is administered is at risk of developing PVD. In some
aspects, the subject has elevated sLOX serum levels and/or elevated
LOX1 activity. In additional aspects, the subject has
atherosclerosis.
[0051] The disclosure also provides methods of treating,
preventing, and/or ameliorating inflammation or a condition
associated with inflammation. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject with inflammation. In further
aspects, the subject has chronic inflammation. In some aspects, the
subject has elevated oxLDL and/or sLOX serum levels and/or elevated
LOX1 activity. In additional aspects, the subject has
atherosclerosis.
[0052] The disclosure also provides methods of treating,
preventing, and/or ameliorating preeclampsia or a condition
associated with preeclampsia. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject with preeclampsia or eclampsia. In
further aspects, the subject has high blood pressure and large
amounts of protein in the urine. In some aspects, the subject has
elevated oxLDL and/or sLOX serum levels and/or elevated LOX1
activity. In additional aspects, the subject has swelling in the
feet, legs and/or hands.
[0053] In additional aspects, the disclosure provides methods of
stabilizing an atherosclerotic plaque in a subject. In some
instances, the method comprises administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody such as, a full length
LOX1-antibody, a LOX1-binding antibody fragment, and variants and
derivatives thereof) to a subject in need thereof. In some aspects
the method reduces the signaling of the RhoA/Racl, nitrogen
monoxide, p38MAPK, protein kinase B and C, ERK1/2, and/or
NF.kappa.B signal transduction pathway in the plaque. In other
aspects, the method decreases apoptosis in the plaque. In further
aspects, the method decreases caspase 8, caspase 9 and/or BAX
activity and/or increases BCL-2 activity in the plaque. In other
aspects, the method decreases the levels of an adhesion molecule or
cytokine produced by the plaque. In further aspects, the method
decreases E-selectin, P-selectin, ICAM-1, VCAM-1, MCP1 and/or
CD40/CD40L expression by the plaque. In additional aspects, the
method decreases atherosclerotic plaque size or formation,
macrophage accumulation and/or MMP (e.g., MMP9) expression in the
atherosclerotic plaque. In additional aspects, the method results
in decreased progression or regression of the plaque.
[0054] Also provided are methods of reducing the loss of vascular
tone in a subject. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
such as, a full length LOX1-antibody and a LOX1-binding antibody
fragment, and variants and derivatives thereof) to a subject in
need thereof. In some aspects the method reduces the loss of
vascular tone. In further aspects, the method reduces the loss of
vascular tone in a subject through regulating HDL driven NO
production (ability of antibody to stimulate endothelial NO
production. Additionally provided are methods of improving vascular
tone in a subject. In some instances, the method comprises
administering a LOX1-binding protein to a subject in need
thereof.
[0055] Additionally provided are methods of treating, preventing,
and/or ameliorating cancer. In some instances the disclosure
provides a method of treating, preventing, and/or ameliorating
cancer in a subject that comprises administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof) to a
subject having cancer. In some aspects, the subject has a cancer
selected from the group consisting of: breast cancer, colon cancer,
ovarian cancer, melanoma, cervical cancer, lung cancer, uterine
cancer, kidney cancer, and pancreatic cancer.
[0056] Also provided are methods of inhibiting tumor cell
proliferation, migration or invasion. In some instances the
disclosure provides a method of antagonizing LOX1 activity that
comprises contacting a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof) with a tumor cell expressing LOX1. In
some aspects the tumor cell is from a cancer selected from the
group consisting of: breast cancer, colon cancer, ovarian cancer,
melanoma, cervical cancer, lung cancer, uterine cancer, kidney
cancer, and pancreatic cancer. In some aspects the tumor cell is
from a cancer line.
[0057] The disclosure additionally provides methods of reducing or
inhibiting angiogenesis. In some aspects the method of reducing or
inhibiting angiogenesis comprises administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof) to a
subject in need thereof. In some aspects the subject has a
condition associated with pathological angiogenesis. In additional
aspects the disclosure provides a method of inhibiting angiogenesis
that comprises contacting a LOX1-binding protein (e.g., an
anti-LOX1 antibody or fragment thereof) with a cell expressing
LOX1. In some aspects the cell is an endothelial cell. In further
aspects the endothelial cell is a coronary endothelial cell. In
some aspects the method is performed in vitro. In other aspects the
method is performed in vivo.
[0058] Additionally provided are methods of blocking or reducing
LOX1 activity. In some aspects the disclosure provides methods of
blocking LOX1 activity comprising administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof) that
reduces or inhibits the interaction between LOX1 and a LOX1-binding
protein such as, oxLDL, AGEs, and/or CRP. In some aspects, LOX1 is
expressed on the surface of an endothelial cell, macrophage, smooth
muscle vascular cell and/or platelet. In some aspects the cell is
an endothelial cell such as, a coronary endothelial cell. In
additional aspects, the cell is a vascular smooth muscle cell,
macrophage, or platelet. In other aspects the cell is part of an
atherosclerotic tissue. In some aspects, the method is performed in
vivo. In other aspects, the method is performed in vitro. In some
aspects the blocked or reduced LOX1 activity is the binding and/or
taking up (e.g. internalization) of oxLDL. In additional aspects,
the blocked or reduced LOX1 activity is the induction of the p38
(MAPK), p44/42 MAPK, protein kinase C (PKC), protein kinase B
(PKB), protein tyrosine kinase (PTK), transcription factor NF-KB
and/or AP1 signaling pathway. In additional aspects the blocked or
reduced LOX1 activity is the induction of apoptosis. In further
embodiments, the induction of apoptosis is mediated by caspase-9,
caspase-3 and/or Bcl-2. In additional aspects the blocked or
reduced LOX1 activity is the expression of the A and B chains of
PDFG and/or heparin-binding EGF-like protein (HB-EGF) in
endothelial cells expressing LOX1. In some aspects, the blocked or
reduced LOX1 activity is a LOX1 activity induced by oxLDL binding
to LOX1.
[0059] Additionally provided are methods of blocking or reducing
LOX1 activity in a pathological condition associated with increased
LOX1 activity levels or LOX1 expression levels (e.g. sLOX1 serum
protein levels). In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof) to a subject having increased LOX1 activity or
LOX1 expression levels (e.g. sLOX1 serum protein levels). In some
aspects the pathological condition is systemic lupus erythematosus
(SLE), diabetes, hypertension, hyperglycemia, heart failure,
vascular injury, transplantation, dyslipidemia (hyperlipidemia),
inflammation, (e.g., chronic inflammation and endotoxin induced
inflammation) or bacterial infection. In some aspects, the subject
has elevated serum levels of OxLDL. In some aspects, the subject
has elevated serum levels of OxLDL, 15 lipoxygenase modified LDL,
15 lipoxygenase modified HDL, glyoxidized LDL,
lysophosphatidylcholinesterase (LPC) and/or palmitic acid. In
additional aspects, the subject has elevated serum levels of TNF
alpha, ILL interferon gamma, LPS (lipopolysaccharide), CRP,
angiotensin II, endothelin I, and/or AGEs. In additional aspects,
the subject has elevated serum levels of soluble LOX1 (sLOX1). In
some aspects, the subject has a single nucleotide polymorphism
(SNP) in the LOX1 gene. In some aspects, the SNP in the LOX1 gene
is the LOX1 K167N variant.
[0060] Also provided are methods of agonizing or increasing a
high-density lipoprotein (HDL) activity. In some aspects, the
disclosure provides a method of increasing or agonizing an HDL
activity by administering a LOX1-binding protein (e.g., an
anti-LOX1 antibody or fragment thereof) to a subject in need
thereof. In some aspects the increased HDL activity is the
promotion of HDL-mediated endothelial NO production. In some
aspects, the increased HDL activity is the inhibition of the
NF.kappa.B signaling activity of the endothelial cell. In some
aspects, the increase HDL activity is the promotion of endothelial
cell repair. In some aspects, the increase HDL activity is the
reduction of inflammation.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0061] FIG. 1A-D shows inhibition of oxLDL, AGE-BSA and CRP binding
to human LOX1 (hLOX1) by antibodies LOX514 and LOX696. Binding of
DyLight 649 labeled ox-LDL (FIG. 1A) or DyLight 649 labeled AGE-BSA
(FIG. 1B) to hLOX1 transfected cells or binding of biotin labeled
C-Reactive Protein (CRP) to recombinant hLOX1 (FIG. 1C) was
measured in the presence of LOX514 ("LOX10514-IgG1-TM") (diamonds),
LOX696 ("LOX10696-IgG1-TM") (circles) or 23C11 (squares), a
commercially available mouse anti-LOX-1 antibody. Representative
plots are shown in FIGS. 1A, 1B and 1C illustrating dose-dependent
inhibition of oxLDL, AGE-BSA and CRP binding, respectively, by
LOX514 and LOX696. In addition, LOX514 and LOX696 also block the
binding of DyLight 649 labeled ox-LDL to hLOX1 K167N transfected
cells (FIG. 1D) confirming that these antibodies bind and block
oxLDL binding to the LOX1 SNP K167N variant. These results
demonstrate specific, multi-ligand inhibition of LOX1 binding to
oxLDL, AGE-BSA and CRP by antibodies LOX514 and LOX696; and that
LOX514 and LOX696 functionally cross react with the common LOX1 SNP
K167N variant. M=molar concentration of antibody; bars denote
standard error.
[0062] FIG. 2A-B shows inhibition of oxLDL internalization and
oxLDL-dependent reactive oxygen species (ROS) generation by
antibodies LOX514 and LOX696. Cypher 5E labeled ox-LDL
internalization (FIG. 2A) or oxLDL-dependent ROS generation (FIG.
2B) in human LOX1 transfected cells was measured in the presence of
LOX514 ("LOX10514-IgG1-TM") (diamonds), LOX696 ("LOX10696-IgG1-TM")
(circles) or 23C11 (squares), a commercially available mouse
anti-LOX-1 antibody. Representative plots are shown in FIGS. 2A and
2B illustrating dose-dependent inhibition of oxLDL internalization
and oxLDL-dependent signaling, respectively, by LOX514 and LOX696.
For oxLDL-dependent ROS generation in hLOX1 transfected cells, the
relative fluorescent units (RFU) of the amount of
Carboxy-dichlorofluorescein (DCF) generated in the assay with
LOX514, LOX696 and 23C11, as averaged for three replicates, is
shown (FIG. 2B). These results demonstrate that antibodies LOX514
and LOX696 inhibit oxLDL internalization and oxLDL-dependent LOX-1
signaling. M=molar concentration of antibody; bars denote standard
error.
[0063] FIG. 3A-B shows the species cross reactivity of anti-LOX1
antibodies, LOX514 and LOX696. Cross-reactivity of anti-human LOX1
antibodies to various LOX1 species orthologs was assessed using a
scFv binding ELISA. As shown in FIG. 3A, LOX514 ("LOX10514") and
LOX696 ("LOX10696") bind to human and cynomolgus LOX1 but not to
mouse, rat or rabbit LOX1 orthologs or Bovine Serum Albumin
(negative control). The specificity of LOX514 and LOX696 to other
human C type lectin and scavenger receptors related to LOX-1 was
also assessed using an IgG binding ELISA. As shown in FIG. 3B,
LOX514 ("LOX10514 IgG1-TM") and LOX696 ("LOX10696 IgG1-TM") bind
only to human LOX1 and do not bind to human CLEC-7A, CLEC-1A,
CLEC-4L, CLEC-1B, SR-A1 or SR-B3. As expected, CAT252 IgG1-TM, an
isotype control antibody, did not bind to any of the human C type
lectin and scavenger receptors tested. CLEC-7A=C-type lectin domain
family 7 member A (also known as Dectin-1); CLEC-1A=C-type lectin
domain family 1 member A; CLEC-4L=C-type lectin domain family 4
member L (also known as DC-SIGN); CLEC-1B=C-type lectin domain
family 1 member B (also known as CLEC-2); SR-A1=Macrophage
scavenger receptor types I and II (also known as MSR);
SR-B3=Platelet glycoprotein 4 (also known as CD36). Bars denote
standard error.
[0064] FIG. 4A-B shows a comparison between LOX514 and several
optimized LOX514 antibodies. Amino acid sequence alignments of the
heavy chain variable region (VH) (FIG. 4A) or the light chain
variable region (VL) (FIG. 4B) between LOX514 and optimized LOX514
antibodies are shown. Differences from LOX514 VH or VL are
highlighted. FW=framework; CDR=complementary determining
regions.
[0065] FIG. 5A-B shows a comparison between LOX696 and several
optimized LOX696 antibodies. Amino acid sequence alignments of the
heavy chain variable region (VH) (FIG. 5A) or the light chain
variable region (VL) (FIG. 5B) between LOX696 and optimized LOX696
antibodies are shown. Differences from LOX696 VH or VL are
highlighted. FW=framework; CDR=complementary determining
regions.
[0066] FIG. 6 shows the specificity of anti-LOX1 antibodies for
human LOX1 compared to several human C type lectin and scavenger
receptors related to LOX-1. The specificity of anti-LOX1 antibodies
LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D,
LX6960073_gl ("LX6960073") and LX6960073_G82bS_gl
("LX6960073G82bS") in an IgG1-TM format to human LOX-1 and other
human C type lectin and scavenger receptors related to LOX-1 was
assessed using an IgG binding ELISA. LX5140108, LX5140110,
LX5140092_N>D, LX5140093_N>D, LX6960073_gl and
LX6960073_G82bS_gl bind only to human LOX1 and do not bind to human
CLEC-7A, CLEC-1A, CLEC-4L, CLEC-1B, SR-A1 or SR-B3. NIP228, an
isotype human IgG1-TM control antibody, did not bind to human LOX-1
or any of the human C type lectin and scavenger receptors tested.
CLEC-7A=C-type lectin domain family 7 member A (also known as
Dectin-1); CLEC-1A=C-type lectin domain family 1 member A;
CLEC-4L=C-type lectin domain family 4 member L (also known as
DC-SIGN); CLEC-1B=C-type lectin domain family 1 member B (also
known as CLEC-2); SR-A1=Macrophage scavenger receptor types I and
II (also known as MSR); SR-B3=Platelet glycoprotein 4 (also known
as CD36). Bars denote standard error.
[0067] FIG. 7A-B shows the cross reactivity of anti-LOX1 antibodies
to various LOX1 species orthologs. Cross-reactivity of anti-LOX1
antibodies LX5140108, LX5140110, LX5140092_N>D,
LX5140093_N>D, LX6960073_gl and LX6960073_G82bS_gl in an IgG1-TM
format to various LOX1 species orthologs was assessed using an IgG
binding ELISA. As shown in FIG. 7A, anti-LOX1 antibodies LX5140108,
LX5140110, LX5140092_N>D, LX5140093_N>D, LX6960073_gl and
LX6960073_G82bS_gl bind to human and cynomolgus LOX1 but not to
mouse or rat LOX1 orthologs or CD86 (negative control). Only
LX5140108, LX5140110 and LX5140092_N>D also bind rabbit LOX-1.
NIP228, an isotype human IgG1-TM control antibody, did not bind
CD86 or any of the LOX-1 orthologs tested. In addition, cross
reactivity characterization between human (huLOX) and cynomolgus
(cyLOX) LOX1 were performed for the anti-LOX1 IgG1-TM antibodies
LX5140108, LX5140110 and LX6960073_G82bS_gl ("LX6960073_G82bS")
using a competition ELISA. As shown in FIG. 7B, LX5140108
(circles), LX5140110 (squares) and LX6960073_G82bS_gl (triangles)
compete with both cynomolgus LOX1 and human LOX1 further confirming
the cynomolgus cross-reactivity of anti-LOX1 antibodies LX5140108,
LX5140110 and LX6960073_G82bS_gl. Bars denote standard error.
[0068] FIG. 8A-B shows inhibition of oxLDL, AGE-BSA and CRP binding
to human LOX1 (hLOX1), inhibition of oxLDL internalization and
inhibition of oxLDL-dependent LOX-1 signaling by antibodies
LX5140110 and LX6960073_G82bS_gl. Binding of DyLight 649 labeled
ox-LDL (FIG. 8A) or DyLight 649 labeled AGE-BSA (FIG. 8B) to hLOX1
transfected cells or binding of biotin labeled C-Reactive Protein
(CRP) to recombinant hLOX1 (FIG. 8C) was measured in the presence
of LX5140110-IgG1-TM ("LOX514110"; circles FIGS. 8A and 8C, squares
FIG. 8B), or LX6960073_G82bS_gl-IgG1-TM ("LX6960073_G82bS"; squares
FIGS. 8A and 8C, circles FIG. 8B). Representative plots are shown
in FIGS. 8A, 8B and 8C illustrating dose-dependent inhibition of
oxLDL, AGE-BSA and CRP binding to hLOX1, respectively, by LX5140110
and LX6960073_G82bS_gl. In addition, LX5140110 and
LX6960073_G82bS_gl also block the binding of DyLight 649 labeled
ox-LDL to hLOX1 K167N transfected cells (FIG. 8D) confirming that
these antibodies bind and block oxLDL binding to the LOX1 SNP K167N
variant. To examine the ability of LX5140110 and LX6960073_G82bS_gl
to block oxLDL internalization and oxLDL-dependent LOX-1 signaling,
cypher 5E labeled ox-LDL internalization (FIG. 8E) or
oxLDL-dependent ROS generation (FIG. 8F) in human LOX1 transfected
cells was measured in the presence of LX5140110-IgG1-TM ("514_110",
circles FIG. 8E; & "LOX10514_110", diamonds FIG. 8F), or
LX6960073_G82bS_gl-IgG1-TM ("LX6960073_G82bS", squares FIG. 8E;
& "LOX10696_73", squares FIG. 8F). Representative plots are
shown in FIGS. 8E and 8F illustrating dose-dependent inhibition of
oxLDL internalization and oxLDL-dependent ROS production,
respectively, by LX5140110 and LX6960073_G82bS_gl. These results
demonstrate: (1) specific, multi-ligand inhibition of LOX1 binding
to oxLDL, AGE-BSA and CRP by antibodies LX5140110 and
LX6960073_G82bS_gl; (2) LX5140110 and LX6960073_G82bS_gl
functionally cross react with the common LOX1 SNP K167N variant;
and (3) LX5140110 and LX6960073_G82bS_gl inhibit oxLDL
internalization and oxLDL-dependent LOX-1 signaling. M=molar
concentration of antibody; bars denote standard error.
[0069] FIG. 9A shows the inhibition of OxLDL uptake in human aortic
endothelial cells (HAECs) by LX5140110. AlexaFluor568-OxLDL uptake
by HAECs incubated with 0, 0.5, 1, 5, or 10 nM of anti-LOX1
antibody LX5140110 ("514") or 10 nM of control antibody (NIP) was
measured using fluorescence microscopy to test the ability of
LX5140110 to block binding and internalization of oxidized low
density lipoprotein (OxLDL) by HAECs. Approximately 12 images for
each set were analyzed and the average number of
Alexafluor-568-conjugated-OxLDL red fluorescent vesicles in each
cell following an 1 hour incubation was determined. Number of
vesicles per cell with 1 standard derivation is reported. These
results demonstrate that 5 nM or 10 nM LX5140110 significantly
inhibits OxLDL uptake by HAECs (p=0.0003 or p=0.0002 for 5 nM and
10 nM, respectively). * indicates that P<0.05 as compared with
untreated controls (cells incubated only with Alexa-Fluor tagged
OxLD, and without antibody); bars denote standard deviation.
[0070] FIG. 9B shows reduction of OxLDL-dependent NFkB signaling in
human aortic endothelial cells (HAECs) by LX5140110. HAECs
co-expressing NF.kappa.B-luciferase and GFP were serum starved for
24 hours and incubated for 8 hours with: vehicle (control); OxLDL
alone (50 .mu.g/ml); OxLDL (50 .mu.g/ml)+LX5140110 ("514") (10 nM);
or OxLDL (50 .mu.g/ml)+NIP (10 nM). Luciferase activity and
luminescence were measured, while GFP fluorescence was used as a
normalization control. These results demonstrate that addition of
10 mM LX5140110, but not NIP (control antibody), significantly
reduces OxLDL-dependent NFkB signaling in HAECs. N=5 for each
group; * indicates P<0.05 (compared with vehicle-only control);
# indicates P<0.05 (compared with OxLDL alone); bars denote
standard error.
[0071] FIG. 9C shows inhibition of OxLDL-dependent augmentation of
arginase activity in human aortic endothelial cells (HAECs) by
LX5140110. HAECs were serum starved for 24 hours and incubated for
3 hours with: vehicle alone (control); OxLDL (50 .mu.g/ml); OxLDL
(50 .mu.g/ml)+LX5140110 ("514") (1 nM); OxLDL (50
.mu.g/ml)+LX5140110 ("514") (3 nM); OxLDL (50 .mu.g/ml)+LX5140110
("514") (10 nM); or OxLDL (50 .mu.g/ml)+NIP (10 nM). Cells were
lysed and arginase activity was determined using the urea assay.
These results demonstrate that addition of 3 nM or 10 nM LX5140110,
but not 10 nM NIP (control antibody), significantly reduces
OxLDL-dependent arginase activity in HAECs in a dose-dependent
manner. N=3 for each group; * indicates that P<0.05 as compared
with vehicle-only controls; # indicates that P<0.05 as compared
with OxLDL (50 .mu.g/ml) control (without antibody); bars denote
standard error.
[0072] FIG. 9D shows that LX5140110 blocks OxLDL-dependent
reduction in nitric oxide production by HAECs. HAECs were serum
starved (1% serum) overnight and incubated for 24 hours with:
vehicle (control); OxLDL (50 .mu.g/ml); OxLDL (50
.mu.g/ml)+LX5140110 ("514") (0.5 nM); OxLDL (50 .mu.g/ml)+LX5140110
("514") (1 nM); OxLDL (50 .mu.g/ml)+LX5140110 ("514") (5 nM); OxLDL
(50 .mu.g/ml)+LX5140110 ("514") (10 nM); or OxLDL (50 .mu.g/ml)+NIP
(10 nM) prior to adding DAF-FM DA (5 .mu.M) in fresh media and
measuring total fluorescence of DAF-FM DA. These results
demonstrate that addition of 5 nM or 10 nM LX5140110, but not 10 nM
NIP (control antibody), significantly inhibits OxLDL-dependent
reduction in nitric oxide production of HAECs in a dose-dependent
manner. To confirm that nitric oxide (NO) was produced by eNOS, the
NOS inhibitor L-NAME was used as a control (data not shown). N=5
for each group; * indicates that P<0.05 (compared with
vehicle-only control); bars denote standard error.
[0073] FIG. 9E shows that LX5140110 blocks OxLDL-dependent
increased reactive oxygen species (ROS) production by HAECs. HAECs
were serum starved (1% serum) overnight and incubated for 24 hours
with: vehicle (control); OxLDL (50 .mu.g/ml); OxLDL (50
.mu.g/ml)+LX5140110 ("514") (0.5 nM); OxLDL (50 .mu.g/ml)+LX5140110
("514") (1 nM); OxLDL (50 .mu.g/ml)+LX5140110 ("514") (5 nM); OxLDL
(50 .mu.g/ml)+LX5140110 ("514") (10 nM); or OxLDL (50 .mu.g/ml)+NIP
(10 nM), prior to incubating the cells with fresh phenol-free media
containing 400 .mu.M of the luminol analogue L-012. Relative light
units (RLU) quantified from the luminescence of the luminol
analogue L-012 indicate changes in production of ROS. These results
demonstrate that addition of 0.5 nM, 1 nM, 5 nM or 10 nM LX5140110,
but not 10 nM NIP (control antibody), inhibits OxLDL-dependent ROS
production in HAECs. To confirm that superoxide was produced by
eNOS, the NOS inhibitor L-NAME was used as a control (data now
shown). N=5 for each group; * indicates P<0.05 as compared with
vehicle-only controls; # indicates P<0.05 as compared with OxLDL
(50 .mu.g/ml) control (without antibody); bars denote standard
error.
[0074] FIG. 9F shows that the luminol analogue L-012 is specific
for reactive oxygen species (ROS). HAECs were serum starved (1%
serum) overnight and incubated for 24 hours with: vehicle (Veh) or
superoxide scavenger SOD (5 mM). As shown, addition of the
superoxide scavenger SOD resulted in virtually undetectable levels
of luminescence thereby confirming the specificity of L-012 for
measuring superoxide (ROS) in FIG. 9E. * indicates that P<0.05
(compared with vehicle); bars denote standard error.
[0075] FIGS. 9G and 9H show that LX5140110 blocks OxLDL-mediated
phosphorylation of Focal Adhesion Kinase (FAK) at tyrosine (Y) 397.
HAECs were serum starved for 18 hours and incubated for 1 hour
with: 0, 0.1, 0.5, 1, 5, or 10 nM of anti-LOX1 antibody LX5140110
(514) or 10 nM of control antibody (NIP), prior to incubating cells
for 1 hour in fresh media with 50 .mu.g/ml of OxLDL and running 10
.mu.g of protein lysates on 4-15% gradient polyacrylamide gels.
Protein samples subjected to SDS-PAGE were transferred to
nitrocellulose membranes for Western blotting. A representative
blot showing changes in FAK phosphorylation at Tyr397 and
expression of GAPDH is shown in FIG. 9G, while quantification of
the percentage increase of FAK phosphorylation at Tyr397
(pY397-FAK) normalized to GAPDH expression is shown in FIG. 9H.
These results demonstrate that addition of 5 nM or 10 nM LX5140110,
but not 10 nM NIP (control antibody), inhibits OxLDL-mediated FAK
phosphorylation at Tyr397 in HAECs. * indicates that P<0.05
(compared with untreated control); # indicates that P<0.05 as
compared with 0 nM LX5140110 (514); bars denote standard error.
[0076] FIG. 9I shows that LOX1 is necessary for OxLDL signaling in
HAECs. Human Aortic Endothelial Cells (HAECs) were co-transduced
with adenoviruses encoding NF.kappa.B-LUC and either a non-targeted
shRNA (Ad-NTsh) or Lox-1 shRNA (Ad-Lox-lsh) to specifically inhibit
LOX1 expression. 24 hours post-transduction, cells were treated
with or without OxLDL (50 .mu.g/mL) and incubated at 37.degree. C.
for 24 hours. Firefly Luciferase activity was measured in cell
lysates using chemiluminescence. Ad-LOX-lsh significantly inhibited
OxLDL-mediated NFkB signaling in HAECs compared to cells incubated
with a control, non-targeted shRNA (Ad-NTsh) virus. * indicates
P<0.05 (compared with untreated control); # indicates P<0.05
as compared with OxLDL+Ad-NTsh.
[0077] FIG. 9J shows that a viral vector expressing interfering
short hairpin RNAs (shRNA) directed to the 5'UTR region of Human
LOX1 gene (LOX1-shRNA) reduces LOX1 protein expression. LOX1
protein expression was monitored by immunoblotting using anti-Loxl
(IB:LOX-1) and anti-GAPDH (IB:GAPDH, used here as protein loading
control) antibodies from the following samples: HAEC lysates
transduced with non-targeted shRNA (NT shRNA) (lanes 1 and 2), or
HAEC lysates transduced with Lox-1 shRNA (lane 3). Lysates in lanes
2 and 3 were treated with OxLDL (50 .mu.g/mL), while lysates in
lane 1 were not exposed to OxLDL. Addition of LOX1-shRNA
significantly reduced LOX1 protein expression (lane 3) compared to
levels that were measured in lysates from cells treated with
non-targeted shRNA (lanes 1 and 2). These data confirm that
LOX1-shRNA effectively inhibits LOX1 expression in HAECs.
[0078] FIG. 9K shows that a viral vector expressing interfering
short hairpin RNAs (shRNA) directed to the 5'UTR region of Human
LOX1 gene (Ad-LOX-1shRNA) reduces LOX1 protein expression in a
dose-dependent manner. HAECs transduced with increasing
concentrations of Lox-1 shRNA (Ad-LOX-1shRNA) (0, 10, 20, 30 and
100 MOI for lanes 1-5, respectively) were stimulated with OxLDL (50
.mu.g/mL) (with the exception of the control, unstimulated sample
shown in lane 1). Cell lysates were obtained and subjected to
immunoblotting with anti-Lox-1 antibody. Data are representative of
at least 3 independent experiments. Ad-LOX-1shRNA significantly
reduced LOX1 protein expression in a dose-dependent manner.
[0079] FIG. 10 shows some of the signaling pathways involved in
LOX1 receptor signaling that are blocked by the anti-LOX1
antibodies disclosed herein, including LX5140110 ("514Ab").
DETAILED DESCRIPTION OF THE INVENTION
[0080] The present disclosure provides LOX1-binding proteins. In
some aspects, the disclosure provides antagonists of LOX1 activity
that are anti-LOX1 antibodies such as, full length anti-LOX1
antibodies, LOX1-binding antibody fragments, and variants and
derivatives thereof. Related nucleic acids, compositions comprising
LOX1-binding proteins, and methods of making the LOX1-binding
proteins are also provided. Methods of using the LOX1-binding
proteins in, for example, improving the LOX1-binding protein
associated diseases or conditions such as: atherosclerosis,
thrombosis, coronary artery disease (CAD), ischemia (e.g.,
myocardial ischemia), infarction (e.g., myocardial infarction),
acute coronary syndrome (ACS), stroke, reperfusion injury,
restenosis, peripheral vascular disease, hypertension, heart
failure, inflammation (e.g., chronic inflammation), angiogenesis,
preeclampsia or cancer in a subject and diagnostic uses, are
further provided.
[0081] In order that the present disclosure can be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
I. Definitions
[0082] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. The terms "a" (or "an"), as
well as the terms "one or more," and "at least one" can be used
interchangeably herein.
[0083] The term "about" is used herein to mean approximately, in
the region of, roughly, or around. When the term "about" is used in
conjunction with a numerical value or range, it modifies that value
or range by extending the boundaries above and below the numerical
values or ranges set forth. In general, the term "about" is used
herein to modify a numerical value or range above and below the
stated value or range by a variance of 10%.
[0084] Furthermore, "and/or" is to be taken as specific disclosure
of each of the two specified features or components with or without
the other. Thus, the term "and/or" as used in a phrase such as "A
and/or B" is intended to include "A and B," "A or B," "A" (alone),
and "B" (alone). Likewise, the term "and/or" as used in a phrase
such as "A, B, and/or C" is intended to encompass each of the
following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C;
A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[0085] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0086] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, amino acid sequences are written left to right in amino
to carboxy orientation. The headings provided herein are not
limitations of the various aspects of the disclosure, which can be
had by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification in its entirety.
[0087] Wherever aspects are described with the language
"comprising," otherwise analogous aspects described in terms of
"consisting of" and/or "consisting essentially of" are also
provided.
[0088] Amino acids are referred to herein by their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
codes.
[0089] The term "LOX1" and "Lectin-like oxidized low density
lipoprotein receptor-1" are used interchangeably herein and refer
to LOX1 and/or biologically active fragments of LOX1. The cDNA and
amino acid sequences of three hLOX1 isoforms are provided at
GenBank Acc. Nos.: NP_002534.1, NP_001166103.1, and NP_001166104.1,
each of which is incorporated herein by reference in its
entirety.
[0090] The terms "inhibit," "block," "reduce," and "suppress" are
used interchangeably herein and refer to any statistically
significant decrease in biological activity, including full
blocking of the activity. For example, "inhibition" can refer to a
decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100% in LOX1 biological activity. Accordingly, when the terms
"inhibition" or "suppression" are applied to describe for example,
an effect on a LOX1-mediated signal transduction pathway in a cell
expressing cell surface LOX1 (e.g., an endothelial cell, smooth
muscle cell, macrophage, and platelet) and in the presence of a
LOX1 ligand (e.g., oxLDL, CRP and AGEs), the terms refer to the
ability of a LOX1-binding protein, e.g., an anti-LOX1 antibody, to
decrease a LOX1-mediated induced signal transduction in the cell at
a statistically significant level (e.g., with a p value less than
or equal to 0.05). In some aspects, the LOX1-mediated signal
transduction pathway is a member selected from RhoA/Racl, nitrogen
monoxide, p38MAPK, protein kinase B and C, ERK1/2, and/or
NF.kappa.B. In additional aspects, the inhibited or blocked
LOX1-mediated biological activity is programmed cell death (i.e.,
apoptosis). In further embodiments, the decreased, inhibited or
blocked LOX1-mediated biological activity is LOX1-mediated
increased caspase 8, caspase 9, and/or decreased BAX activity. The
cell which expresses cell surface LOX1 can be a naturally occurring
cell (e.g., human endothelial cells, smooth muscle cells, and
macrophage), a cell from a cell line or a recombinant cell produced
by introducing a nucleic acid encoding LOX1 into the host cell.
[0091] The terms "antibody" or "immunoglobulin," as used
interchangeably herein, include whole antibodies and any
antigen-binding fragment or single chains thereof. A typical
antibody comprises at least two heavy (H) chains and two light (L)
chains interconnected by disulfide bonds. Each heavy chain is
comprised of a heavy chain variable region (abbreviated herein as
VH) and a heavy chain constant region. The heavy chain constant
region is comprised of three domains, CH1, CH2, and CH3. Each light
chain is comprised of a light chain variable region (abbreviated
herein as VL) and a light chain constant region. The light chain
constant region is comprised of one domain, Cl. The VH and VL
regions can be further subdivided into regions of hypervariablity,
termed Complementarity Determining Regions (CDRs), interspersed
with regions that are more conserved, termed framework regions
(FW). Each VH and VL is composed of three CDRs and four FWs,
arranged from amino-terminus to carboxy-terminus in the following
order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions
of the heavy and light chains contain a binding domain that
interacts with an antigen. The constant regions of the antibodies
can mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g.,
effector cells) and the first component (C1q) of the classical
complement system. Exemplary antibodies of the present disclosure
include typical antibodies, scFvs, and combinations thereof where,
for example, an scFv is covalently linked (for example, via
peptidic bonds or via a chemical linker) to the N-terminus of
either the heavy chain and/or the light chain of a typical
antibody, or intercalated in the heavy chain and/or the light chain
of a typical antibody.
[0092] The term "antibody" can refer to an immunoglobulin molecule
that recognizes and specifically binds to a target, such as a
protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid,
or combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. The term "antibody" includes monoclonal antibodies,
recombinant antibodies, human antibodies, humanized antibodies,
chimeric antibodies, bi-specific antibodies, multi-specific
antibodies, or antibody fragments thereof.
[0093] The term "antibody" encompasses intact polyclonal
antibodies, intact monoclonal antibodies, antibody fragments (such
as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv)
mutants, multispecific antibodies such as bispecific antibodies
generated from at least two intact antibodies, chimeric antibodies,
humanized antibodies, human antibodies, fusion proteins comprising
an antigen determination portion of an antibody, and any other
modified immunoglobulin molecule comprising an antigen recognition
site so long as the antibodies exhibit the desired biological
activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses
(isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2),
based on the identity of their heavy-chain constant domains
referred to as alpha, delta, epsilon, gamma, and mu, respectively.
The different classes of immunoglobulins have different and
well-known subunit structures and three-dimensional configurations.
Antibodies can be naked or conjugated to other molecules such as
toxins, radioisotopes, etc.
[0094] The term "germlining" means that amino acids at specific
positions in an antibody are mutated back to those amino acids
occurring at the same position as found in the germ line.
[0095] The term "antigen-binding antibody fragment" or
"LOX1-binding antibody fragment" refers to a portion of an intact
antibody and refers to the complementarity determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, single chain antibodies (e.g.,
ScFvs), and multispecific antibodies formed from antibody
fragments. The disclosure further provides LOX1-binding antibody
fragments wherein the antibody fragment is a Fab fragment, a Fab'
fragment, a F(ab')2 fragment, a Fv fragment, a diabody, or a single
chain antibody molecule.
[0096] A "monoclonal antibody" refers to a homogeneous antibody
population involved in the highly specific recognition and binding
of a single antigenic determinant, or epitope. This is in contrast
to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal antibody" encompasses both intact and full-length
monoclonal antibodies as well as antibody fragments (such as Fab,
Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins
comprising an antibody portion, and any other modified
immunoglobulin molecule comprising an antigen recognition site.
Furthermore, "monoclonal antibody" refers to such antibodies made
in any number of ways including, but not limited to, by hybridoma,
phage selection, recombinant expression, and transgenic
animals.
[0097] The term "humanized antibody" refers to an antibody derived
from a non-human (e.g., murine) immunoglobulin, which has been
engineered to contain minimal non-human (e.g., murine) sequences.
Typically, humanized antibodies are human immunoglobulins in which
residues from the complementary determining region (CDR) are
replaced by residues from the CDR of a non-human species (e.g.,
mouse, rat, rabbit, or hamster) that have the desired specificity,
affinity, and capability (Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)). In some instances, the Fv framework
region (FW) residues of a human immunoglobulin are replaced with
the corresponding residues in an antibody from a non-human species
that has the desired specificity, affinity, and capability.
[0098] Humanized antibodies can be further modified by the
substitution of additional residues either in the Fv framework
region and/or within the replaced non-human residues to refine and
optimize antibody specificity, affinity, and/or capability. In
general, humanized antibodies will comprise substantially all of at
least one, and typically two or three, variable domains containing
all or substantially all of the CDR regions that correspond to the
non-human immunoglobulin whereas all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence.
Humanized antibody can also comprise at least a portion of an
immunoglobulin constant region or domain (Fc), typically that of a
human immunoglobulin. Examples of methods used to generate
humanized antibodies are described in U.S. Pat. No. 5,225,539 or
5,639,641.
[0099] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FW) connected by three complementarity-determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FW regions and, with
the CDRs from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al., J. Molec. Biol. 273:927-948 (1997)). In addition,
combinations of these two approaches are sometimes used in the art
to determine CDRs.
[0100] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)
herein incorporated by reference). The amino acid position
numbering as in Kabat, refers to the numbering system used for
heavy chain variable domains or light chain variable domains of the
compilation of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence can contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FW or CDR of the variable domain. For example, a
heavy chain variable domain can include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g., residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FW residue 82.
[0101] The Kabat numbering of residues can be determined for a
given antibody by alignment at regions of homology of the sequence
of the antibody with a "standard" Kabat numbered sequence. Chothia
refers instead to the location of the structural loops (Chothia and
Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia
CDR-H1 loop when numbered using the Kabat numbering convention
varies between H32 and H34 depending on the length of the loop
(this is because the Kabat numbering scheme places the insertions
at H35A and H35B; if neither 35A nor 35B is present, the loop ends
at 32; if only 35A is present, the loop ends at 33; if both 35A and
35B are present, the loop ends at 34). The AbM hypervariable
regions represent a compromise between the Kabat CDRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software.
[0102] IMGT (ImMunoGeneTics) also provides a numbering system for
the immunoglobulin variable regions, including the CDRs. See, e.g.,
Lefranc, M. P. et al., Dev. Comp. Immunol. 27: 55-77(2003), which
is herein incorporated by reference. The IMGT numbering system was
based on an alignment of more than 5,000 sequences, structural
data, and characterization of hypervariable loops and allows for
easy comparison of the variable and CDR regions for all species.
According to the IMGT numbering schema VH-CDR1 is at positions 26
to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93
to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions
50 to 52, and VL-CDR3 is at positions 89 to 97.
[0103] As used throughout the specification the VH CDRs sequences
described correspond to the classical Kabat numbering locations,
namely Kabat VH-CDR1 is at positions 31-35, VH-CDR2 is a positions
50-65, and VH-CDR3 is at positions 95-102. VL-CDR1, VL-CDR2 and
VL-CDR3 also correspond to classical Kabat numbering locations,
namely positions 24-34, 50-56 and 89-97, respectively
[0104] The term "human antibody" means an antibody produced by a
human or an antibody having an amino acid sequence corresponding to
an antibody produced by a human made using any technique known in
the art. This definition of a human antibody includes intact or
full-length antibodies, fragments thereof, and/or antibodies
comprising at least one human heavy and/or light chain polypeptide
such as, for example, an antibody comprising murine light chain and
human heavy chain polypeptides.
[0105] The term "chimeric antibodies" refers to antibodies wherein
the amino acid sequence of the immunoglobulin molecule is derived
from two or more species. Typically, the variable region of both
light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g., mouse, rat,
etc.) with the desired specificity, affinity, and capability while
the constant regions are homologous to the sequences in antibodies
derived from another (usually human) to avoid eliciting an immune
response in that species.
[0106] The terms "TM" or "TM mutant" refer to a mutation in the
IgG1 constant region that results in a decreased effector function
(e.g., ADCC) of an antibody having the mutation. A TM mutant
comprises a combination of three mutations L234F/L235E/P331S
resulting in an effector null human IgG1 (EU numbering Kabat et al.
(1991) Sequences of Proteins of Immunological Interest, U.S. Public
Health Service, National Institutes of Health, Washington, D.C.),
introduced into the heavy chain of an IgG1.
[0107] The terms "YTE" or "YTE mutant" refer to a mutation in IgG1
Fc that results in an increase in the binding to human FcRn and
improves the serum half-life of the antibody having the mutation. A
YTE mutant comprises a combination of three mutations,
M252Y/S254T/T256E (EU numbering Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, U.S. Public Health Service,
National Institutes of Health, Washington, D.C.), introduced into
the heavy chain of an IgG1. See U.S. Pat. No. 7,658,921, which is
incorporated by reference herein. The YTE mutant has been shown to
increase the serum half-life of antibodies approximately four-times
as compared to wild-type versions of the same antibody (Dall'Acqua
et al., J. Biol. Chem. 281:23514-24 (2006)). See also U.S. Pat. No.
7,083,784, which is incorporated by reference herein in its
entirety.
[0108] The terms "LOX1-binding protein", "anti-LOX1 antibody," or
"an antibody that specifically binds LOX1" refer to a LOX1-binding
protein such as an anti-LOX1 antibody that is capable of binding
LOX1 with sufficient affinity such that the antibody is useful as a
therapeutic agent or diagnostic reagent in targeting LOX1. The
extent of binding of an anti-LOX1 antibody to an unrelated,
non-LOX1 protein is less than about 10% of the binding of the
antibody to LOX1 as measured, e.g., by a radioimmunoassay (RIA),
BIACORE.RTM. (using recombinant LOX1 as the analyte and antibody as
the ligand, or vice versa), Kinetic Exclusion Assay (KINEXA.RTM.),
or other binding assays known in the art. In certain aspects, the
LOX1-binding protein is a full-length antibody or a LOX1-binding
antibody fragment that has a dissociation constant (KD) of
.ltoreq.1 nM, .ltoreq.0.5 nM, .ltoreq.0.1 nM, .ltoreq.10 pM, or
.ltoreq.1 pM, or in some instances, a KD of about 150 pM to about
600 pM or about 400 pM to about 600 pM. In certain aspects, the
LOX1-binding protein is a full-length antibody or a LOX1-binding
antibody fragment that has a dissociation constant (KD) of
.ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM,
.ltoreq.0.1 nM, .ltoreq.10 pM, .ltoreq.1 pM, or .ltoreq.0.1 pM, or
in some instances, a KD of about 150 pM to about 600 pM.
[0109] An "antagonist" or "blocking" LOX1-binding protein is one
that inhibits or reduces the biological activity of LOX1. In some
aspects, the antagonist LOX1-binding protein inhibits the ability
of LOX1 to bind oxLDL, AGEs, and/or CRP. In some aspects, the
LOX1-binding protein inhibits the ability of LOX1 to bind oxHDL,
HSP60, leukocytes and/or activated platelets. In certain aspects a
LOX1-binding protein substantially or completely inhibits the
biological activity of LOX1. Desirably, the LOX1 biological
activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or
even 100%. In particular aspects, the LOX1-binding protein is an
anti-LOX1 antibody, such as a full length antibody or a
LOX1-binding antibody fragment. In further aspects, the anti-LOX1
antibody inhibits or reduces the biological activity of LOX1 by
10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.
[0110] "Binding affinity" generally refers to the strength of the
sum total of non-covalent interactions between a single binding
site of a molecule (e.g., an antibody) and its binding partner
(e.g., an antigen). Unless indicated otherwise, as used herein,
"binding affinity" refers to intrinsic binding affinity which
reflects a 1:1 interaction between members of a binding pair (e.g.,
antibody and antigen). The affinity of a molecule X for its partner
Y can generally be represented by the dissociation constant (KD).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present disclosure.
[0111] "Potency" is normally expressed as an IC.sub.50 value, in nM
or pM unless otherwise stated. IC.sub.50 is the median inhibitory
concentration of an antibody molecule. In functional assays,
IC.sub.50 is the concentration that reduces a biological response
by 50% of its maximum. In ligand-binding studies, IC.sub.50 is the
concentration that reduces receptor binding by 50% of maximal
specific binding level. IC.sub.50 can be calculated by means known
in the art.
[0112] The fold improvement in potency for the LOX1-binding protein
disclosed herein (e.g., an antibody such as, a full length
LOX1-antibody and a LOX1-binding antibody fragment, and variants
and derivatives thereof) as compared to a reference antibody can be
at least about 2-fold, at least about 4-fold, at least about
6-fold, at least about 8-fold, at least about 10-fold, at least
about 20-fold, at least about 30-fold, at least about 40-fold, at
least about 50-fold, at least about 60-fold, at least about
70-fold, at least about 80-fold, at least about 90-fold, at least
about 100-fold, at least about 110-fold, at least about 120-fold,
at least about 130-fold, at least about 140-fold, at least about
150-fold, at least about 160-fold, at least about 170-fold, or at
least about 180-fold or more.
[0113] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enables these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
Specific high-affinity IgG antibodies directed to the surface of
target cells "arm" the cytotoxic cells and are absolutely required
for such killing. Lysis of the target cell is extracellular,
requires direct cell-to-cell contact, and does not involve
complement. It is contemplated that, in addition to antibodies,
other proteins comprising Fc regions, specifically Fc fusion
proteins, having the capacity to bind specifically to an
antigen-bearing target cell will be able to effect cell-mediated
cytotoxicity. For simplicity, the cell-mediated cytotoxicity
resulting from the activity of an Fc fusion protein is also
referred to herein as ADCC activity.
[0114] A LOX1-binding protein (e.g., a LOX1 antibody, including an
antigen-binding fragment, variant, and derivative thereof),
polynucleotide, vector, cell, or composition that is "isolated" is
a polypeptide, antibody, polynucleotide, vector, cell, or
composition that is in a form not found in nature. Isolated
polypeptides (e.g., anti-LOX1 antibodies including full-length
antibodies and LOX1-binding antibody fragments), polynucleotide,
vector, polynucleotides, vectors, cells or compositions include
those which have been purified to a degree that they are no longer
in a form in which they are found in nature. In some aspects, an
antibody, polynucleotide, vector, cell, or composition that is
isolated is substantially pure.
[0115] By "subject" or "individual" or "animal" or "patient" or
"mammal," is meant any subject, particularly a mammalian subject,
for whom diagnosis, prognosis, or therapy is desired. Mammalian
subjects include humans, domestic animals, farm animals, sports
animals, and zoo animals including, e.g., humans, non-human
primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses,
cattle, bears, and so on.
[0116] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient (e.g., a LOX1-binding protein
disclosed herein) to be effective, and which contains no additional
components which are unacceptably toxic to a subject to which the
composition would be administered. Such composition can be
sterile.
[0117] An "effective amount" or "pharmaceutically effective amount"
of a LOX1-binding protein such as, an anti-LOX1 antibody, or
another therapeutic agent, is an amount sufficient to carry out a
specifically stated purpose. An "effective amount" can be
determined empirically and in a routine manner, in relation to the
stated purpose. The disclosure provides therapeutics to treat,
prevent or ameliorate diseases and conditions associated with LOX1
and/or decreased HDL-mediated signaling. These diseases and
conditions include, for example, atherosclerosis, thrombosis, CAD,
ischemia (e.g., myocardial ischemia), infarction (e.g., myocardial
infarction), acute coronary syndrome (ACS), stroke, reperfusion
injury, restenosis, peripheral vascular disease, hypertension,
heart failure, inflammation (e.g., chronic inflammation),
angiogenesis, preeclampsia and cancer.
[0118] The word "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
can be detectable by itself (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, can
catalyze chemical alteration of a substrate compound or composition
that is detectable.
[0119] Terms such as "treating" or "treatment" or "to treat" or
"ameliorating" or "to ameliorate" refer to both (1) therapeutic
measures that cure, slow down, lessen conditions associated with,
and/or halt progression of a diagnosed disease or condition and (2)
prophylactic or preventative measures that prevent and/or slow the
development of a targeted disease or condition. Thus, those in need
of treatment include those already with the disease or condition;
those at risk of developing the disease or condition; and those in
whom the disease or condition is to be prevented. In certain
aspects, a subject is successfully "treated" according to the
methods provided herein if the subject shows, e.g., total, partial,
or transient amelioration or elimination of a symptom associated
with the disease or condition. Such diseases or conditions include,
for example, atherosclerosis, thrombosis, CAD, ischemia (e.g.,
myocardial ischemia), infarction (e.g., myocardial infarction),
acute coronary syndrome (ACS), stroke, reperfusion injury,
restenosis, peripheral vascular disease, hypertension, heart
failure, inflammation (e.g., chronic inflammation), angiogenesis,
preeclampsia and cancer.
[0120] A "nucleic acid" or "polynucleotide," as used herein can
include one or more "polynucleotides," "polynucleotide molecules,"
or "polynucleotide sequences," and refers to a polymer of
nucleotides of any length, and includes DNA (genomic or cDNA) and
RNA. The nucleic acids can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase. A "nucleic acid" or "polynucleotide," can
comprise modified nucleotides, such as methylated nucleotides and
their analogs. The preceding description applies to all
polynucleotides referred to herein, including RNA and DNA (genomic
or cDNA).
[0121] The term "vector" means a construct, which is capable of
delivering, and in some aspects, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0122] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer can be linear or branched, it can comprise
modified amino acids, and non-amino acids can interrupt it. The
terms also encompass an amino acid polymer that has been modified
naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. It is understood that,
because the polypeptides of this disclosure are based upon
antibodies, in certain aspects, the polypeptides can occur as
single chains or associated chains.
[0123] The terms "identical" or percent sequence "identity" in the
context of two or more nucleic acids or proteins, refer to two or
more sequences or subsequences that are the same or have a
specified percentage of nucleotides or amino acid residues that are
the same, when compared and aligned (introducing gaps, if
necessary) for maximum correspondence, not considering any
conservative amino acid substitutions as part of the sequence
identity. The percent identity can be measured using sequence
comparison software or algorithms or by visual inspection. Various
algorithms and software are known in the art that can be used to
obtain alignments of amino acid or nucleotide sequences.
[0124] One such non-limiting example of a sequence alignment
algorithm is the algorithm described in Karlin et al., Proc. Natl.
Acad. Sci., 87:2264-2268 (1990), as modified in Karlin et al.,
Proc. Natl. Acad. Sci. 90:5873-5877 (1993), and incorporated into
the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids
Res., 25:3389-3402 (1991)). In certain aspects, Gapped BLAST can be
used as described in Altschul et al., Nucleic Acids Res.
25:3389-3402 (1997), BLAST-2, WU-BLAST-2 (Altschul et al., Methods
in Enzymology 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South
San Francisco, Calif.) or Megalign (DNASTAR) are additional
publicly available software programs that can be used to align
sequences. In certain aspects, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (e.g., using a NWSgapdna.CMP matrix and a gap
weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4,
5, or 6). In certain alternative aspects, the GAP program in the
GCG software package, which incorporates the algorithm of Needleman
and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to
determine the percent identity between two amino acid sequences
(e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a
gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,
2, 3, 4, 5). Alternatively, in certain aspects, the percent
identity between nucleotide or amino acid sequences is determined
using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
For example, the percent identity can be determined using the ALIGN
program (version 2.0) and using a PAM120 with residue Table, a gap
length penalty of 12 and a gap penalty of 4. One skilled in the art
can determine appropriate parameters for maximal alignment by
particular alignment software. In certain aspects, the default
parameters of the alignment software are used.
[0125] In certain aspects, the percent sequence identity "X" of a
first amino acid sequence to a second sequence amino acid is
calculated as 100.times.(Y/Z), where Y is the number of amino acid
residues scored as identical matches in the alignment of the first
and second sequences (as aligned by visual inspection or a
particular sequence alignment program) and Z is the total number of
residues in the second sequence. If the length of a first sequence
is longer than the second sequence, the percent identity of the
first sequence to the second sequence will be higher than the
percent identity of the second sequence to the first sequence.
[0126] A "conservative amino acid substitution" is one in which one
amino acid residue is replaced with another amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art, including basic
side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., glycine, alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). For example, substitution of
a phenylalanine for a tyrosine is a conservative substitution. In
certain aspects, conservative substitutions in the sequences of the
LOX1-binding proteins of the disclosure do not abrogate the binding
of the protein containing the substituted amino acid sequence to
LOX1. Methods of identifying nucleotide and amino acid conservative
substitutions which do not eliminate antigen-binding are well-known
in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187
(1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and
Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
[0127] The term "epitope" as used herein refers to a LOX1, e.g.,
human LOX1 (hLOX1) or monkey LOX1 (e.g. M. cynomolgus), protein
determinant capable of binding to a LOX1-binding protein (e.g., an
antibody) of the disclosure. Epitopes usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three-dimensional structural
characteristics, as well as specific charge characteristics.
Conformational and non-conformational epitopes are distinguished in
that the binding to the former but not the latter is lost in the
presence of denaturing solvents. Such LOX1-binding proteins can be
identified based on their ability to cross-compete (e.g., to
competitively inhibit the binding of, in a statistically
significant manner) with antibodies comprising for example, a VH
sequence of SEQ ID NO:4 and a VL sequence of SEQ ID NO:33, or a VH
sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:58 in
standard antigen-binding or activity assays.
[0128] A LOX1-binding protein (e.g., an antibody) is said to
"compete" with a reference molecule for binding to LOX1 if it binds
to LOX1 to the extent that it blocks, to some degree, binding of
the reference molecule to LOX1. The ability of proteins to compete
for binding to LOX1 can be determined by any method known in the
art including, for example, a competition ELISA assay. As used
herein, LOX1-binding protein may be said to competitively inhibit
binding of the reference molecule to LOX1, for example, by at least
90%, at least 80%, at least 70%, at least 60%, or at least 50%.
II. LOX1-Binding Proteins
[0129] The present disclosure provides LOX1-binding proteins that
specifically bind LOX1. In some aspects, a LOX1-binding protein is
an antibody (e.g., a full length LOX1-antibody, an antigen-binding
antibody fragment, and a variant and derivative thereof). In
further aspects, the antibody is a monoclonal antibody, a
recombinant antibody, a human antibody, a humanized antibody, a
chimeric antibody, a bi-specific antibody, a multi-specific
antibody, or an antibody fragment thereof. In certain aspects, the
LOX1-antibody is a full-length antibody.
[0130] In some aspects, the LOX1-antibody is a LOX1-binding
antibody fragment. In some aspects, the LOX1-binding antibody
fragment is a: Fab, Fab', F(ab')2, Fv fragment, diabody, or single
chain antibody molecule. In additional aspects, the LOX1-antibody
is a Fd, single chain Fv(scFv), disulfide linked Fv, V-NAR domain,
IgNar, intrabody, IgGACH2, minibody, F(ab).sub.3, tetrabody,
triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb.sup.2,
(scFv).sub.2, or a scFv-Fc.
[0131] In additional aspects, the LOX1 binding protein is an
antibody that includes a VH and a VL. In some aspects, the LOX1
binding protein (e.g. a LOX1 antibody or fragment thereof) further
includes a heavy chain constant region or fragment thereof. In some
aspects, the antibody comprises a heavy chain immunoglobulin
constant region selected from the group consisting of: (a) a human
IgA constant region, or fragment thereof; (b) a human IgD constant
region, or fragment thereof, (c) a human IgE constant domain, or
fragment thereof; (d) a human IgG1 constant region, or fragment
thereof, (e) a human IgG2 constant region, or fragment thereof, (f)
a human IgG3 constant region, or fragment thereof; (g) a human IgG4
constant region, or fragment thereof, and (h) a human IgM constant
region, or fragment thereof. In further aspects, the LOX1-binding
protein (e.g. a LOX1 antibody or fragment thereof) comprises a
heavy chain immunoglobulin constant domain that has, or has been
mutated to have, reduced ADCC activity. In particular aspects, the
LOX1-binding protein (e.g. a LOX1 antibody or fragment thereof)
comprises an IgG1 heavy chain constant region containing a mutation
that decreases effector function. In some aspects, the IgG1
constant region comprises a mutation at positions 234, 235 and 331,
wherein the position numbering is according to the EU index as in
Kabat. In further aspects, the IgG1 constant region comprises
triple mutations L234F/L235E/P331S (TM), wherein the position
numbering is according to the EU index as in Kabat, resulting in an
effector null human IgG1. In some aspects, the IgG1 constant region
comprises the triple mutation mutant YTE, as disclosed supra in the
Definitions section. In further aspects, the LOX1-binding protein
is an antibody containing an IgG1 constant region that comprises
both the triple mutation TM and the triple mutation YTE.
[0132] In certain aspects a heavy chain constant region or fragment
thereof, e.g., a human IgG constant region or fragment thereof, can
include one or more amino acid substitutions relative to a
wild-type IgG constant domain wherein the modified IgG has an
increased half-life compared to the half-life of an IgG having the
wild-type IgG constant domain. For example, the IgG constant domain
can contain one or more amino acid substitutions of amino acid
residues at positions 251-257, 285-290, 308-314, 385-389, and
428-436, wherein the amino acid position numbering is according to
the EU index as set forth in Kabat. In certain aspects the IgG
constant domain can contain one or more of a substitution of the
amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine
(F), Tryptophan (W), or Threonine (T), a substitution of the amino
acid at Kabat position 254 with Threonine (T), a substitution of
the amino acid at Kabat position 256 with Serine (S), Arginine (R),
Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine
(T), a substitution of the amino acid at Kabat position 257 with
Leucine (L), a substitution of the amino acid at Kabat position 309
with Proline (P), a substitution of the amino acid at Kabat
position 311 with Serine (S), a substitution of the amino acid at
Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine
(F), or Serine (S), a substitution of the amino acid at Kabat
position 433 with Arginine (R), Serine (S), Isoleucine (I), Proline
(P), or Glutamine (Q), or a substitution of the amino acid at Kabat
position 434 with Tryptophan (W), Methionine (M), Serine (S),
Histidine (H), Phenylalanine (F), or Tyrosine. More specifically,
the IgG constant domain can contain amino acid substitutions
relative to a wild-type human IgG constant domain including as
substitution of the amino acid at Kabat position 252 with Tyrosine
(Y), a substitution of the amino acid at Kabat position 254 with
Threonine (T), and a substitution of the amino acid at Kabat
position 256 with Glutamic acid (E).
[0133] In additional aspects, the LOX1-binding protein (e.g. a LOX1
antibody or fragment thereof) comprises a light chain
immunoglobulin constant domain selected from the group consisting
of: (a) a human Ig kappa constant domain; and (b) a human Ig lambda
constant domain. In additional aspects, the LOX1-binding protein
e.g. a LOX1 antibody or fragment thereof) comprises a human heavy
chain IgG1 constant domain containing triple mutations
L234F/L235E/P331S ("IgG-TM") resulting in an effector null human
IgG1 and a human light chain lambda constant domain.
[0134] The disclosure provides an isolated LOX1-binding protein
comprising a VH and/or a VL which has a total of one, two, three,
four, five, six, seven, eight or fewer amino acid substitutions,
deletions, and/or insertions from a reference VH or VL disclosed
herein. In some aspects, the LOX1-binding protein has a VH and/or
VL as shown in Table 1.
[0135] Exemplary LOX-1 binding proteins are provided in Table
1.
TABLE-US-00001 TABLE 1 Exemplary LOX-1 binding proteins Lox514 VH
CDR1 ELSMH (SEQ ID NO: 1) VH CDR2 GFDPEDGETIYAQKFQG (SEQ ID NO: 5)
VH CDR3 PNGQQGKGVRGWDYYYG MDV (SEQ ID NO: 14) VH QVQLVQSGAEVKKPGAS
VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF DPEDGETIYAQKFQGRV
TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGQ QGKGVRGWDYYYGMDVW GRGTTVTVSS
(SEQ ID NO: 29) VL CDR1 TGSSSNIGAGYDVH (SEQ ID NO: 30) VL CDR2
GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV (SEQ ID NO: 32) VL
QSVVTQPPSVSGAPGQ RVTISCTGSSSNIGAG YDVHWYQQLPGTAPKL LIYGNSNRPSGVPDRF
SGSKSGTSASLAITGL QAEDEADYYCQSYDSS LSGWVFGGGTKLTVL (SEQ ID NO: 33)
LX5140011 VH CDR1 ELSMH (SEQ ID NO: 1) VH CDR2 GFDPEDWEYAYDQKFQG
(SEQ ID NO: 6) VH CDR3 PNGQQGKGVRGWDYYYG MDV (SEQ ID NO: 14) VH
QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF
DPEDWEYAYDQKFQGRV TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGQ
QGKGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 19) VL CDR1 TGSSSNIGAGYDVH
(SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV
(SEQ ID NO: 32) VL QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD
VHWYQQLPGTAPKLLIY GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE
ADYYCQSYDSSLSGWVF GGGTKLTVL (SEQ ID NO: 33) LX5140014 VH CDR1 ELSMH
(SEQ ID NO: 1) VH CDR2 GFDPEDYTIRVGQKFQG (SEQ ID NO: 7) VH CDR3
PNGQQGKGVRGWDYYYG MDV (SEQ ID NO: 14) VH QVQLVQSGAEVKKPGAS
VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF DPEDYTIRVGQKFQGRV
TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGQ QGKGVRGWDYYYGMDVW GQGTTVTVSS
(SEQ ID NO: 20) VL CDR1 TGSSSNIGAGYDVH (SEQ ID NO: 30) VL CDR2
GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV (SEQ ID NO: 32) VL
QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIY
GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE ADYYCQSYDSSLSGWVF GGGTKLTVL
(SEQ ID NO: 33) LX5140016 VH CDR1 ELSMH (SEQ ID NO: 1) VH CDR2
GFDPEDWQTHTAQKFQG (SEQ ID NO: 8) VH CDR3 PNGQQGKGVRGWDYYYG MDV (SEQ
ID NO: 14) VH QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF
DPEDWQTHTAQKFQGRV TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGQ
QGKGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 21) VL CDR1 TGSSSNIGAGYDVH
(SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV
(SEQ ID NO: 32) VL QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD
VHWYQQLPGTAPKLLIY GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE
ADYYCQSYDSSLSGWVF GGGTKLTVL (SEQ ID NO: 33) LX5140038 VH CDR1 ELSMH
(SEQ ID NO: 1) VH CDR2 GFDPEDWTIHVDQKFQG (SEQ ID NO: 9) VH CDR3
PNGQQGKGVRGWDYYYG MDV (SEQ ID NO: 14) VH QVQLVQSGAEVKKPGAS
VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF DPEDWTIHVDQKFQGRV
TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGQ QGKGVRGWDYYYGMDVW GQGTTVTVSS
(SEQ ID NO: 22) VL CDR1 TGSSSNIGAGYDVH (SEQ ID NO: 30) VL CDR2
GNSNRPS (SEQ ID NO: 3l) VL CDR3 QSYDSSLSGWV (SEQ ID NO: 32) VL
QSVVTQPPSVSGAPGQ RVTISCTGSSSNIGAG YDVHWYQQLPGTAPKL LIYGNSNRPSGVPDRF
SGSKSGTSASLAITGL QAEDEADYYCQSYDSS LSGWVFGGGTKLTVL (SEQ ID NO: 33)
LX5140094 VH CDR1 ELSMH (SEQ ID NO: 1) VH CDR2 GFDPEDWQYHVSQKFQG
(SEQ ID NO: 10) VH CDR3 PNGQQGKGVRGWDYYYG MDV (SEQ ID NO: 14) VH
QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF
DPEDWQYHVSQKFQGRV TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGQ
QGKGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 23) VL CDR1 TGSSSNIGAGYDVH
(SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSMYRFG
(SEQ ID NO: 34) VL QSVVTQPPSVSGAPGQ RVTISCTGSSSNIGAG
YDVHWYQQLPGTAPKL LIYGNSNRPSGVPDRF SGSKSGTSASLAITGL QAEDEADYYCQSYDSM
YRFGFGGGTKLTVL (SEQ ID NO: 36) LX5140108 VH CDR1 ELSMH (SEQ ID NO:
1) VH CDR2 GFDPEDWSNHVSQKFQG (SEQ ID NO: 11) VH CDR3
STGRQGKGVRGWDYYYG MDV (SEQ ID NO: 15)
VH QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF
DPEDWSNHVSQKFQGRV TMTEDTSTDTAYMELSS LRSEDTAVYYCLTSTGR
QGKGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 24) VL CDR1 TGSSSNIGAGYDVH
(SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV
(SEQ ID NO: 32) VL QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD
VHWYQQLPGTAPKLLIY GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE
ADYYCQSYDSSLSGWVF GGGTKLTVL (SEQ ID NO: 33) LX5140110 VH CDR1 ELSMH
(SEQ ID NO: 1) VH CDR2 GFDPEDFKYHTHQKFQG (SEQ ID NO: 2) VH CDR3
VWGTQGKGVRGWDYYYG MDV (SEQ ID NO: 3) VH QVQLVQSGAEVKKPGAS
VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF DPEDFKYHTHQKFQGRV
TMTEDTSTDTAYMELSS LRSEDTAVYYCALVWGT QGKGVRGWDYYYGMDVW GQGTTVTVSS
(SEQ ID NO: 4) VL CDR1 TGSSSNIGAGYDVH (SEQ ID NO: 30) VL CDR2
GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV (SEQ ID NO: 32) VL
QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIY
GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE ADYYCQSYDSSLSGWVF GGGTKLTVL
(SEQ ID NO: 33) LX5140092 VH CDR1 ELSMH (SEQ ID NOT) VH CDR2
GFDPEDWKYHLSQKFQG (SEQ ID NOT2) VH CDR3 PNGTHQGGVRGWDYYYG MDV (SEQ
ID NO: 17) VH QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF
DPEDWKYHLSQKFQGRV TMTEDTSTDTAYMELSS LRSEDTAVYYCATPNGT
HQGGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 26) VL CDR1 TGSSSNIGAGYDVH
(SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV
(SEQ ID NO: 32) VL QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD
VHWYQQLPGTAPKLLIY GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE
ADYYCQSYDSSLSGWVF GGGTKLTVL (SEQ ID NO: 33) LX5140092_D VH CDR1
ELSMH (SEQ ID NO: 1) VH CDR2 GFDPEDWKYHLSQKFQG (SEQ ID NO: 12) VH
CDR3 PDGTHQGGVRGWDYYYG MDV (SEQ ID NO: 16) VH QVQLVQSGAEVKKPGAS
VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF DPEDWKYHLSQKFQGRV
TMTEDTSTDTAYMELSS LRSEDTAVYYCATPDGT HQGGVRGWDYYYGMDVW GQGTTVTVSS
(SEQ ID NO: 25) VL CDR1 TGSSSNIGAGYDVH (SEQ ID NO: 30) VL CDR2
GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSLSGWV (SEQ ID NO: 32) VL
QSVVTQPPSVSGAPGQR VTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIY
GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE ADYYCQSYDSSLSGWVF GGGTKLTVL
(SEQ ID NO: 33 LX5140093 VH CDR1 ELSMH (SEQ ID NO: 1) VH CDR2
GFDPEDWAYHQAQKFQG (SEQ ID NO: 13) VH CDR3 PNGQQGKGVRGWDYYYG MDV
(SEQ ID NO: 14) VH QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM
HWVRQAPGKGLEWMGGF DPEDWAYHQAQKFQGRV TMTEDTSTDTAYMELSS
LRSEDTAVYYCATPNGQ QGKGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 27) VL
CDR1 TGSSSNIGAGYDVH (SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31)
VL CDR3 QSYDSSHRAWA (SEQ ID NO: 35) VL QSVVTQPPSVSGAPGQR
VTISCTGSSSNIGAGYD VHWYQQLPGTAPKLLIY GNSNRPSGVPDRFSGSK
SGTSASLAITGLQAEDE ADYYCQSYDSSHRAWAF GGGTKLTVL (SEQ ID NO: 37
LX5140093_D VH CDR1 ELSMH (SEQ ID NO: 1) VH CDR2 GFDPEDWAYHQAQKFQG
(SEQ ID NO: 13) VH CDR3 PDGQQGKGVRGWDYYYG MDV (SEQ ID NO: 18) VH
QVQLVQSGAEVKKPGAS VKVSCKVSGYTLTELSM HWVRQAPGKGLEWMGGF
DPEDWAYHQAQKFQGRV TMTEDTSTDTAYMELSS LRSEDTAVYYCATPDGQ
QGKGVRGWDYYYGMDVW GQGTTVTVSS (SEQ ID NO: 28) VL CDR1 TGSSSNIGAGYDVH
(SEQ ID NO: 30) VL CDR2 GNSNRPS (SEQ ID NO: 31) VL CDR3 QSYDSSHRAWA
(SEQ ID NO: 35) VL QSVVTQPPSVSGAPGQR VTISCTGSSSN1GAGYD
VHWYQQLPGTAPKLLIY GNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDE
ADYYCQSYDSSHRAWAF GGGTKLTVL (SEQ ID NO: 37 Lox696 VH CDR1 DYAMH
(SEQ ID NO: 38) VH CDR2 GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3
EGNWNYDAFDI (SEQ ID NO: 44) VH QVQLVQSGGGLVQPGRS LRLSCAASGFTFDDYAM
HWVRQAPGKGLEWVSGI SWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNS
LRAEDTAVYYCAREGNW NYDAFDIWGRGTTVTVS S (SEQ ID NO: 54) VL CDR1
TGTSSDVGGYNYVS (SEQ ID NO: 55) VL CDR2 DVSNRPS (SEQ ID NO: 60) VL
CDR3 SSYTSSSTNWV (SEQ ID NO: 61)
VL QSALTQPASVSGSPGQS ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSNRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCSSYTSSSTNWVF GGGTKLTVL
(SEQ ID NO: 70) LX6960067_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GVSLQELYTGYADSVKG (SEQ ID NO: 42) VH CDR3 EGSWNYDAFDI (SEQ ID
NO: 45) VH QVQLVQSGGGLVQPGRS LRLSCAASGFTFDDYAM HWVRQAPGKGLEWVSGV
SLQELYTGYADSVKGRF TVSGDNAKNSLYLQMNS LRAEDTAVYYCAREGSW
NYDAFDIWGRGTTVTVS S (SEQ ID NO: 48) VL CDR1 TGTSSDVGGYNYVS (SEQ ID
NO: 55) VL CDR2 DVSNRPS (SEQ ID NO: 60) VL CDR3 LGRTWSSTNWV (SEQ ID
NO: 62) VL QSALTQPASVSGSPGQS ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSNRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCLGRTWSSTNWVF GGGTKLTVL
(SEQ ID NO: 65) LX6960071_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGNWNYDAFDI (SEQ ID
NO: 44) VH QVQLVQSGGGLVQPGRSL RLSCAASGFTFDDYAMHW VRQAPGKGLEWVSGISWN
SGSIGYADSVKGRFTISR DNAKNSLYLQMDSLRAED TAVYYCAREGNWNYDAFD
IWGRGTTVTVSS (SEQ ID NO: 49) VL CDR1 TGTSSDVGGYNYVS (SEQ ID NO: 55)
VL CDR2 DVSNRPS (SEQ ID NO: 60) VL CDR3 MGGMGRSTNWV (SEQ ID NO: 57)
VL QSALTQPASVSGSPGQPI TISCTGTSSDVGGYNYVS WYQQHPGKAPKLMIYDVS
NRPSGVSNRFSGSKSGNT ASLT1SGLQAEDEADYYC MGSMGRSTNWVFGGGTKL TVL (SEQ
ID NO: 66) LX6960073_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH CDR2
GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGSWNYDALDI (SEQ ID NO:
46) VH QVQLVQSGGGLVQPGRSL RLSCAASGFTSDDYAMHW VRQAPGKGLEWVSGISWN
SGSIGYADSVKGRFTISR DNAKNSLYLQMNGLRAED TAVYYCAREGSWNYDALD
IWGRGTTVTVSS (SEQ ID NO: 50) VL CDR1 TGTSSDVGGYNYVS (SEQ ID NO: 55)
VL CDR2 DVSKRPS (SEQ ID NO: 56) VL CDR3 MGGMGRSTNWV (SEQ ID NO: 57)
VL QSALTQPASVSGSPGQP ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSKRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCMGGMGRSTNWVF GGGTKLTVL
(SEQ ID NO: 67) LX6960086_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GISWNSPDRYMDDSVKG (SEQ ID NO: 43) VH CDR3 EGNWNYDAFDI (SEQ ID
NO: 44) VH QVQLVQSGGGLVQPGRS LRLSCAASGFTFDDYAM HWVRQAPGKGLEWVSGI
SWNSPDRYMDDSVKGRF TISRDNAQNSLYLQMDS LRAEDTAVYYCAREGNW
NYDAFDIWGRGTTVTVS S (SEQ ID NO: 51) VL CDR1 TGTSSDVGGYNYVS (SEQ ID
NO: 55) VL CDR2 DVSNRPS (SEQ ID NO: 60) VL CDR3 LGRTWSSTNWV (SEQ ID
NO: 62) VL QSALTQPASVSGSPGQS ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSNRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCLGRTWSSTNWVF GGGTKLTVL
(SEQ ID NO: 65) LX6960094_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGNWNYDAFDI (SEQ ID
NO: 44) VL QVQLVQSGGGLVQPGRS LRLSCAASGFTFDDYAM HWVRQAPGKGLEWVSGI
SWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNS LRAEDTAVYYCAREGNW
NYDAFDIWGRGTTVTVS S (SEQ ID NO: 54) VL CDR1 TGTSSDVGGYNYVS (SEQ ID
NO: 55) VL CDR2 DVSNRPS (SEQ ID NO: 60) VL CDR3 AQRTVSSTNWV (SEQ ID
NO: 64) VL QSALTQPASVSGSPGQS ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSNRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCAQRTVSSTNWVF GGGTKLTVL
(SEQ ID NO: 68) LX6960101_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGNWNYDAFDV (SEQ ID
NO: 47) VH QVQLVQSGGGLVQPGRS LRLSCAASGFTFDDYAM HWVRQAPGKGLEWVSGI
SWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNG LRAEDTAVYYCAREGNW
NYDAFDVWGRGTTVTVS S (SEQ ID NO: 52) VL CDR1 TGTSSDVGGYNYVS (SEQ ID
NO: 55) VL CDR2 DVSNRPS (SEQ ID NO: 60) VL CDR3 MGGMGRSTNWV (SEQ ID
NO: 57) VL QSALTQPASVSGSPGQP ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSKRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCMGGMGRSTNWVF GGGTKLTVL
(SEQ ID NO: 67) LX6960102_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGNWNYDAFDI (SEQ ID
NO: 44) VH QVQLVQSGGGLVQPGRS LRLSCAASGFTFDDYAM HWVRQAPGKGLEWVSGI
SWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNS
LRAEDTAVYYCAREGNW NYDAFDIWGRGTTVTVS S (SEQ ID NO: 54) VL CDR1
TGTSSDVGGYNYVS (SEQ ID NO: 55) VL CDR2 DVSNRPS (SEQ ID NO: 60) VL
CDR3 MGGMGRSTNWV (SEQ ID NO: 57) VL QSALTQPASVSGSPGQS
ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY DVSNRPSGVSNRFSGSK
SGNTASLTISGLQAEDE ADYYCSSYTSSSTNWVF GGGTKLTVL (SEQ ID NO: 70)
LX6960116_ngl1 VH CDR1 DYAMH (SEQ ID NO: 38) VH CDR2
GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGNWNYDAFDI (SEQ ID NO:
44) VH QVQLVQSGGGLVQPGR SLRLSCAASGFTFDDY AMHWVRQAPGKGLEWV
SGISWNSGSIGYADSV KGRFTISRDNAKNSLY LQMNSLRAEDTAVYYC AREGNWNYDAFDIWGR
GTTVTVSS (SEQ ID NO: 54) VL CDR1 TGTSNDVGGYNYVS (SEQ ID NO: 59) VL
CDR2 DVSNRPS (SEQ ID NO: 60) VL CDR3 SSYTSSSTNWV (SEQ ID NO: 61) VL
QSALTQPASVSGSPGQ SITISCTGTSNDVGGY NYVSWYQQHPGKAPKL MIYDVSNRPSGVSNRF
SGSKSGNTASLTISGL QAEDEADYYCMGSMGR STNWVFGGGTKLTVL (SEQ ID NO: 69)
LX6960073_gl VH CDR1 DYAMH (SEQ ID NO: 38) VH CDR2
GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGSWNYDALDI (SEQ ID NO:
40) VH EVQLVESGGGLVQPGRS LRLSCAASGFTSDDYAM HWVRQAPGKGLEWVSGI
SWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNG LRAEDTAVYYCAREGSW
NYDALDIWGQGTMVTVS S (SEQ ID NO: 53) VL CDR1 TGTSSDVGGYNYVS (SEQ ID
NO: 55) VL CDR2 DVSKRPS (SEQ ID NO: 56) VL CDR3 MGGMGRSTNWV (SEQ ID
NO: 57) VL QSALTQPASVSGSPGQ PITISCTGTSSDVGGY NYVSWYQQHPGKAPKL
MIYDVSKRPSGVSNRF SGSKSGNTASLTISGL QAEDEADYYCMGGMGR STNWVFGGGTKLTVL
(SEQ ID NO: 58) LX6960073_G82bS_gl VH CDR1 DYAMH (SEQ ID NO: 38) VH
CDR2 GISWNSGSIGYADSVKG (SEQ ID NO: 39) VH CDR3 EGSWNYDALDI (SEQ ID
NO: 40) VH EVQLVESGGGLVQPGRS LRLSCAASGFTSDDYAM HWVRQAPGKGLEWVSGI
SWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNS LRAEDTAVYYCAREGSW
NYDALDIWGQGTMVTVS S (SEQ ID NO: 41) VL CDR1 TGTSSDVGGYNYVS (SEQ ID
NO: 55) VL CDR2 DVSKRPS (SEQ ID NO: 56) VL CDR3 MGGMGRSTNWV (SEQ ID
NO: 57) VL QSALTQPASVSGSPGQP ITISCTGTSSDVGGYNY VSWYQQHPGKAPKLMIY
DVSKRPSGVSNRFSGSK SGNTASLTISGLQAEDE ADYYCMGGMGRSTNWVF GGGTKLTVL
(SEQ ID NO: 58)
[0136] In some aspects the isolated LOX1-binding protein comprises
a VH sequence that has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions and/or
insertions from a reference VH sequence of SEQ ID NO:4. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0137] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VH of SEQ ID NO:4. The disclosure
also provides nucleic acids encoding the LOX1-binding protein,
vectors containing these nucleic acids and host cells transformed
with these nucleic acids and vectors, and methods of making and
using the LOX1-binding protein.
[0138] In some aspects the isolated LOX1-binding protein comprises
a VL sequence that has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions and/or
insertions from a reference VL sequence of SEQ ID NO:33. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0139] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VL of SEQ ID NO:33. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0140] In some aspects, the LOX1-binding protein comprises a VH
selected from a VH containing a VH-CDR1 having the amino acid
sequence of SEQ ID NO:1, a VH-CDR2 having the amino acid sequence
of SEQ ID NO:2, 5-12 or 13, and a VH-CDR3 having the amino acid
sequence of SEQ ID NO:3, 14-17 or 18; and a light chain VL selected
from a VL containing a VL-CDR1 having the amino acid sequence of
SEQ ID NO:30, a VL-CDR2 having the amino acid sequence of SEQ ID
NO:31, and a VL-CDR3 having the amino acid sequence of SEQ ID
NO:32, 34, or 35. The disclosure also provides nucleic acids
encoding the LOX1-binding protein, vectors containing these nucleic
acids and host cells transformed with these nucleic acids and
vectors, and methods of making and using the LOX1-binding
protein.
[0141] The disclosure also provides an isolated LOX1-binding
protein comprising a VH and a VL sequence that each have a total of
one, two, three, four, five, six, seven, eight or fewer amino acid
substitutions, deletions, and/or insertions from a reference VH and
VL LOX1-binding proteins disclosed herein. The disclosure also
provides nucleic acids encoding the LOX1-binding protein, vectors
containing these nucleic acids and host cells transformed with
these nucleic acids and vectors, and methods of making and using
the LOX1-binding protein.
[0142] In some aspects, the isolated LOX1-binding protein has a VH
comprising SEQ ID NO:4, 19-28, or 29, and a VL comprising SEQ ID
NO:33, 36, or 37. The disclosure also provides nucleic acids
encoding the LOX1-binding protein, vectors containing these nucleic
acids and host cells transformed with these nucleic acids and
vectors, and methods of making and using the LOX1-binding
protein.
[0143] In some aspects, the isolated LOX1-binding protein comprises
a set of complementary determining regions (CDRs): heavy chain
variable region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain
variable region (VL)-CDR1, VL-CDR2 and VL-CDR3 wherein the set of
CDRs is identical to, or has a total of 18 or fewer (e.g. 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) amino acid
substitutions, deletions, and/or insertions from a reference set of
CDRs in which: (a) VH-CDR1 has the amino acid sequence of SEQ ID
NO:1; (b) VH-CDR2 has the amino acid sequence of SEQ ID NO:5; (c)
VH-CDR3 has the amino acid sequence of SEQ ID NO:14; (d) VL-CDR1
has the amino acid sequence of SEQ ID NO:30; (e) VL-CDR2 has the
amino acid sequence of SEQ ID NO:31; and (0 VL-CDR3 has the amino
acid sequence of SEQ ID NO:32 The disclosure also provides nucleic
acids encoding the LOX1-binding protein, vectors containing these
nucleic acids and host cells transformed with these nucleic acids
and vectors, and methods of making and using the LOX1-binding
protein.
[0144] In further aspects, the LOX1-binding protein comprises a set
of CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3
wherein: (a) VH-CDR1 has the amino acid sequence of SEQ ID NO:1;
(b) VH-CDR2 has the amino acid sequence of SEQ ID NO:2; (c) VH-CDR3
has the amino acid sequence of SEQ ID NO:3; (d) VL-CDR1 has the
amino acid sequence of SEQ ID NO:30; (e) VL-CDR2 has the amino acid
sequence of SEQ ID NO:31; and (0 VL-CDR3 has the amino acid
sequence of SEQ ID NO:32. The disclosure also provides nucleic
acids encoding the LOX1-binding protein, vectors containing these
nucleic acids and host cells transformed with these nucleic acids
and vectors, and methods of making and using the LOX1-binding
protein.
[0145] The disclosure also provides an isolated LOX1-binding
protein comprising a heavy chain variable region (VH) having at
least 90, 95, 97, 98 or 99% sequence identity to a reference VH
sequence disclosed herein and/or a light chain variable region (VL)
having at least 90, 95, 97, 98 or 99% sequence identity to a
reference VL sequence disclosed herein (e.g., the VH and VL
sequences disclosed in Table 1, FIG. 4 or FIG. 5). In further
aspects, the disclosure provides an isolated LOX1-binding protein
comprising a VH having at least 90, 95, 97, 98 or 99% sequence
identity to SEQ ID NO:4 and/or a VL having at least 90, 95, 97, 98
or 99% sequence identity to SEQ ID NO:33. The disclosure also
provides nucleic acids encoding the LOX1-binding protein, vectors
containing these nucleic acids and host cells transformed with
these nucleic acids and vectors, and methods of making and using
the LOX1-binding protein.
[0146] In some aspects, the LOX1-binding protein comprises a heavy
chain variable region (VH) having at least 90, 95, 97, 98 or 99%
sequence identity and a light chain variable region (VL) having at
least 90, 95, 97, 98 or 99% sequence identity to a VH and a VL
selected from the group consisting of: (a) a VH having the amino
acid sequence of SEQ ID NO:4 and a VL having the amino acid
sequence of SEQ ID NO:33; (b) a VH having the amino acid sequence
of SEQ ID NO:29 and a VL having the amino acid sequence of SEQ ID
NO:33; (c) a VH having the amino acid sequence of SEQ ID NO:41 and
a VL having the amino acid sequence of SEQ ID NO:58; and (d) a VH
having the amino acid sequence of SEQ ID NO:54 and a VL having the
amino acid sequence of SEQ ID NO:70. The disclosure also provides
nucleic acids encoding the LOX1-binding protein, vectors containing
these nucleic acids and host cells transformed with these nucleic
acids and vectors, and methods of making and using the LOX1-binding
protein.
[0147] In some aspects, the LOX1-binding protein comprises a VH
having at least 90, 95, 97, 98 or 99% sequence identity and a VL
having at least 90, 95, 97, 98 or 99% sequence identity to a VH and
a VL selected from the group consisting of: (a) a VH having the
amino acid sequence of SEQ ID NO:19 and a VL having the amino acid
sequence of SEQ ID NO:33; (b) a VH having the amino acid sequence
of SEQ ID NO:20 and a VL having the amino acid sequence of SEQ ID
NO:33; (c) a VH having the amino acid sequence of SEQ ID NO:21 and
a VL having the amino acid sequence of SEQ ID NO:33; (d) a VH
having the amino acid sequence of SEQ ID NO:22 and a VL having the
amino acid sequence of SEQ ID NO:33; (e) a VH having the amino acid
sequence of SEQ ID NO:23 and a VL having the amino acid sequence of
SEQ ID NO:33; (f) a VH having the amino acid sequence of SEQ ID
NO:24 and a VL having the amino acid sequence of SEQ ID NO:33; (g)
a VH having the amino acid sequence of SEQ ID NO:25 and a VL having
the amino acid sequence of SEQ ID NO:33; (h) a VH having the amino
acid sequence of SEQ ID NO:26 and a VL having the amino acid
sequence of SEQ ID NO:33; (i) a VH having the amino acid sequence
of SEQ ID NO:27 and a VL having the amino acid sequence of SEQ ID
NO:37; (j) a VH having the amino acid sequence of SEQ ID NO:28 and
a VL having the amino acid sequence of SEQ ID NO:37; (k) a VH
having the amino acid sequence of SEQ ID NO:48 and a VL having the
amino acid sequence of SEQ ID NO:65; (1) a VH having the amino acid
sequence of SEQ ID NO:49 and a VL having the amino acid sequence of
SEQ ID NO:66; (m) a VH having the amino acid sequence of SEQ ID
NO:50 and a VL having the amino acid sequence of SEQ ID NO:67; (n)
a VH having the amino acid sequence of SEQ ID NO:51 and a VL having
the amino acid sequence of SEQ ID NO:65; (o) a VH having the amino
acid sequence of SEQ ID NO:54 and a VL having the amino acid
sequence of SEQ ID NO:68; (p) a VH having the amino acid sequence
of SEQ ID NO:52 and a VL having the amino acid sequence of SEQ ID
NO:67; (q) a VH having the amino acid sequence of SEQ ID NO:54 and
a VL having the amino acid sequence of SEQ ID NO:69; (r) a VH
having the amino acid sequence of SEQ ID NO:53 and a VL having the
amino acid sequence of SEQ ID NO:58; and (s) a VH having the amino
acid sequence of SEQ ID NO:41 and a VL having the amino acid
sequence of SEQ ID NO:58. The disclosure also provides nucleic
acids encoding the LOX1-binding protein, vectors containing these
nucleic acids and host cells transformed with these nucleic acids
and vectors, and methods of making and using the LOX1-binding
protein.
[0148] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VH-CDR3 having the amino acid
sequence of SEQ ID NO:3. In further aspects, the LOX1-binding
protein comprises a VH-CDR3 having the amino acid sequence of SEQ
ID NO:3 and a VH-CDR2 having the amino acid sequence of SEQ ID
NO:2. In other aspects, the LOX1-binding protein comprises a
VH-CDR3 having the amino acid sequence of SEQ ID NO:3 and a VH-CDR2
having the amino acid sequence of SEQ ID NO:5-12 or 13. In
additional aspects, the LOX1-binding protein comprises a VH-CDR3
having the amino acid sequence of SEQ ID NO:3, 14-17 or 18 and a
VH-CDR2 having the amino acid sequence of SEQ ID NO:2. In further
aspects, the LOX1-binding protein comprises a VH-CDR3 having the
amino acid sequence of SEQ ID NO: 3, 14-17 or 18 and a VH-CDR2
having the amino acid sequence of SEQ ID NO:2, 5-12 or 13. In
further aspects, the LOX1-binding protein comprises a VH-CDR3
having the amino acid sequence of SEQ ID NO: 3, 14-17 or 18 and a
VH-CDR2 having the amino acid sequence of SEQ ID NO:2, 5-12 or 13
and a VH-CDR1 having the amino acid sequence of SEQ ID NO:1. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0149] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VLH-CDR3 having the amino acid
sequence of SEQ ID NO:32. In further aspects, the LOX1-binding
protein comprises a VL-CDR3 having the amino acid sequence of SEQ
ID NO:32 and a VL-CDR2 having the amino acid sequence of SEQ ID
NO:31. In other aspects, the LOX1-binding protein comprises a
VL-CDR3 having the amino acid sequence of SEQ ID NO:32, a VL-CDR2
having the amino acid sequence SEQ ID NO:31 and a VL-CDR1 having
the amino acid sequence of SEQ ID NO:30. In other aspects, the
LOX1-binding protein comprises a VL-CDR3 having the amino acid
sequence of SEQ ID NO:32, 34, or 35, a VL-CDR2 having the amino
acid sequence SEQ ID NO:31. In further aspects, the LOX1-binding
protein comprises a VL-CDR3 having the amino acid sequence of SEQ
ID NO:32, 34, or 35, a VL-CDR2 having the amino acid sequence SEQ
ID NO:31 and a VL-CDR1 having the amino acid sequence of SEQ ID
NO:30. The disclosure also provides nucleic acids encoding the
LOX1-binding protein, vectors containing these nucleic acids and
host cells transformed with these nucleic acids and vectors, and
methods of making and using the LOX1-binding protein.
[0150] In additional aspects, the LOX1-binding protein comprises
one, two, or three VH-CDRs such as a VH-CDR1 identical to, or that
has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO:1, a VH-CDR2 identical to, or
that has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO:2, 5-12 or 13, or a VH-CDR3
identical to, or that has a total of one, two, three, four, or
fewer amino acid substitutions, deletions, or insertions to SEQ ID
NO:3, 14-17 or 18.
[0151] In additional aspects, the LOX1-binding protein comprises
one, two, or three VL-CDRs such as a VL-CDR1 identical to, or that
has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO:30, a VL-CDR2 identical to,
or that has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO:31, or a VH-CDR3 identical
to, or that has a total of one, two, three, four, or fewer amino
acid substitutions, deletions, or insertions to SEQ ID NO:32, 34,
or 35.
[0152] In some aspects the isolated LOX1-binding protein comprises
a VH sequence that has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions and/or
insertions from a reference VH sequence of SEQ ID NO:41. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0153] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VH of SEQ ID NO:41. The
disclosure also provides nucleic acids encoding the LOX1-binding
proteins, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making an using the LOX1-binding proteins.
[0154] In some aspects the isolated LOX1-binding protein comprises
a VL sequence that has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions and/or
insertions from a reference VL sequence of SEQ ID NO:58. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0155] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VL of SEQ ID NO:58. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0156] In some aspects, the LOX1-binding protein comprises a VH
selected from a VH containing a VH-CDR1 having the amino acid
sequence of SEQ ID NO:38, a VH-CDR2 having the amino acid sequence
of SEQ ID NO:39, 42 or 43, and a VH-CDR3 having the amino acid
sequence of SEQ ID NO:40, 44-46 or 47; and a light chain VL
selected from a VL containing a VL-CDR1 having the amino acid
sequence of SEQ ID NO:55 or 59, a VL-CDR2 having the amino acid
sequence of SEQ ID NO:56 or 60, and a VL-CDR3 having the amino acid
sequence of SEQ ID NO:57, 61-63, or 64.
[0157] In some aspects, the isolated LOX1-binding protein has a VH
comprising a sequence of SEQ ID NO:41, 48-53, or 54, and a VL
comprising a sequence of SEQ ID NO:58, 65-69, or 70. The disclosure
also provides nucleic acids encoding the LOX1-binding protein,
vectors containing these nucleic acids and host cells transformed
with these nucleic acids and vectors, and methods of making and
using the LOX1-binding protein.
[0158] In some aspects, the isolated LOX1-binding protein comprises
a set of complementary determining regions (CDRs): heavy chain
variable region (VH)-CDR1, VH-CDR2, VH-CDR3, light chain variable
region (VL)-CDR1, VL-CDR2 and VL-CDR3 wherein the set of CDRs is
identical to, or has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions, and/or
insertions from a reference set of CDRs in which: (a) VH-CDR1 has
the amino acid sequence of SEQ ID NO:38; (b) VH-CDR2 has the amino
acid sequence of SEQ ID NO:39; (c) VH-CDR3 has the amino acid
sequence of SEQ ID NO:44; (d) VL-CDR1 has the amino acid sequence
of SEQ ID NO:55; (e) VL-CDR2 has the amino acid sequence of SEQ ID
NO:60; and (0 VL-CDR3 has the amino acid sequence of SEQ ID NO:61.
The disclosure also provides nucleic acids encoding the
LOX1-binding protein, vectors containing these nucleic acids and
host cells transformed with these nucleic acids and vectors, and
methods of making and using the LOX1-binding protein.
[0159] In some aspects, the isolated LOX1-binding protein comprises
a set of complementary determining regions (CDRs): heavy chain
variable region (VH)-CDR1, VH-CDR2, VH-CDR3, light chain variable
region (VL)-CDR1, VL-CDR2 and VL-CDR3 wherein the set of CDRs is
identical to, or has a total of one, two, three, four, five, six,
seven, eight or fewer amino acid substitutions, deletions, and/or
insertions from a reference set of CDRs in which: (a) VH-CDR1 has
the amino acid sequence of SEQ ID NO:38, (b) VH-CDR2 has the amino
acid sequence of SEQ ID NO:39; (c) VH-CDR3 has the amino acid
sequence of SEQ ID NO:40; (d) VL-CDR1 has the amino acid sequence
of SEQ ID NO:55; (e) VL-CDR2 has the amino acid sequence of SEQ ID
NO:56; and (0 VL-CDR3 has the amino acid sequence of SEQ ID NO:57.
The disclosure also provides nucleic acids encoding the
LOX1-binding protein, vectors containing these nucleic acids and
host cells transformed with these nucleic acids and vectors, and
methods of making and using the LOX1-binding protein.
[0160] In further aspects, the LOX1-binding protein comprises a set
of CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3
wherein: (a) VH-CDR1 has the amino acid sequence of SEQ ID NO:38;
(b) VH-CDR2 has the amino acid sequence of SEQ ID NO:39; (c)
VH-CDR3 has the amino acid sequence of SEQ ID NO:40; (d) VL-CDR1
has the amino acid sequence of SEQ ID NO:55; (e) VL-CDR2 has the
amino acid sequence of SEQ ID NO:56; and (0 VL-CDR3 has the amino
acid sequence of SEQ ID NO:57. The disclosure also provides nucleic
acids encoding the LOX1-binding protein, vectors containing these
nucleic acids and host cells transformed with these nucleic acids
and vectors, and methods of making and using the LOX1-binding
protein.
[0161] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VH-CDR3 having the amino acid
sequence of SEQ ID NO:40. In further aspects, the LOX1-binding
protein comprises a VH-CDR3 having the amino acid sequence of SEQ
ID NO:40 and a VH-CDR2 having the amino acid sequence of SEQ ID
NO:39. In other aspects, the LOX1-binding protein comprises a
VH-CDR3 having the amino acid sequence of SEQ ID NO:40 and a
VH-CDR2 having the amino acid sequence of SEQ ID NO:42 or 43. In
additional aspects, the LOX1-binding protein comprises a VH-CDR3
having the amino acid sequence of SEQ ID NO:40, 44-46, or 47 and a
VH-CDR2 having the amino acid sequence of SEQ ID NO:39. In further
aspects, the LOX1-binding protein comprises a VH-CDR3 having the
amino acid sequence of SEQ ID NO: 40, 44-46, or 47 and a VH-CDR2
having the amino acid sequence of SEQ ID NO:39, 42, or 43. In
further aspects, the LOX1-binding protein comprises a VH-CDR3
having the amino acid sequence of SEQ ID NO: 40, 44-46, or 47 and a
VH-CDR2 having the amino acid sequence of SEQ ID NO: 39, 42, or 43,
and a VH-CDR1 having the amino acid sequence of SEQ ID NO:38. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0162] In further aspects, the disclosure provides an isolated
LOX1-binding protein comprising a VLH-CDR3 having the amino acid
sequence of SEQ ID NO:57. In further aspects, the LOX1-binding
protein comprises a VL-CDR3 having the amino acid sequence of SEQ
ID NO:57 and a VL-CDR2 having the amino acid sequence of SEQ ID
NO:56. In other aspects, the LOX1-binding protein comprises a
VL-CDR3 having the amino acid sequence of SEQ ID NO:57, a VL-CDR2
having the amino acid sequence SEQ ID NO:56 and a VL-CDR3 having
the amino acid sequence of SEQ ID NO:55. In other aspects, the
LOX1-binding protein comprises a VL-CDR3 having the amino acid
sequence of SEQ ID NO:57, a VL-CDR2 having the amino acid sequence
SEQ ID NO:56 or 60, and a VL-CDR1 having the amino acid sequence of
SEQ ID NO:55 or 59. The disclosure also provides nucleic acids
encoding the LOX1-binding protein, vectors containing these nucleic
acids and host cells transformed with these nucleic acids and
vectors, and methods of making and using the LOX1-binding
protein.
[0163] In additional aspects, the LOX1-binding protein comprises
one, two, or three VH-CDRs such as a VH-CDR1 identical to, or that
has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO:38, a VH-CDR2 identical to,
or that has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO:39,42 or 43, or a VH-CDR3
identical to, or that has a total of one, two, three, four, or
fewer amino acid substitutions, deletions, or insertions to SEQ ID
NO:40, 44-46 or 47. The disclosure also provides nucleic acids
encoding the LOX1-binding protein, vectors containing these nucleic
acids and host cells transformed with these nucleic acids and
vectors, and methods of making and using the LOX1-binding
protein.
[0164] In additional aspects, the LOX1-binding protein comprises
one, two, or three VL-CDRs such as a VL-CDR1 identical to, or that
has a total of one, two or fewer amino acid substitutions,
deletions, or insertions to SEQ ID NO: 55 or 59, a VL-CDR2
identical to, or that has a total of one, two or fewer amino acid
substitutions, deletions, or insertions to SEQ ID NO:56 or 60, or a
VH-CDR3 identical to, or that has a total of one, two, three, four,
or fewer amino acid substitutions, deletions, or insertions to SEQ
ID NO: 57, 61-63 or 64. The disclosure also provides nucleic acids
encoding the LOX1-binding protein, vectors containing these nucleic
acids and host cells transformed with these nucleic acids and
vectors, and methods of making and using the LOX1-binding
protein.
[0165] In some aspects, the LOX1-binding protein comprises a set of
CDRs: VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR3 and VL-CDR3 as
described in Table 1, FIG. 4 or FIG. 5. The disclosure also
provides nucleic acids encoding the LOX1-binding protein, vectors
containing these nucleic acids and host cells transformed with
these nucleic acids and vectors, and methods of making and using
the LOX1-binding protein.
[0166] In some additional aspects, the LOX1-binding protein
comprises a heavy chain variable region (VH) and light chain
variable region (VL) as described in Table 1, FIG. 4 or FIG. 5. The
disclosure also provides nucleic acids encoding the LOX1-binding
protein, vectors containing these nucleic acids and host cells
transformed with these nucleic acids and vectors, and methods of
making and using the LOX1-binding protein.
[0167] In some aspects, the isolated LOX1-binding protein comprises
a VH-CDR2 having the amino acid sequence of GF DP ED HX.sub.1
HX.sub.2 HX.sub.3 HX.sub.4 HX.sub.5 HX.sub.6 Q K F Q G (Gly Phe Asp
Pro Glu Asp HX.sub.1 HX.sub.2 HX.sub.3 HX.sub.4 HX.sub.5 HX.sub.6
Gln Lys Phe Gln Gly), wherein HX.sub.1 is Gly, Trp, Tyr, or Phe;
HX.sub.2 is Glu, Thr, Gln, Ser, Lys, or Ala; HX.sub.3 is Thr, Tyr,
Ile, or Asn; HX.sub.4 is Ile, Ala, Arg, or His; HX.sub.5 is Tyr,
Val, Thr, Leu, or Gln, and HX.sub.6 is Ala, Asp, Gly, Ser, or His
(SEQ ID NO:71).
[0168] In additional aspects, the isolated LOX1-binding protein
comprises a VH-CDR3 having the amino acid sequence of HX.sub.7
HX.sub.8 G HX.sub.9 HX.sub.10 HX.sub.11 HX.sub.12 G V R G W D Y Y Y
G M D V (HX.sub.7 HX.sub.8 Gly HX.sub.9 HX.sub.10 HX.sub.11
HX.sub.12 Gly Val Arg Gly Trp Asp Tyr Tyr Tyr Gly Met Asp Val Trp)
wherein HX.sub.7 is Pro, Ser, or Val; HX.sub.8 is Asn, Thr, Trp, or
Asp; HX.sub.9 is Gln, Arg, or Thr; HX.sub.10 is Gln or His;
HX.sub.11 is Gly or Gln; HX.sub.12 is Lys or Gly (SEQ ID
NO:72).
[0169] In other aspects, the isolated LOX1-binding protein
comprises a VL-CDR3 having the amino acid sequence of Q S Y D S
LX.sub.1 LX.sub.2 LX.sub.3 LX.sub.4 LX.sub.5 LX.sub.6 (Gln Ser Tyr
Asp Ser LX.sub.1 LX.sub.2 LX.sub.3 LX.sub.4 LX.sub.5 LX.sub.6),
wherein LX.sub.1 is Ser or Met; LX.sub.2 is Leu, Tyr, or His;
LX.sub.3 is Ser or Arg; LX.sub.4 is Gly, Ala, or no amino acid;
LX.sub.5 is Trp or Phe; and LX.sub.6 is Val, Gly, or Ala (SEQ ID
NO:73).
[0170] In some aspects, the isolated LOX1-binding protein comprises
a set of complementary determining regions (CDRs): heavy chain
variable region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain
variable region (VL)-CDR1, VL-CDR2 and VL-CDR3 wherein: (a) VH-CDR1
comprises the amino acid sequence: EL S MH (SEQ ID NO: 1); (b)
VH-CDR2 comprises the amino acid sequence: G F D P E D HX.sub.1
HX.sub.2 HX.sub.3 HX.sub.4 HX.sub.5 HX.sub.6 Q K F Q G, wherein
HX.sub.1 is selected from the group consisting of G, W, Y and F,
HX.sub.2 is selected from the group consisting of E, T, Q, K, A,
and S, HX.sub.3 is selected from the group consisting of T, Y, I,
and N, HX.sub.4 is selected from the group consisting of I, A, R
and H, HX.sub.5 is selected from the group consisting of Y, V, T, L
and Q, and HX.sub.6 is selected from the group consisting of A, D,
G, S and H (SEQ ID NO: 71); (c) VH-CDR3 comprises the amino acid
sequence: HX.sub.7 HX.sub.8 G HX.sub.9 HX.sub.10 HX.sub.11
HX.sub.12 G V R G W D Y Y Y G M D V, wherein HX.sub.7 is selected
from the group consisting of P, S and V, HX.sub.8 is selected from
the group consisting of N, W, D, and T, HX.sub.9 is selected from
the group consisting of Q, R and T, HX.sub.10 is selected from the
group consisting of Q and H, HX.sub.11 is selected from the group
consisting of G and Q, and HX.sub.12 is selected from the group
consisting of K and G (SEQ ID NO: 72); (d) VL-CDR1 comprises the
amino acid sequence: T G S S S N I G A G Y D V H (SEQ ID NO: 30);
(e) VL-CDR2 comprises the amino acid sequence: G N S N R P S (SEQ
ID NO: 31); and (0 VL-CDR3 comprises the amino acid sequence: Q S Y
D S LX.sub.1 LX.sub.2 LX.sub.3 LX.sub.4 LX.sub.5 LX.sub.6, wherein
LX.sub.1 is selected from the group consisting of M and S, LX.sub.2
is selected from the group consisting of L, Y and H, LX.sub.3 is
selected from the group consisting of S and R, LX.sub.4 is selected
from the group consisting of A and G or is omitted (no amino acid),
LX.sub.5 is selected from the group consisting of W and F, and
LX.sub.6 is selected from the group consisting of V, G and A (SEQ
ID NO: 73).
[0171] In additional aspects, the isolated LOX1-binding protein
comprises a VH-CDR2 having the amino acid sequence of G HX.sub.1 S
HX.sub.2 HX.sub.3 HX.sub.4 HX.sub.5 HX.sub.6 HX.sub.7 HX.sub.8
HX.sub.9 HX.sub.10 D S V K G (Gly HX.sub.1 Ser HX.sub.2 HX.sub.3
HX.sub.4 HX.sub.5 HX.sub.6 HX.sub.7 HX.sub.8 HX.sub.9 HX.sub.10 Asp
Ser Val Lys Gly), wherein HX.sub.1 is Ile or Val; HX.sub.2 is Trp
or Leu; HX.sub.3 is Asn or Gln; HX.sub.4 is Ser or Glu; HX.sub.5 is
Gly, Leu, or Pro; HX.sub.6 is Ser, Tyr, or Asp; HX.sub.7 is Ile,
Thr, or Arg; HX.sub.8 is Gly or Tyr; HX.sub.9 is Tyr or Met; and
HX.sub.10 is Ala or Asp (SEQ ID NO:74).
[0172] In additional aspects, the isolated LOX1-binding protein
comprises a VH-CDR3 having the amino acid sequence of E G HX.sub.11
W N Y D A HX.sub.12 D HX.sub.13 (Glu Gly HX.sub.11 Trp Asn Tyr Asp
Ala HX.sub.12 Asp HX.sub.13), wherein HX.sub.11 is Asn or Ser;
HX.sub.12 is Phe or Leu; and HX.sub.13 is Ile or Val (SEQ ID
NO:75).
[0173] In additional aspects, the isolated LOX1-binding protein
comprises a VL-CDR1 having the amino acid sequence of TGTS LX.sub.1
DV G G Y N Y V S (Thr Gly Thr Ser LX.sub.1 Asp Val Gly Gly Tyr Asn
Tyr Val Ser), wherein LX.sub.1 is Ser or Asn (SEQ ID NO:76).
[0174] In additional aspects, the isolated LOX1-binding protein
comprises a VL-CDR2 having the amino acid sequence of D V S
LX.sub.2 R P S (Asp Val Ser X4 Arg Pro Ser), wherein LX.sub.2 is
Asn or Lys (SEQ ID NO:77).
[0175] In additional aspects, the isolated LOX1-binding protein
comprises a VL-CDR3 having the amino acid sequence of LX.sub.3
LX.sub.4 LX.sub.5 LX.sub.6 LX.sub.7 LX.sub.8 S T N W V (LX.sub.3
LX.sub.4 LX.sub.5 LX.sub.6 LX.sub.7 LX.sub.8 Ser Thr Asn Trp Val),
wherein LX.sub.3 is Ser, Leu, Met, or Ala; LX.sub.4 is Ser, Gly, or
Gln; LX.sub.5 is Tyr, Arg, Ser, or Gly; LX.sub.6 is Thr or Met;
LX.sub.7 is Ser, Trp, Gly, or Val; and LX.sub.8 is Ser or Arg (SEQ
ID NO:78).
[0176] In some aspects, the isolated LOX1-binding protein comprises
a set of complementary determining regions (CDRs): heavy chain
variable region (VH)-CDR1, VH-CDR2, VH-CDR3, and light chain
variable region (VL)-CDR1, VL-CDR2 and VL-CDR3 wherein: (a) VH-CDR1
comprises the amino acid sequence: D Y A M H (SEQ ID NO: 38); (b)
VH-CDR2 comprises the amino acid sequence: G HX.sub.1 S HX.sub.2
HX.sub.3 HX.sub.4 HX.sub.5 HX.sub.6 HX.sub.7 HX.sub.8 HX.sub.9
HX.sub.10 D S V K G, wherein HX.sub.1 is selected from the group
consisting of I and V, HX.sub.2 is selected from the group
consisting of W and L, HX.sub.3 is selected from the group
consisting of N and Q, HX.sub.4 is selected from the group
consisting of S and E, HX.sub.5 is selected from the group
consisting of G, L and P, HX.sub.6 is selected from the group
consisting of S, Y and D, HX.sub.7 is selected from the group
consisting of I, T and R, HX.sub.8 is selected from the group
consisting of G and Y, HX.sub.9 is selected from the group
consisting of M and Y, and HX.sub.10 is selected from the group
consisting of A and D (SEQ ID NO:74); (c) VH-CDR3 comprises the
amino acid sequence: E G HX.sub.11 W N Y D A HX.sub.12 D HX.sub.13,
wherein HX.sub.11 is selected from the group consisting of N and S,
HX.sub.12 is selected from the group consisting of F and L, and
HX.sub.13 is selected from the group consisting of I and V (SEQ ID
NO:75); (d) VL-CDR1 comprises the amino acid sequence: T G T S
LX.sub.1 D V G G Y N Y V S, wherein LX.sub.1 is selected from the
group consisting of N and S (SEQ ID NO:76); (e) VL-CDR2 comprises
the amino acid sequence: D V S LX.sub.2 R P S, wherein LX.sub.2 is
selected from the group consisting of N and K (SEQ ID NO:77); and
(f) VL-CDR3 comprises the amino acid sequence: LX.sub.3 LX.sub.4
LX.sub.5 LX.sub.6 LX.sub.7 LX.sub.8 S T N W V, wherein LX.sub.3 is
selected from the group consisting of L, M, A and S, LX.sub.4 is
selected from the group consisting of S, Q and G, LX.sub.5 is
selected from the group consisting of Y, S, G and R, LX.sub.6 is
selected from the group consisting of T and M, LX.sub.7 is selected
from the group consisting of S, W, G and V, and LX.sub.8 is
selected from the group consisting of S and R (SEQ ID NO:78).
[0177] In some aspects, the set of CDRs of a LOX1-binding protein
disclosed herein is provided within antibody framework regions or
other protein scaffolds known in the art. Exemplary antibody
framework regions include: germline framework regions, such as
VH1-24 (DP-5), J146, VH3-09 (DP-31), and JH3 for the antibody
framework region of the heavy chain and V.lamda.1e (DPL-8),
V.lamda.2a2 (DPL-11), and JL3 for the antibody framework region of
the light chain and/or any suitable framework regions well known to
one of skilled in the art.
[0178] In some aspects, the LOX1-binding protein contains one or
more framework regions from a heavy chain variable region (VH)
and/or a light chain variable region (VL) that is a germline
framework. Frameworks regions of the heavy chain domain may be
selected from VH1-24 (DP-5), JH6, VH3-09 (DP-31), JH3 frameworks
and/or any suitable framework regions or protein scaffolds well
known in the art. Framework regions of the light chain may be
selected from V.lamda.1e (DPL-8), V.lamda.2a2 (DPL-11), and JL3
frameworks, and/or any suitable framework regions or protein
scaffolds well known in the art. One or more CDRs may be taken from
the LOX1-binding proteins disclosed herein (e.g. a LOX1-binding
protein described in Table 1, FIG. 4 or FIG. 5) and incorporated
into a suitable framework and/or protein scaffold.
[0179] In additional aspects, the disclosure provides an isolated
LOX1-binding protein that binds to the same LOX1 epitope as an
antibody comprising a VH and VL of SEQ ID NO:4 and SEQ ID NO:33,
respectively.
[0180] In other aspects, the disclosure provides LOX1-binding
proteins (e.g., antibodies such as, full length LOX1-antibodies,
LOX1-binding antibody fragments, and variants and derivatives
thereof) that compete or cross-compete for binding to LOX1 with an
antibody comprising a VH sequence of SEQ ID NO:4 and a VL sequence
of SEQ ID NO:33. In a particular aspect, the LOX1-binding protein
is able to competitively inhibit an antibody comprising a VH of SEQ
ID NO:4 and a VL of SEQ ID NO:33, to bind to LOX1.
[0181] Also provided is a LOX1-binding protein such as a LOX1
antibody (e.g., a full length anti-LOX1 antibody, a LOX1-binding
antibody fragment, and variants, and derivatives thereof) which can
bind to LOX1 with a greater affinity than a LOX1-binding protein
(e.g. a LOX1 antibody or fragment thereof) comprising the VH of SEQ
ID NO:29 and the VL of SEQ ID NO:33. The disclosure also provides
nucleic acids encoding the LOX1-binding protein, vectors containing
these nucleic acids and host cells transformed with these nucleic
acids and vectors, and methods of making and using the LOX1-binding
protein.
[0182] In additional aspects, the disclosure provides an isolated
LOX1-binding protein that binds to the same LOX1 epitope as an
antibody comprising a VH and VL of SEQ ID NO:41 and SEQ ID NO:58,
respectively.
[0183] In other aspects, the disclosure provides LOX1-binding
proteins (e.g., antibodies such as, full length LOX1-antibodies and
LOX1-binding antibody fragments, and variants and derivatives
thereof) that compete or cross-compete for binding to LOX1 with an
antibody comprising a VH sequence of SEQ ID NO:41 and a VL sequence
of SEQ ID NO:58. In a particular aspect, the LOX1-binding protein
is able to competitively inhibit an antibody comprising a VH of SEQ
ID NO:41 and a VL of SEQ ID NO:58 to bind to LOX1.
[0184] Also provided is a LOX1-binding protein such as, a LOX1
antibody (e.g., a full length anti-LOX1 antibody and a LOX1-binding
antibody fragment, and variants, and derivatives thereof), which
can bind to LOX1 with a greater affinity than a full-length
antibody comprising the VH of SEQ ID NO:54 and the VL of SEQ ID
NO:70. The disclosure also provides nucleic acids encoding the
LOX1-binding protein, vectors containing these nucleic acids and
host cells transformed with these nucleic acids and vectors, and
methods of making and using the LOX1-binding protein.
[0185] In some aspects, the anti-LOX1 antibody provided herein is a
murine antibody, a human antibody, a humanized antibody, a chimeric
antibody, a monoclonal antibody, a polyclonal antibody, a
recombinant antibody, a bi-specific antibody, a multispecific
antibody, or any combination thereof. In some aspects the anti-LOX1
antibody is an Fv fragment, an Fab fragment, an F(ab')2 fragment,
an Fab' fragment, a dsFv fragment, an scFv fragment, or an sc(Fv)2
fragment.
[0186] The disclosure provides a LOX1-binding protein (e.g., an
isolated anti-LOX1 antibody such as, a full length LOX1-antibody, a
LOX1-binding antibody fragment, and a variant and derivative
thereof) that can bind to LOX1 molecules across species. In some
aspects, the LOX1-binding protein binds to human LOX1 (hLOX1) and
cynomolgus LOX1 (cynoLOX1). In additional aspects, the LOX1-binding
protein binds to human LOX1 (hLOX1) and rabbit LOX1. In further
aspects, the LOX1-binding protein binds to hLOX1, cynoLOX1 and
rabbit LOX1. In certain aspects provided herein, a LOX1-binding
protein (e.g., an anti-LOX1 antibody) specifically binds to LOX1
(e.g., hLOX1, cynoLOX1 and/or rabbit LOX1) and does not bind to one
or more of: CLEC-7A, CLEC-1A, CLEC-4L, CLEC-1B, SR-A1 and/or SR-B3.
See, e.g., FIGS. 3, 6 and 7.
[0187] In some aspects, the LOX1-binding protein (e.g., an isolated
anti-LOX1 antibody or thereof) further comprises a human IgG1 TM
mutant heavy chain, wherein the heavy chain variable region (VH)
comprises an amino acid sequence having at least 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% sequence identity to the
reference amino acid sequence SEQ ID NO:4, 19-28 or 29.
[0188] In some aspects, the LOX1-binding protein (e.g., an isolated
anti-LOX1 antibody or thereof) further comprises a human IgG1 TM
mutant heavy chain, wherein the heavy chain variable region (VH)
comprises an amino acid sequence having at least 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% sequence identity to the
reference amino acid sequence SEQ ID NO:41, 48-53 or 54.
[0189] In some aspects, the LOX1-binding protein (e.g., a full
length LOX1-antibody, a LOX1-binding antibody fragment, and a
variant and derivative thereof) includes in addition to a heavy
chain variable region (VH) and a light chain variable region (VL),
and optionally a heavy chain constant region or fragment thereof, a
light chain constant region or fragment thereof. In certain aspects
the light chain constant region is a kappa lambda light chain
constant region, e.g., a human kappa constant region or a human
lambda constant region. In a specific aspect, the light chain
constant region is a human kappa constant region.
[0190] In additional aspects, the LOX1-binding protein (e.g., a
full length LOX1-antibody, a LOX1-binding antibody fragment, and a
variant and derivative thereof) has a light chain variable region
(VL) that contains a human kappa constant region, e.g., the VL
comprises a VL amino acid sequence having at least 70%, 75%, 80%,
85%, 90%, 95%, or 100% sequence identity to the reference amino
acid sequence SEQ ID NO:33, 36, or 37. In certain aspects, the
disclosure provides a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof) further comprising a human IgG1 TM
mutant heavy chain and a human kappa light chain, wherein the heavy
chain variable region (VH) and the light chain variable region (VL)
comprise: SEQ ID NO:4 and SEQ ID NO:33; SEQ ID NO:29 and SEQ ID
NO:33; SEQ ID NO:41 and SEQ ID NO:58; or SEQ ID NO:54 and SEQ ID
NO:70, respectively.
[0191] In additional aspects, the LOX1-binding protein (e.g., a
full length LOX1-antibody, a LOX1-binding antibody fragment, and a
variant and derivative thereof) has a light chain variable region
(VL) that further comprises a human kappa constant region, e.g.,
the light chain can comprise a light chain amino acid sequence
having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence
identity to the reference amino acid sequence SEQ ID NO:58, 65-69
or 70. In certain aspects, the disclosure provides a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof) further
comprising a human IgG1 TM mutant heavy chain and a human kappa
light chain, wherein the heavy chain variable region (VH) and the
light chain variable region (VL) comprise: SEQ ID NO:41 and SEQ ID
NO:58, SEQ ID NO:48 and SEQ ID NO:65, SEQ ID NO:49 and SEQ ID
NO:66, SEQ ID NO:50 and SEQ ID NO:67, or SEQ ID NO:53 and SEQ ID
NO:58, respectively.
[0192] In some aspects, the disclosure provides an isolated
LOX1-binding protein such as an anti-LOX1 antibody (e.g., a full
length LOX1-antibody, a LOX1-binding antibody fragment, and a
variant and derivative thereof), wherein the LOX1-binding protein
has at least one property selected from the group consisting of:
(a) reduces or inhibits binding of oxLDL, C-reactive protein (CRP)
and/or advanced glycation end products (AGEs) to LOX1 as determined
by any suitable assay including an assay disclosed herein (see,
e.g., Example 10, assays 1, 2 and/or 3 or Example 11); (b)
decreases or inhibits RhoA/Racl, nitrogen monoxide (NO), p38MAPK,
protein kinase B and C, ERK1/2, and/or NF.kappa.B signaling in an
endothelial cell expressing cell surface LOX1 as determined by any
suitable assay including an assay disclosed herein (see, e.g.,
Example 11); (c) decreases or inhibits caspase-8, caspase-9, and/or
BAX activity in an endothelial cell expressing cell surface LOX1 as
determined by any suitable assay including an assay disclosed
herein; (d) binds to LOX1 having the single nucleotide polymorphism
K167N as determined by any suitable assay including an assay
disclosed herein (see, e.g., Example 10, assays 1, 2 and/or 3 or
Example 11); (e) reduces or inhibits oxLDL internalization as
determined by any suitable assay including an assay disclosed
herein (see, e.g., Example 10, assay 4 or Example 11); (f) reduces
or inhibits oxLDL-induced LOX1 signaling as determined by any
suitable assay including an assay disclosed herein (see, e.g.,
Example 10, assay 5 or Example 11); (g) binds to LOX1 with a
dissociation constant (KD) of about 150 pM to about 600 pM (e.g.
about 400 pM) as determined by BIACORE or KinExA; (h) binds to LOX1
with a Kon rate of about 1.times.10.sup.5 M.sup.-1 s.sup.-1 to
about 6.times.10.sup.6 M.sup.-1 s.sup.-1 (e.g. about
5.times.10.sup.5 M.sup.-1 s.sup.-1) as determined by BIACORE; and
(i) binds to LOX1 with a Koff rate of about 1.times.10.sup.-4
s.sup.-1 to about 3.times.10.sup.-4 s.sup.-1 (e.g. about
2.3.times.10.sup.-4 s.sup.-1) as determined by BIACORE.
[0193] In certain aspects, the blocking of LOX1 biological activity
by a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment
thereof) described herein, reduces atherosclerosis and/or decreases
one or more conditions associated with atherosclerosis. In
particular aspects, the LOX1 antagonist (e.g. an anti-LOX1 antibody
or fragment thereof) inhibits or decreases LOX1-mediated
development of atherosclerotic lesions, coronary artery disease,
stroke, ischemia, infarction, peripheral vascular disease,
reperfusion, injury, or other clinical symptoms, associated with
the development of atherosclerosis.
[0194] In certain aspects, a LOX1-binding protein such as, an
anti-LOX1 antibody (e.g., a full length LOX1-antibody, a
LOX1-binding antibody fragment, and a variant and derivative
thereof) can specifically bind to LOX1, e.g., human LOX1 (hLOX1)
and/or cynomolgus LOX1 (cynoLOX1), and antigenic fragments thereof
with a dissociation constant or KD of less than 10.sup.-6 M, or of
less than 10.sup.-7 M, or of less than 10.sup.-8 M, or of less than
10.sup.-9 M, or of less than 10.sup.-10 M, or of less than
10.sup.-11 M, of less than 10.sup.-12 M, of less than 10.sup.-13 M,
of less than 10.sup.-14 M, or of less than 10.sup.-15M as measured,
e.g., by KINEXA.RTM. or BIACORE.RTM.. In one aspect, the anti-LOX1
antibody can bind to hLOX1 and cynoLOX1 with a KD of less than
about 1.times.10.sup.-8 M to about 1.times.10.sup.-1.degree. M as
measured by BIACORE.RTM..
[0195] In another aspect, a LOX1-binding protein such as, an
anti-LOX1 antibody (e.g., a full length LOX1-antibody, a
LOX1-binding antibody fragment, and a variant and derivative
thereof) can bind to LOX1 with a Koff of less than
1.times.10.sup.-3 s.sup.-1, or less than 2.times.10.sup.-3
s.sup.-1. In other aspects, the LOX1-binding protein binds to LOX1
with a Koff of less than 10.sup.-3 s.sup.-1, less than
5.times.10.sup.-3 s.sup.-1, less than 10.sup.-4 s.sup.-1, less than
5.times.10.sup.-4 s.sup.-1, less than 10.sup.-5 s.sup.-1, less than
5.times.10.sup.-5 s.sup.-1, less than 10.sup.-6 s.sup.-1, less than
5.times.10.sup.-5 s.sup.-1, less than less than 5.times.10.sup.-5
s.sup.-1, less than 10.sup.-8 s.sup.-1, less than 5.times.10.sup.-8
s.sup.-1, less than 10.sup.-9 s.sup.-1, less than 5.times.10.sup.-9
s.sup.-1, or less than 10.sup.-10 s.sup.-1 as measured, e.g., by
KINEXA.RTM. or BIACORE.RTM.. In one aspect, the anti-LOX1 antibody
can bind to hLOX1 and cynLOX1 with a Koff of 1 to
10.times.10.sup.-4 s.sup.-1 as measured by BIACORE.RTM..
[0196] In another aspect, a LOX1-binding protein such as, an
anti-LOX1 antibody (e.g., a full length LOX1-antibody, a
LOX1-binding antibody fragment, and a variant and derivative
thereof) can bind to LOX1, e.g., human LOX1 (h LOX1) and/or
cynomolgus LOX1, with an association rate constant or k.sub.on rate
of at least 10.sup.5 M.sup.-1 s.sup.-1, at least 5.times.10.sup.5
M.sup.-1 s.sup.-1, at least 10.sup.6 M.sup.-1 s.sup.-1 at least
5.times.10.sup.6 M.sup.-1 s.sup.-1 at least 10.sup.7 M.sup.-1
s.sup.-1 at least 5.times.10.sup.7 M.sup.-1 s.sup.-1 or at least
10.sup.8 M.sup.-1 s.sup.-1, or at least 10.sup.9 M.sup.-1 s.sup.-1
as measured, e.g., by KINEXA.RTM. or BIACORE.RTM.. In one aspect,
the anti-LOX1 antibody can bind to hLOX1 and cynLOX1 with a
k.sub.on rate of about 1.times.10.sup.5 M.sup.-1 s.sup.-1 to about
20.times.10.sup.5 M.sup.-1 s.sup.-1 as measured by
BIACORE.RTM..
[0197] As noted above, a LOX1-binding protein (e.g., a full length
LOX1-antibody, a LOX1-binding antibody fragment, and a variant and
derivative thereof) containing a VH and/or VL amino acid sequence
that binds LOX1 can have at least 85%, 90%, 95%, 96%, 97%, 98% or
99% sequence identity to a sequence set forth herein (see, e.g.,
Table 1, FIG. 4 or FIG. 5). In some aspects, the VH and/or VL amino
acid sequence(s) that binds LOX1 comprise 15 or fewer (e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acid
additions, substitutions (e.g., conservative substitutions) or
deletions relative to a sequence set forth herein. In some aspects,
the VH and/or VL amino acid sequence(s) that binds LOX1 comprise 8
or fewer (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) amino acid additions,
substitutions (e.g., conservative substitutions) or deletions
relative to a sequence set forth herein. In additional aspects, the
VH and/or VL amino acid sequence that binds LOX1 comprise 5 or
fewer (e.g., 1, 2, 3, 4 or 5) amino acid additions, substitutions
(e.g., conservative substitutions) or deletions relative to a
sequence set forth herein. A LOX1-binding protein (e.g. an
anti-LOX1 antibody or fragment thereof) containing VH and VL
regions having a certain percent similarity to a VH region or VL
region, or having one or more substitutions, deletions and/or
insertions (e.g., conservative substitutions) can be obtained by
mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of
nucleic acid molecules encoding VH and/or VL regions described
herein, followed by testing of the encoded altered antibody for
binding to LOX1 and optionally testing for retained function using
the functional assays described herein or an assay known in the art
that can be routinely modified to test the retained function.
[0198] The affinity or avidity of a LOX1-binding protein such as,
an anti-LOX1 antibody (e.g., a full length LOX1-antibody, a
LOX1-binding antibody fragment, and a variant and derivative
thereof), for LOX1 can be determined experimentally using any
suitable method known in the art, e.g., flow cytometry,
enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay
(RIA), or kinetics (e.g., KINEXA.RTM. or BIACORE.RTM. analysis).
Direct binding assays as well as competitive binding assay formats
can be readily employed. (See, for example, Berzofsky et al.,
"Antibody-Antigen Interactions," In Fundamental Immunology, Paul,
W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology,
W. H. Freeman and Company: New York, N.Y. (1992); and methods
described herein). The measured affinity of a particular
antibody-antigen interaction can vary if measured under different
conditions (e.g., salt concentration, pH, temperature). Thus,
measurements of affinity and other LOX1-binding parameters (e.g.,
K.sub.D or Kd, K.sub.on, K.sub.off) are made with standardized
solutions of LOX1-binding proteins and LOX1, and a standardized
buffer, as known in the art such as, the buffer described
herein.
[0199] The disclosure further provides a LOX1-binding protein such
as, an anti-LOX1 antibody (e.g., a full length LOX1-antibody, a
LOX1-binding antibody fragment, and a variant and derivative
thereof), as described herein (see, e.g., Table 1, FIG. 4 or FIG.
5), wherein the antibody is conjugated to a heterologous agent. In
certain aspects the agent is an antimicrobial agent, a therapeutic
agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a
biological response modifier, a pharmaceutical agent, a lymphokine,
a heterologous antibody or antibody fragment, a detectable label,
or a polyethylene glycol (PEG). Heteroconjugate LOX1-binding
proteins are discussed in more detail elsewhere herein.
[0200] In certain aspects, the LOX1-binding protein is not an
anti-LOX1 antibody. A variety of methods for identifying and
producing non-antibody polypeptides that bind with high affinity to
a protein target are known in the art. See, e.g., Skerra, Curr.
Opin. Biotech. 18:295-304 (2007), Hosse et al., Protein Science
15:14-27 (2006), Gill et al., Curr. Opin. Biotechnol. 17:653-658
(2006), Nygren, FEBS J. 275:2668-76 (2008), and Skerra, FEBS J.
275:2677-83 (2008), each of which is incorporated by reference
herein in its entirety. In certain aspects, phage display
technology can been used to identify/produce a LOX1-binding
protein. In certain aspects, the polypeptide comprises a protein
scaffold of a type selected from the group consisting of ankyrin,
protein A, a lipocalin, a fibronectin domain, an ankyrin consensus
repeat domain, and thioredoxin.
[0201] Proteins that Bind the Same Epitope as a LOX1-Binding
Protein
[0202] In certain aspects the disclosure provides LOX1-binding
proteins (e.g., anti-LOX1 antibodies such as, full length anti-LOX1
antibodies, LOX1-binding antibody fragments, and variants and
derivatives thereof) that bind to the same epitope as one or more
LOX1-binding proteins disclosed herein (see, e.g., Table 1, FIG. 4
or FIG. 5). The term "epitope" as used herein refers to a target
protein determinant capable of binding to an antibody of the
disclosure. Epitopes usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
usually have specific three-dimensional structural characteristics,
as well as specific charge characteristics. Conformational and
non-conformational epitopes are distinguished in that the binding
to the former but not the latter is lost in the presence of
denaturing solvents. Such antibodies can be identified based on
their ability to cross-compete (e.g., to competitively inhibit the
binding of, in a statistically significant manner) with antibodies
comprising a VH sequence of SEQ ID NO:4 and a VL sequence of SEQ ID
NO:33, and/or antibodies comprising a VH sequence of SEQ ID NO:41
and a VL sequence of SEQ ID NO:58, in standard antigen-binding or
activity assays.
[0203] The disclosure also provides LOX1-binding proteins such as,
anti-LOX1 antibodies (e.g., full length LOX1-antibodies,
LOX1-binding antibody fragments, and variants and derivatives
thereof), that bind the same epitope as an isolated LOX1-binding
protein disclosed herein (see, e.g., Table 1, FIG. 4 or FIG.
5).
[0204] The ability of a test LOX1-binding protein to inhibit the
binding of, e.g., an antibody comprising a VH sequence of SEQ ID
NO:4 and a VL sequence of SEQ ID NO:33 and/or antibodies comprising
a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:58,
demonstrates that the test LOX1-binding protein can compete with
that antibody for binding to LOX1; such a LOX1-binding protein can,
according to non-limiting theory, bind to the same or a related
(e.g., a structurally similar or spatially proximal) epitope on
LOX1 as the LOX1-binding proteins (e.g., anti-LOX1 antibodies such
as, full length anti-LOX1 antibodies, LOX1-binding antibody
fragments, and variants and derivatives thereof) with which it
competes. In one aspect, the LOX1-binding protein binds to the same
epitope on LOX1 as an antibody comprising a VH sequence of SEQ ID
NO:4 and a VL sequence of SEQ ID NO:33. In another aspect, the
LOX1-binding protein binds to the same epitope on LOX1 as an
antibody comprising a VH sequence of SEQ ID NO:41 and a VL sequence
of SEQ ID NO:58.
[0205] In one aspect, the disclosure provides LOX1-binding proteins
(e.g., antibodies such as, full length LOX1-antibodies,
LOX1-binding antibody fragments, and variants and derivatives
thereof), that compete for binding to LOX1 with an antibody
comprising a VH sequence of SEQ ID NO:4 and a VL sequence of SEQ ID
NO:33. In another aspect, the disclosure provides LOX1-binding
proteins (e.g., antibodies such as, full length LOX1-antibodies,
LOX1-binding antibody fragments, and variants and derivatives
thereof), that compete for binding to LOX1 with an antibody
comprising a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ
ID NO:58.
IV. Activity of LOX1-Binding Proteins
[0206] In some aspects, a LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody, a LOX1-binding
antibody fragment, and variants and derivatives thereof) has at
least one property selected from the group consisting of: (a)
reduces or inhibits binding of oxLDL, C-reactive protein (CRP)
and/or advanced glycation end products (AGEs) to LOX1 as determined
by any suitable assay including an assay disclosed herein (see,
e.g., Example 10, assays 1, 2 and/or 3 or Example 11); (b)
decreases or inhibits RhoA/Racl, nitrogen monoxide (NO), p38MAPK,
protein kinase B and C, ERK1/2, and/or NF.kappa.B signaling in an
endothelial cell expressing cell surface LOX1 as determined by any
suitable assay including an assay disclosed herein (see, e.g.,
Example 11); (c) decreases or inhibits caspase-8, caspase-9, and/or
BAX activity in an endothelial cell expressing cell surface LOX1 as
determined by any suitable assay including an assay disclosed
herein; (d) binds to LOX1 having the single nucleotide polymorphism
K167N as determined by any suitable assay including an assay
disclosed herein (see, e.g., Example 10, assays 1, 2 and/or 3 or
Example 11); (e) reduces or inhibits oxLDL internalization as
determined by any suitable assay including an assay disclosed
herein (see, e.g., Example 10, assay 4 or Example 11); (f) reduces
or inhibits oxLDL-induced LOX1 signaling as determined by any
suitable assay including an assay disclosed herein (see, e.g.,
Example 10, assay 5 or Example 11); (g) binds to LOX1 with a
dissociation constant (1(D) of about 150 pM to about 600 pM (e.g.
about 400 pM) as determined by BIACORE or KinExA; (h) binds to LOX1
with a Kon rate of about 1.times.10.sup.5 M.sup.-1 s.sup.-1 to
about 6.times.10.sup.6 M.sup.-1 s.sup.-1 (e.g. about
5.times.10.sup.5 M.sup.-1 s.sup.-1) as determined by BIACORE; and
(i) binds to LOX1 with a Koff rate of about 1.times.10.sup.4
s.sup.-1 to about 3.times.10.sup.4 s.sup.-1 (e.g. about
2.3.times.10.sup.4 s.sup.-1) as determined by BIACORE.
[0207] In some aspects, a LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody, a LOX1-binding
antibody fragment, and variants and derivatives thereof) can
suppress, inhibit or reduce LOX1-mediated signal transduction in
cells expressing LOX1. In some aspects, a LOX1-binding protein can
suppress, inhibit or reduce LOX1-mediated activation of the
RhoA/Racl, nitrogen monoxide, p38MAPK, protein kinase B and C,
ERK1/2, and/or NF.kappa.B signal transduction pathway as measured
using a cell-based assay for example as described herein, with an
IC50 lower than about 500 pM, lower than about 450 pM, lower than
about 450 pM, lower than about 350 pM, lower than about 300 pM,
lower than about 250 pM, lower than about 150 pM, lower than about
100 pM, lower than about 75 pM, lower than about 100 nM, lower than
about 75 nM, lower than about 50 nM, lower than about 30 nM, lower
than about 20 pM, lower than about 10 nM, or lower than about 5
nM.
[0208] In certain aspects, a LOX1-binding protein (e.g., an
anti-LOX1 antibody such as, a full length anti-LOX1 antibody,
LOX1-binding antibody fragment, and variants and derivatives
thereof) can suppress, inhibit or reduce cynomolgus LOX1-mediated
activation of the RhoA/Racl, nitrogen monoxide, p38MAPK, protein
kinase B and C, ERK1/2, and/or NF.kappa.B signaling pathway in
cynomolgus endothelial cells, smooth muscle cells, and/or
macrophages expressing LOX1 with an IC.sub.50 of about 700 pM,
about 550 pM, about 500 pM, about 300 pM, about 250 pM, about 220
pM, about, 100 pM, about 1 pM, about 0.1, about 1 nM, about 10 nM,
about 20 nM, about 30 nM, about 50 nM or about 100 nM. In certain
aspects, a LOX1-binding protein can suppress, inhibit or reduce
cynomolgus LOX1-mediated activation in cynomolgus endothelial
cells, smooth muscle cells, and/or macrophages expressing LOX1 with
an IC.sub.50 of about 1 nM to about 500 pM.
[0209] In additional aspects, an antagonist LOX1-binding protein
(e.g., an anti-LOX1 antibody such as, a full length anti-LOX1
antibody, LOX1-binding antibody fragment, and variants and
derivatives thereof) can suppress, inhibit or reduce human
LOX1-mediated activation of the RhoA/Racl, nitrogen monoxide,
p38MAPK, protein kinase B and C, ERK1/2, and/or NF.kappa.B signal
transduction pathway as measured using a cell-based assay for
example as described herein, in endothelial cells, smooth muscle
cells, and/or macrophages expressing LOX1 with an IC.sub.50 of
about 300 pM, about 250 pM, about 100 pM, about 55 pM, about 44 pM,
about 1 pM, about 0.1 pM, about 100 nm, about 50 nm about 44 nM,
about 10 nM, or about 3 nM.
[0210] In some aspects, a LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody, a LOX1-binding
antibody fragment, and variants and derivatives thereof) inhibits
or reduces LOX1-binding to oxLDL. In some aspects the LOX1-binding
protein inhibits or reduces LOX1-binding to multiple LOX1 ligands.
In some aspects the LOX1-binding protein inhibits or reduces
LOX1-binding to oxLDL and AGEs. In some aspects the LOX1-binding
protein inhibits or reduces LOX1-binding to oxLDL and CRP. In some
aspects the LOX1-binding protein inhibits or reduces LOX1-binding
to oxLDL, AGEs and CRP. In further aspects, the LOX1-binding
protein inhibits or reduces LOX1-binding to oxLDL, CRP,
phosphatidylserine, advanced AGEs, small dense lipoproteins
(sdLDL), oxidized HDL, N4-oxononanoyl lysine (ONL), heat shock
proteins (hsp, e.g., HSP60), Chlamydia pneumoniae, platelets,
leukocytes and/or apoptotic cells. Methods of measuring inhibition
or reduction of LOX-1 binding to one or more ligands include those
described herein in the Examples and any other suitable method
known in the art.
V. Preparation of Anti-LOX1 Antibodies
[0211] In certain aspects, the LOX1-binding proteins are anti-LOX1
antibodies such as, a full length anti-LOX1 antibody, LOX1-binding
antibody fragments, and variants, and derivatives thereof.
[0212] Monoclonal anti-LOX1 antibodies can be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature 256:495 (1975). Using the hybridoma method, a mouse,
hamster, or other appropriate host animal, is immunized as
described above to elicit the production by lymphocytes of
antibodies that will specifically bind to an immunizing antigen.
Lymphocytes can also be immunized in vitro. Following immunization,
the lymphocytes are isolated and fused with a suitable myeloma cell
line using, for example, polyethylene glycol, to form hybridoma
cells that can then be selected away from unfused lymphocytes and
myeloma cells. Hybridomas that produce monoclonal antibodies
directed specifically against LOX1 such as hLOX1, as determined by
immunoprecipitation, immunoblotting, or by an in vitro binding
assay (e.g. radioimmunoassay (RIA); enzyme-linked immunosorbent
assay (ELISA)) can then be propagated either in in vitro culture
using standard methods (Goding, Monoclonal Antibodies: Principles
and Practice, Academic Press, 1986) or in vivo as ascites tumors in
an animal. The monoclonal antibodies can then be purified from the
culture medium or ascites fluid as described for polyclonal
antibodies above.
[0213] Alternatively anti-LOX1 monoclonal antibodies can also be
made using recombinant DNA methods as described in U.S. Pat. No.
4,816,567. The polynucleotides encoding a monoclonal antibody are
isolated from mature B-cells or hybridoma cell, such as by RT-PCR
using oligonucleotide primers that specifically amplify the genes
encoding the heavy and light chains of the antibody, and their
sequence is determined using conventional procedures. The isolated
polynucleotides encoding the heavy and light chains are then cloned
into suitable expression vectors, which when transfected into host
cells such as E. coli cells, simian COS cells, Chinese hamster
ovary (CHO) cells, Per.C6 cells, or myeloma cells (e.g. NSO cells)
that do not otherwise produce immunoglobulin protein, monoclonal
antibodies are generated by the host cells. Also, recombinant
anti-LOX1 monoclonal antibodies can be isolated from phage display
libraries expressing CDRs of the desired species as described
(McCafferty et al., Nature 348:552-554 (1990); Clackson et al.,
Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol.
222:581-597 (1991)).
[0214] The nucleic acid(s) encoding an anti-LOX1 antibody, such as
a full length anti-LOX1 antibody and a LOX1-binding antibody
fragment, and variants and derivatives thereof, can further be
modified in a number of different manners using recombinant DNA
technology to generate alternative antibodies. In some aspects, the
constant domains of the light and heavy chains of, for example, a
mouse monoclonal antibody can be substituted (1) for those regions
of, for example, a human antibody to generate a chimeric antibody
or (2) for a non-immunoglobulin polypeptide to generate a fusion
antibody. In some aspects, the constant regions are truncated or
removed to generate the desired antibody fragment of a monoclonal
antibody. Site-directed or high-density mutagenesis of the variable
region can be used to optimize specificity, affinity, etc. of a
monoclonal antibody.
[0215] In certain aspects, the anti-LOX1-binding protein binds
human LOX1 such as, a full length anti-hLOX1 antibody and a
hLOX1-binding human antibody fragment, and variants, and
derivatives thereof. Human antibodies can be directly prepared
using various techniques known in the art. Immortalized human B
lymphocytes immunized in vitro or isolated from an immunized
individual that produce an antibody directed against a target
antigen can be generated (See, e.g., Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer
et al., J. Immunol. 147 (1):86-95(1991); and U.S. Pat. No.
5,750,373).
[0216] Also, the anti-LOX1 human antibody (e.g., a LOX1-binding
human antibody fragment) can be selected from a phage library,
where that phage library expresses human antibodies, as described,
for example, in Vaughan et al., Nat. Biotech., 14:309-314 (1996),
Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162 (1998),
Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), and Marks et
al., J. Mol. Biol. 222:581 (1991)). Techniques for the generation
and use of antibody phage libraries are also described in U.S. Pat.
Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731;
6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484;
and 7,264,963; and Rothe et al., J. Mol. Biol. 376(4): 1182-200
(2008) (each of which is incorporated by reference herein in its
entirety).
[0217] Affinity maturation strategies and chain shuffling
strategies (Marks et al., Bio/Technology 10:779-783 (1992), which
is incorporated by reference herein in its entirety) are known in
the art and can be employed to generate high affinity anti-LOX1
human antibodies.
[0218] In some aspects, a LOX1 antibody, such as a full length
anti-LOX1 antibody and a LOX1-binding antibody fragment, and
variants and derivatives thereof), can be a humanized antibody.
Methods for engineering, humanizing or resurfacing non-human or
human antibodies can also be used and are known in the art. A
humanized, resurfaced or similarly engineered antibody can have one
or more amino acid residues from a source that is non-human, e.g.,
but not limited to, mouse, rat, rabbit, non-human primate or other
mammal. These non-human amino acid residues are replaced by
residues that are often referred to as "import" residues, which are
typically taken from an "import" variable, constant or other domain
of a known human sequence. Such imported sequences can be used to
reduce immunogenicity or reduce, enhance or modify binding,
affinity, on-rate, off-rate, avidity, specificity, half-life, or
any other suitable characteristic, as known in the art. In general,
the CDR residues are directly and most substantially involved in
influencing LOX1 binding. Accordingly, part or all of the non-human
or human CDR sequences are maintained while the non-human sequences
of the variable and constant regions can be replaced with human or
other amino acids.
[0219] Anti-LOX1 antibodies can also optionally be humanized,
resurfaced, engineered or human antibodies engineered with
retention of high affinity for the antigen LOX1 and other favorable
biological properties. To achieve this goal, humanized (or human)
or engineered LOX1 antibody, such as a full length anti-LOX1
antibody and a LOX1-binding antibody fragment, and variants, and
derivatives thereof such as, resurfaced antibodies can be
optionally prepared by a process of analysis of the parental
sequences and various conceptual humanized and engineered products
using three-dimensional models of the parental, engineered, and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen, such as LOX1. In this way,
framework (FW) residues can be selected and combined from the
consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved.
[0220] Humanization, resurfacing or engineering of anti-LOX1
antibodies of the disclosure can be performed using any known
method including, but not limited to, those described in Jones et
al., Nature 321:522 (1986); Riechmann et al., Nature 332:323
(1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J.
Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901
(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285
(1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos.
5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;
5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;
5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567, 7,557,189;
7,538,195; and 7,342,110; Intl. Appl. Nos. PCT/US98/16280;
PCT/US96/18978; PCT/US91/09630; PCT/US91/05939; PCT/US94/01234;
PCT/GB89/01334; PCT/GB91/01134; PCT/GB92/01755; Intl. Appl. Publ.
Nos. WO90/14443; WO90/14424; WO90/14430; and EP Pat. Publ. No. EP
229246; each of which is entirely incorporated herein by reference,
including the references cited therein.
[0221] Antagonist human anti-LOX1 antibodies can also be made in
transgenic mice containing human immunoglobulin loci that are
capable upon immunization of producing the full repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0222] In certain aspects the anti-LOX1 antibody is a LOX1-binding
antibody fragment. Various techniques are known for the production
of antibody fragments. Traditionally, these fragments are derived
via proteolytic digestion of intact antibodies (for example
Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993);
Brennan et al., Science 229:81 (1985)). In certain aspects,
LOX1-binding antibody fragments are produced recombinantly. Fab,
Fv, and scFv antibody fragments can all be expressed in and
secreted from E. coli or other host cells, thus allowing the
production of large amounts of these fragments. Such LOX1-binding
antibody fragments can also be isolated from the antibody phage
libraries discussed above. The LOX1-binding antibody fragments can
also be linear antibodies as described in U.S. Pat. No. 5,641,870.
Other techniques for the production of antibody fragments are known
in the art.
[0223] According to the present disclosure, techniques known in the
art can be adapted for the production of single-chain antibodies
specific to LOX1 (see, e.g., U.S. Pat. No. 4,946,778). In addition,
methods can be adapted for the construction of Fab expression
libraries (see, e.g., Huse et al., Science 246:1275-1281 (1989)) to
allow rapid and effective identification of monoclonal Fab
fragments with the desired specificity for LOX1. Anti-LOX1-binding
antibody fragments can be produced by techniques known in the art
including, but not limited to: (a) a F(ab')2 fragment produced by
pepsin digestion of an antibody molecule; (b) a Fab fragment
generated by reducing the disulfide bridges of an F(ab')2 fragment,
(c) a Fab fragment generated by the treatment of the anti-LOX1
antibody with papain and a reducing agent, and (d) Fv
fragments.
[0224] In certain aspects, a LOX1-binding protein (e.g., a LOX1
antibody, such as a full length anti-LOX1 antibody and a
LOX1-binding antibody fragment, and variants and derivatives
thereof) can be modified in order to increase its serum half-life.
This can be achieved, for example, by incorporation of a salvage
receptor binding epitope into the LOX1-binding protein by mutation
of the appropriate region in the LOX1-binding protein or by
incorporating the epitope into a peptide tag that is then fused to
the LOX1-binding protein at either end or in the middle (e.g., by
DNA or peptide synthesis), or by YTE mutation. Other methods to
increase the serum half-life of a LOX1-binding protein (e.g., a
LOX1 antibody, such as a full length anti-LOX1 antibody and a
LOX1-binding antibody fragment, and variants, and derivatives
thereof), e.g., conjugation to a heterologous molecule such as PEG
are known in the art.
[0225] Heteroconjugate LOX1-binding proteins (e.g., anti-LOX1
antibodies, such as a full length anti-LOX1 antibodies and
LOX1-binding antibody fragments, and variants and derivatives
thereof) are also within the scope of the disclosure.
Heteroconjugate LOX1-binding proteins are composed of two
covalently joined proteins. Such proteins have, for example, been
proposed to target immune cells to unwanted cells (see, e.g., U.S.
Pat. No. 4,676,980). It is contemplated that the heteroconjugate
LOX1-binding proteins can be prepared in vitro using known methods
in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins can be constructed
using a disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate.
[0226] Modified anti-LOX1 antibodies such as, full length anti-LOX1
antibodies and LOX1-binding antibody fragments, and variants, and
derivatives thereof, as provided herein can comprise any type of
variable region that provides for the association of the antibody
with LOX1. In this regard, the variable region can comprise or be
derived from any type of mammal that can be induced to mount a
humoral response and generate immunoglobulins against the LOX1
antigen. As such, the variable region of an anti-LOX1 antibody can
be, for example, of human, murine, non-human primate (e.g.,
cynomolgus monkeys, macaques, etc.) or lupine origin. In some
aspects both the variable and constant regions of the modified
anti-LOX1 antibodies are human. In other aspects the variable
regions of compatible antibodies (usually derived from a non-human
source) can be engineered or specifically tailored to improve the
binding properties or reduce the immunogenicity of the molecule. In
this respect, variable regions useful according to the disclosure
can be humanized or otherwise altered through the inclusion of
imported amino acid sequences.
[0227] In certain aspects, the variable domains in both the heavy
and light chains of an anti-LOX1 antibody (e.g., a full length
anti-LOX1 antibody and a LOX1-binding antibody fragment, and
variants and derivatives thereof) are altered by at least partial
replacement of one or more CDRs and/or by partial framework region
replacement and sequence changing. Although the CDRs can be derived
from an antibody of the same class or even subclass as the antibody
from which the framework regions are derived, it is envisaged that
the CDRs will be derived from an antibody of different class and in
certain aspects from an antibody from a different species. It is
not necessary to replace all of the CDRs with the complete CDRs
from the donor variable region to transfer the antigen-binding
capacity of one variable domain to another. Rather, it is only
necessary to transfer those residues that are necessary to maintain
the activity of the antigen-binding site. Given the explanations
set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it
will be well within the competence of those skilled in the art,
either by carrying out routine experimentation or by trial and
error testing to obtain a functional antibody with reduced
immunogenicity.
[0228] Alterations to the variable region notwithstanding, those
skilled in the art will appreciate that the modified anti-LOX1
antibodies (e.g., full length anti-LOX1 antibodies and a
LOX1-binding antibody fragments, and variants and derivatives
thereof) of the disclosure will comprise antibodies in which at
least a fraction of one or more of the constant region domains has
been deleted or otherwise altered so as to provide desired
biochemical characteristics such as increased tumor localization or
reduced serum half-life when compared with an antibody of
approximately the same immunogenicity comprising a native or
unaltered constant region. In some aspects, the constant region of
the modified anti-LOX1 antibodies will comprise a human constant
region. Modifications to the constant region can include additions,
deletions or substitutions of one or more amino acids in one or
more domains. That is, the modified anti-LOX1 antibodies disclosed
herein can comprise alterations or modifications to one or more of
the three heavy chain constant domains (CH1, CH2 or CH3) and/or to
the light chain constant domain (CL). In some aspects, the modified
anti-LOX1 antibodies will comprise constant regions wherein one or
more domains are partially or entirely deleted are contemplated. In
some aspects, the modified anti-LOX1 antibodies will comprise
domain deleted constructs or variants wherein the entire CH2 domain
has been removed (ACH2 constructs). In some aspects, the omitted
constant region domain can be replaced by a short amino acid spacer
(e.g., 10 residues) that provides some of the molecular flexibility
typically imparted by the absent constant region.
[0229] Besides their configuration, it is known in the art that the
constant region mediates several effector functions. For example,
binding of the Cl component of complement to antibodies activates
the complement system. Activation of complement is important in the
opsonization and lysis of cell pathogens. The activation of
complement also stimulates the inflammatory response and can also
be involved in autoimmune hypersensitivity. Further, antibodies
bind to cells via the Fc region, with a Fc receptor site on the
antibody Fc region binding to a Fc receptor (FcR) on a cell. There
are a number of Fc receptors that are specific for different
classes of antibody, including IgG (gamma receptors), IgE (eta
receptors), IgA (alpha receptors) and IgM (mu receptors). Binding
of antibody to Fc receptors on cell surfaces triggers a number of
important and diverse biological responses including engulfment and
destruction of antibody-coated particles, clearance of immune
complexes, lysis of antibody-coated target cells by killer cells
(called antibody-dependent cell-mediated cytotoxicity, or ADCC),
release of inflammatory mediators, placental transfer and control
of immunoglobulin production.
[0230] In certain aspects, an anti-LOX1 antibody (e.g., a full
length anti-LOX1 antibody and a LOX1-binding antibody fragment, and
variants and derivatives thereof) has an altered effector function
that, in turn, affects the biological profile of the administered
anti-LOX1 antibody. For example, the deletion or inactivation
(through point mutations or other means) of a constant region
domain can reduce Fc receptor binding of the circulating modified
antibody. In other cases the constant region modifications, can
moderate complement binding and thus reduce the serum half-life and
nonspecific association of a conjugated cytotoxin. Yet other
modifications of the constant region can be used to eliminate
disulfide linkages or oligosaccharide moieties that allow for
enhanced localization due to increased antigen specificity or
antibody flexibility. Similarly, modifications to the constant
region in accordance with this disclosure can easily be made using
well-known biochemical or molecular engineering techniques well
within the purview of the skilled artisan.
[0231] In some aspects, a LOX1-binding protein provided herein is a
LOX1 antibody (e.g., a full length anti-LOX1 antibody and a
LOX1-binding antibody fragment, and variants, and derivatives
thereof) that does not have one or more effector functions. For
instance, in some aspects, the anti-LOX1-antibody has no
antibody-dependent cellular cytoxicity (ADCC) activity and/or no
complement-dependent cytoxicity (CDC) activity. In certain aspects,
the anti-LOX1 antibody does not bind to an Fc receptor and/or
complement factors. In certain aspects, the anti-LOX1 antibody has
no effector function.
[0232] In some aspects, an anti-LOX1 antibody (e.g., a full length
anti-LOX1 antibody and a LOX1-binding antibody fragment, and
variants and derivatives thereof) is engineered to fuse the CH3
domain directly to the hinge region of the respective modified
antibody. In other constructs a peptide spacer is inserted between
the hinge region and the modified CH2 and/or CH3 domains. For
example, compatible constructs can be expressed in which the CH2
domain has been deleted and the remaining CH3 domain (modified or
unmodified) is joined to the hinge region with a 5-20 amino acid
spacer. Such a spacer can be added, for instance, to ensure that
the regulatory elements of the constant domain remain free and
accessible or that the hinge region remains flexible. Amino acid
spacers can, in some cases, prove to be immunogenic and elicit an
unwanted immune response against the construct. Accordingly, in
certain aspects, any spacer added to the construct can be
relatively non-immunogenic, or even omitted altogether, so as to
maintain the desired biochemical qualities of the modified
anti-LOX1.
[0233] Besides the deletion of whole constant region domains,
anti-LOX1 antibodies (e.g., full length anti-LOX1 antibodies and
LOX1-binding antibody fragments, and variants and derivatives
thereof) can be modified by the partial deletion or substitution of
a few or even a single amino acid in a constant region. For
example, the mutation of a single amino acid in selected areas of
the CH2 domain can be enough to substantially reduce Fc binding and
thereby. Similarly one or more constant region domains that control
the effector function (e.g., complement C1Q binding) can be fully
or partially deleted. Such partial deletions of the constant
regions can improve selected characteristics of the antagonist
anti-LOX1 antibody (e.g., serum half-life) while leaving other
desirable functions associated with the corresponding constant
region domain intact. Moreover, the constant regions of the
anti-LOX1 antibodies provided herein can be modified through the
mutation or substitution of one or more amino acids that enhances
the profile of the resulting construct. In this respect it is
possible to disrupt the activity provided by a conserved binding
site (e.g., Fc binding) while substantially maintaining the
configuration and immunogenic profile of the modified anti-LOX1
antibody. The disclosure also provides an anti-LOX1 antibody that
contains the addition of one or more amino acids to the constant
region to enhance desirable characteristics such, as decreasing or
increasing effector function or providing attachments sites for
more cytotoxin, labeling or carbohydrate moieties. In such aspects
it can be desirable to insert or replicate specific sequences
derived from selected constant region domains.
[0234] The disclosure is also directed to variants and equivalents
that are substantially homologous in structure and/or similar in
one or more functions, to anti-LOX1 antibodies disclosed herein
(e.g., murine, chimeric, humanized and human LOX1-binding
proteins). These variants can contain, for example, conservative
amino acid residue substitution mutations. As generally understood
in the art, a conservative amino acid residue substitution refers
to the substitution of an amino acid residue with another within
the same general class such as, for example, one acidic amino acid
with another acidic amino acid, one basic amino acid with another
basic amino acid or one neutral amino acid by another neutral amino
acid. What is intended by a conservative amino acid substitution is
known in the art.
[0235] LOX1-binding proteins such as, anti-LOX1 antibodies provided
herein can be derivatised to contain additional chemical moieties
that are not normally part of the protein. Such derivatization
moieties are known in the art and can function to improve for
example, the solubility, biological half-life, bioavailability, and
to otherwise improve the stability, formulation and/or therapeutic
properties of the LOX1-binding protein. A non-exhaustive overview
for such moieties can be found for example, in Remington's
Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, Pa.
(2000).
VI. Nucleic Acids Encoding LOX1-Binding Proteins and Their
Expression
[0236] This disclosure provides nucleic acid molecules that encode
LOX1-binding proteins (see, e.g., those described in Table 1, FIG.
4 or FIG. 5) including anti-LOX1 antibodies such as full length
anti-LOX1 antibodies, LOX1-binding antibody fragments, and variants
and derivatives thereof. The nucleic acid molecules disclosed
herein can be in the form of RNA or in the form of DNA. DNA
includes cDNA, genomic DNA, and synthetic DNA; and can be
double-stranded or single-stranded, and if single stranded can be
the coding strand or non-coding (anti-sense) strand. In certain
aspects, the nucleic acid molecule is isolated. In additional
aspects, a nucleic acid molecule is substantially pure. In some
aspects the nucleic acid is cDNA or is derived from cDNA. In some
aspects the nucleic acid is recombinantly produced.
[0237] In some aspects, the nucleic acid molecule comprises a
LOX1-binding protein coding sequence operably linked to a control
sequence that controls the expression of the coding sequence in a
host cell or in vitro. In particular aspects, the coding sequence
is a cDNA. The disclosure also relates to vectors containing
nucleic acid molecules comprising a LOX1-binding protein coding
sequence operably linked to a control sequence that controls the
expression of the coding sequence in a host cell or in vitro.
[0238] In some aspects, the nucleic acid molecule comprises a
coding sequence for a mature LOX1-binding protein that is fused in
the same reading frame to a heterologous polynucleotide sequence.
In some aspects, the heterologous polynucleotide sequence encodes a
leader peptide sequence that facilitates the secretion of the
expressed protein from the host cell transformed with the
LOX1-encoding nucleic acid molecule. A protein containing a leader
sequence is referred to as a preprotein and can have the leader
sequence cleaved by the host cell to form the mature form of the
polypeptide. Such leader peptide sequences and their use
facilitating the secretion of recombinant proteins in host cells is
generally known in the art. In additional aspects, the heterologous
polynucleotide sequence encodes additional 5' and/or 3' amino acid
residues that can function, for example, to facilitate
purification, add or improve protein stability and/or therapeutic
or diagnostic properties of the recombinantly expressed
LOX1-binding protein.
[0239] In some aspects the disclosure provides isolated nucleic
acids such as a cDNA molecule sufficient for use as a hybridization
probe, PCR primer or sequencing primer.
[0240] In certain aspects the disclosure provides an isolated
nucleic acid molecule encoding a light chain variable region (VL),
wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid
sequences identical to, or that has a total of one, two, three,
four, five, six, seven, eight or fewer amino acid substitutions in
one or more of the VL-CDRs to: SEQ ID NO:30; SEQ ID NO:31; or SEQ
ID NO:32, 34 or 35, respectively. In other aspects the disclosure
provides an isolated nucleic acid molecule encoding a light chain
variable region (VL), wherein the VL comprises VL-CDR1, VL-CDR2,
and VL-CDR3 amino acid sequences identical to, or that has a total
of one, two, three, four, five, six, seven, eight or fewer amino
acid substitutions in one or more of the VL-CDRs to: SEQ ID NO:55
or 59; SEQ ID NO:56 or 60; or SEQ ID NO:57, 61-63 or 64,
respectively.
[0241] The disclosure further provides an isolated nucleic acid
molecule encoding a heavy chain variable region (VH), wherein the
VH comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences
identical to, or that has a total of one, two, three, four, five,
six, seven, eight or fewer amino acid substitutions in one or more
of the VH-CDRs to: SEQ ID NO: 1; SEQ ID NO:2, 5-12 or 13; and SEQ
ID NO:3, 14-17 or 18, respectively. In other aspects, the
disclosure provides an isolated nucleic acid molecule encoding a
heavy chain variable region (VH), wherein the VH comprises VH-CDR1,
VH-CDR2, and VH-CDR3 amino acid sequences identical to, or that has
a total of one, two, three, four, five, six, seven, eight or fewer
amino acid substitutions in one or more of the VH-CDRs to: SEQ ID
NO:38; SEQ ID NO:39, 42 or 43; and SEQ ID NO:40, 44-46 or 47,
respectively.
[0242] The disclosure further provides an isolated nucleic acid
such as a cDNA, encoding a light chain variable region (VL),
wherein the VL comprises an amino acid sequence having at least
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to a reference amino acid sequence selected from the group
consisting of SEQ ID NO:33, SEQ ID NO:36, and SEQ ID NO:37. In some
aspects, the isolated nucleic acid molecule encodes a light chain
variable region (VL), wherein the VL comprises an amino acid
sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity
to the reference amino acid sequence.
[0243] Moreover, the disclosure provides an isolated nucleic acid
molecule encoding a heavy chain variable region (VH), wherein the
VH comprises an amino acid sequence having at least 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a
reference amino acid sequence selected from the group consisting of
SEQ ID NO:4, 19-28 and 29. In some aspects, the isolated nucleic
acid molecule encodes a heavy chain variable region (VH), wherein
the VH comprises an amino acid sequence having at least 90%, 95%,
97%, 98%, 99% sequence identity to the reference amino acid
sequence.
[0244] In certain aspects the disclosure provides a nucleic acid
molecule or combination of nucleic acid molecules that encode a
LOX1-binding protein (e.g. the LOX-1 binding proteins described in
Table 1, FIG. 4 or FIG. 5) that specifically binds to LOX1. Further
provided is a vector comprising a nucleic molecule such as a cDNA
or combination of nucleic acid molecules, as described herein.
Suitable vectors are described elsewhere herein and are known in
the art.
[0245] In certain aspects, the heavy chain variable region (VH) and
light chain variable region (VL) are encoded by nucleic acid
sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to reference nucleic acid
sequences encoding SEQ ID NO:4 and SEQ ID NO:33, SEQ ID NO:23 and
SEQ ID NO:36, or SEQ ID NO:27 and SEQ ID NO:37, respectively. In a
particular aspect, the isolated LOX1-binding protein (e.g. an
anti-LOX1 antibody or fragment thereof) is encoded by nucleic acid
sequences encoding SEQ ID NO:4 and SEQ ID NO:33, respectively.
[0246] The disclosure further provides an isolated nucleic acid
such as a cDNA, encoding a light chain variable region (VL),
wherein the VL comprises an amino acid sequence having at least
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to a reference amino acid sequence selected from the group
consisting of SEQ ID NO:58, 65-69 and 70. In some aspects, the
isolated nucleic acid molecule encodes a light chain variable
region (VL), wherein the VL comprises an amino acid sequence having
at least 90%, 95%, 97%, 98%, 99% sequence identity to the reference
amino acid sequence.
[0247] The disclosure also provides an isolated nucleic acid
molecule encoding a heavy chain variable region (VH), wherein the
VH comprises an amino acid sequence having at least 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a
reference amino acid sequence selected from the group consisting of
SEQ ID NO:41, 48-53 and 54. In some aspects, the isolated nucleic
acid molecule encodes a heavy chain variable region (VH), wherein
the VH comprises an amino acid sequence having at least 90%, 95%,
97%, 98%, 99% sequence identity to the reference amino acid
sequence.
[0248] In certain aspects, the heavy chain variable region (VH) and
light chain variable region (VL) are encoded by nucleic acid
sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to reference nucleic acid
sequences encoding SEQ ID NO:54 and SEQ ID NO:70, SEQ ID NO:41 and
SEQ ID NO:58, SEQ ID NO:48 and SEQ ID NO:65, SEQ ID NO:49 and SEQ
ID NO:66, SEQ ID NO:50 and SEQ ID NO:67, or SEQ ID NO:53 and SEQ ID
NO:58, respectively. In a particular aspect, the isolated
LOX1-binding protein (e.g. an anti-LOX1 antibody or fragment
thereof) is encoded by nucleic acid sequences encoding SEQ ID NO:41
and SEQ ID NO:58, respectively.
[0249] In some aspects, the isolated nucleic acid encodes a
LOX1-binding protein comprising a set of CDRs: VH-CDR1, VH-CDR2,
VH-CDR3, VL-CDR1, VL-CDR3 and VL-CDR3 described in Table 1, FIG. 4
or FIG. 5. In some additional aspects, the isolated nucleic acid
encodes a LOX1-binding protein comprising a heavy chain variable
region (VH) and light chain variable region (VL) described in Table
1, FIG. 4 or FIG. 5.
[0250] In a nucleic acid molecule composition as described above
the nucleic acid encoding a VH and the nucleic acid encoding a VL
can reside in a single vector, or can be on separate vectors.
Accordingly, the disclosure provides one or more vectors comprising
the nucleic acid molecule composition or combination of nucleic
acid molecules described above.
[0251] In some cases, a polynucleotide composition encoding a VH
and VL as described above can encode an antibody (including a
LOX1-binding antibody fragment) that specifically binds to LOX1,
e.g., human and cynomolgus monkey LOX1. In some aspects the
polynucleotide composition encodes an antibody that specifically
binds to the same epitope as an antibody or antigen-binding
fragment comprising the VH and VL of SEQ ID NO:4 and SEQ ID NO:33,
respectively. In other aspects the polynucleotide composition
encodes an antibody that specifically binds to the same epitope as
an antibody or antigen-binding fragment comprising the VH and VL of
SEQ ID NO:41 and SEQ ID NO:58, respectively. In some additional
aspects, the polynucleotide composition encodes an antibody that
specifically binds to the same epitope as a LOX1-binding protein
comprising a heavy chain variable region (VH) and light chain
variable region (VL) described in Table 1, FIG. 4 or FIG. 5.
[0252] In additional aspects, the disclosure provides a host cell
comprising a nucleic acid or nucleic acids or vector as provided
above, where host cell can, in some instances express a
LOX1-binding protein (e.g., an anti-LOX1 antibody such as, a full
length LOX1-antibody, a LOX1-binding antibody fragment, and
variants and derivatives thereof), that specifically binds to LOX1.
Such a host cell can be utilized in a method of making a
LOX1-binding protein as provided herein, where the method includes
(a) culturing the host cell and (b) isolating the LOX1-binding
proteins expressed from the host cell.
[0253] The disclosure also provides a method for making a
LOX1-binding protein comprising culturing a host cell or hybridoma
capable of expressing the LOX1-binding protein under suitable
conditions and optionally provides a method for isolating the
LOX1-binding protein secreted from the host cell or hybridoma. In
addition, the disclosure additionally provides the LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof), isolated
after being secreted from the host cell or hybridoma using the
disclosed methods.
[0254] In certain aspects the polynucleotides comprise the coding
sequence(s) for the mature LOX1-binding protein(s) (e.g., an
LOX1-antibody, such as a full-length antibody and a LOX1-binding
antibody fragment) fused in the same reading frame to a marker
sequence that allows, for example, for purification of the encoded
polypeptide. For example, the marker sequence can be a
hexa-histidine tag supplied by a pQE-9 vector to provide for
purification of the mature polypeptide fused to the marker in the
case of a bacterial host, or the marker sequence can be a
hemagglutinin (HA) tag derived from the influenza hemagglutinin
protein when a mammalian host (e.g., COS-7 cells) is used.
[0255] Nucleic acid variants encoding a LOX1-binding protein such
as, an anti-LOX1 antibody and a LOX1-binding antibody fragment, are
also provided. Nucleic acid variants can contain alterations in the
coding regions, non-coding regions, or both. In some aspects the
nucleic acid variants contain alterations that produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. In some
aspects, the nucleic acid variants are produced by silent
substitutions due to the degeneracy of the genetic code. Nucleic
acid variants can be produced for a variety of reasons, e.g., to
optimize codon expression for a particular host (change codons in
the human mRNA to those preferred by a bacterial host such as E.
coli). Vectors and cells comprising the nucleic acids described
herein are also provided.
[0256] In some aspects a nucleic acid sequence encoding a
LOX1-binding protein (e.g., an anti-LOX1 antibody such as a
full-length antibody and a LOX1-binding antibody fragment) is
constructed by chemical synthesis using an oligonucleotide
synthesizer. Such oligonucleotides can be designed based on the
amino acid sequence of the desired polypeptide and codon
optimization based on the host cell preferences. Standard methods
can routinely be applied to synthesize an isolate polynucleotide
sequences encoding LOX1-binding proteins.
[0257] Once assembled (by synthesis, site-directed mutagenesis or
another method), the nucleic acid sequences encoding LOX1-binding
proteins can routinely be operably linked to a control sequence
appropriate for expression of the LOX1-binding proteins in a
desired host. In some aspects, the nucleic acid sequences encoding
LOX1-binding proteins is inserted into an expression vector and
operably linked to a control sequence appropriate for expression of
the protein in a desired host. In order to obtain high expression
levels of a transfected gene in a host, the gene can be operably
linked to or associated with transcriptional and translational
expression control sequences that are functional in the chosen
expression host.
[0258] In certain aspects, recombinant expression vectors are used
to amplify and express DNA encoding a LOX1-binding protein, such
as, an anti-LOX1 antibody or a LOX1-binding antibody fragment.
Recombinant expression vectors are replicable DNA constructs which
have synthetic or cDNA-derived DNA fragments encoding a polypeptide
chain of a LOX1-binding protein (e.g., an anti-LOX1 antibody such
as a full-length antibody and a LOX1-binding antibody fragment)
operably linked to suitable transcriptional or translational
regulatory elements derived from mammalian, microbial, viral or
insect genes. A transcriptional unit generally comprises an
assembly of (1) a genetic element or elements having a regulatory
role in gene expression, for example, transcriptional promoters or
enhancers, (2) a structural or coding sequence which is transcribed
into mRNA and translated into protein, and (3) appropriate
transcription and translation initiation and termination sequences,
as described in detail below. Such regulatory elements can include
an operator sequence to control transcription. The ability to
replicate in a host, usually conferred by an origin of replication,
and a selection gene to facilitate recognition of transformants can
additionally be incorporated. DNA regions are operably linked when
they are functionally related to each other. For example, DNA for a
signal peptide (secretory leader) is operably linked to DNA for a
polypeptide if it is expressed as a precursor which participates in
the secretion of the polypeptide; a promoter is operably linked to
a coding sequence if it controls the transcription of the sequence;
or a ribosome binding site is operably linked to a coding sequence
if it is positioned so as to permit translation. Structural
elements intended for use in yeast expression systems include a
leader sequence enabling extracellular secretion of translated
protein by a host cell. Alternatively, where a recombinant protein
is expressed without a leader or transport sequence, the protein
can include an N-terminal methionine residue. This residue can
optionally be subsequently cleaved from the expressed recombinant
protein to provide a final protein. In certain aspects, the
disclosure provides a composition, e.g., a pharmaceutical
composition, comprising a nucleic acid or vector of as described
above or elsewhere herein, optionally further comprising one or
more carriers, diluents, excipients, or other additives.
[0259] Also provided is a host cell transformed with the nucleic
acid molecule or cDNA molecules and/or the vectors disclosed
herein. The disclosure also provides host cells transformed with
the disclosed nucleic acid molecule or molecules operably linked to
a control sequence and optionally inserted into a vector. In some
aspects, the host cell is a mammalian host cell. In further
aspects, the mammalian host cell is a NSO murine myeloma cell, a
PER.C6.RTM. human cell, or a Chinese hamster ovary (CHO) cell. In
other aspects, the host cell is a hybridoma.
[0260] Additionally host cells expressing nucleic acids encoding
LOX1-binding proteins disclosed herein are hybridomas that produce
an LOX1-binding protein.
[0261] In additional aspects, the disclosure provides a method of
making a LOX1-binding protein (e.g., an anti-LOX1 antibody such as,
a full length LOX1-antibody and a LOX1-binding antibody fragment,
and variants and derivatives thereof) provided herein comprising
culturing a transformed host cell or a hybridoma disclosed herein
under suitable conditions for producing the LOX1-binding protein.
The disclosure optionally provides isolating the LOX1-binding
protein secreted from the host cell or hybridoma. The disclosure
also optionally provides the LOX1-binding protein produced using
this method and pharmaceutical compositions comprising the
LOX1-binding protein and a pharmaceutically acceptable carrier.
[0262] The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations can be employed. Useful
expression vectors for eukaryotic hosts, include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from E. coli, including pCR 1, pBR322, pMB9 and their
derivatives, and also wider host range plasmids, such as M13 and
filamentous single-stranded DNA phages.
[0263] Suitable host cells for expression of a LOX1-binding protein
(e.g., an anti-LOX1 antibody such as, a full length LOX1-antibody
and a LOX1-binding antibody fragment, and variants and derivatives
thereof), include prokaryotes, yeast, insect or higher eukaryotic
cells under the control of appropriate promoters. Prokaryotes
include gram negative or gram positive organisms, for example E.
coli or bacilli. Higher eukaryotic cells include established cell
lines of mammalian origin as described below. Cell-free translation
systems could also be employed. Additional information regarding
methods of protein production, including antibody production, can
be found, e.g., in U.S. Appl. Publ. No. 2008/0187954, U.S. Pat.
Nos. 6,413,746 and 6,660,501, and Intl. Appl. Publ. No. WO
04009823, each of which is hereby incorporated by reference herein
in its entirety.
[0264] Various mammalian or insect cell culture systems can also be
advantageously employed to express recombinant LOX1-binding protein
(e.g., an anti-LOX1 antibody such as, a full length LOX1-antibody
and a LOX1-binding antibody fragment, and variants and derivatives
thereof). Expression of recombinant LOX1-binding proteins in
mammalian cells can be performed because such proteins are
generally correctly folded, appropriately modified and completely
functional. Examples of suitable mammalian host cell lines include
HEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells,
described by Gluzman (Cell 23:175 (1981)), and other cell lines
including, for example, L cells, C127, 3T3, Chinese hamster ovary
(CHO), HeLa and BHK cell lines. Mammalian expression vectors can
comprise nontranscribed elements such as an origin of replication,
a suitable promoter and enhancer linked to the gene to be
expressed, and other 5' or 3' flanking nontranscribed sequences,
and 5' or 3' nontranslated sequences, such as necessary ribosome
binding sites, a polyadenylation site, splice donor and acceptor
sites, and transcriptional termination sequences. Baculovirus
systems for production of heterologous proteins in insect cells are
reviewed by Luckow and Summers, BioTechnology 6:47 (1988).
[0265] LOX1-binding proteins (e.g., anti-LOX1 antibodies such as,
full length anti-LOX1 antibodies and LOX1-binding antibody
fragments, and variants and derivatives thereof) produced by a
transformed host or hybridoma can be purified according to any
suitable method. Such standard methods include chromatography
(e.g., ion exchange, affinity and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for protein purification. Affinity tags such as
hexahistidine, maltose binding domain, influenza coat sequence and
glutathione-S-transferase can be attached to the protein to allow
easy purification by passage over an appropriate affinity column.
Isolated LOX1-binding proteins can also be physically characterized
using such techniques as proteolysis, nuclear magnetic resonance
and x-ray crystallography.
[0266] For example, supernatants from systems that secrete
recombinant LOX1-binding proteins into culture media can be first
concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. Following the concentration step, the
concentrate can be applied to a suitable purification matrix.
Alternatively, an anion exchange resin can be employed, for
example, a matrix or substrate having pendant diethylaminoethyl
(DEAE) groups. The matrices can be acrylamide, agarose, dextran,
cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable
cation exchangers include various insoluble matrices comprising
sulfopropyl or carboxymethyl groups. Finally, one or more
reversed-phase high performance liquid chromatography (RP-HPLC)
steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant methyl or other aliphatic groups, can be employed to
further purify an LOX1-binding protein. Some or all of the
foregoing purification steps, in various combinations, can also
routinely be employed to provide a homogeneous recombinant
LOX1-binding proteins.
[0267] A recombinant LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody and a LOX1-binding
antibody fragment, and variants and derivatives thereof) produced
in bacterial culture can be isolated, for example, by initial
extraction from cell pellets, followed by one or more
concentration, salting-out, aqueous ion exchange or size exclusion
chromatography steps. High performance liquid chromatography (HPLC)
can be employed for final purification steps. Microbial cells
employed in expression of a recombinant protein can be disrupted by
any convenient method, including freeze-thaw cycling, sonication,
mechanical disruption, or use of cell lysing agents.
[0268] Methods known in the art for purifying target binding
proteins such as full-length antibodies and antigen-binding
antibody fragments also include, for example, those described in
U.S. Appl. Publ. Nos. 2008/0312425, 2008/0177048, and 2009/0187005,
each of which is hereby incorporated by reference herein in its
entirety.
VII. Treatment Methods Using Therapeutic LOX1-Binding Proteins
[0269] Methods are provided for the use of a LOX1-binding protein
(e.g., an anti-LOX1 antibody such as, a full length LOX1-antibody,
a LOX1-binding antibody fragment, and variants and derivatives
thereof) to treat subjects having a disease associated with LOX1,
LOX1 activity, and/or LOX1 expression. Methods for detecting LOX1
expression are known in the art and include, but are not limited
to, PCR techniques, immunohistochemistry, flow cytometry, Western
blot, ELISA, and the like.
[0270] In additional aspects, the disclosure provides a
pharmaceutical composition containing a LOX1-binding protein
provided herein e.g., an anti-LOX1 antibody or fragment thereof)
and a pharmaceutically acceptable carrier. In some aspects, the
disclosure provides a pharmaceutical composition containing a
LOX1-binding protein and a pharmaceutically acceptable carrier, for
use as a medicament. The disclosure also provides the use of the
pharmaceutical compositions for treating, preventing and/or
ameliorating a disease or condition associated with LOX1, LOX1
activity, LOX1 expression and/or reduced HDL-mediated signaling. In
some aspects, the disease or condition treated using the
pharmaceutical compositions provided herein is atherosclerosis,
thrombosis, CAD, ischemia (e.g., myocardial ischemia), infarction
(e.g., myocardial infarction), acute coronary syndrome (ACS),
stroke, reperfusion injury, restenosis, peripheral vascular
disease, hypertension, heart failure, inflammation (e.g., chronic
inflammation), angiogenesis, preeclampsia and/or cancer.
[0271] In some aspects, a pharmaceutical composition contains a
LOX1-binding protein (e.g., an anti-LOX1 antibody such as, a full
length LOX1-antibody and a LOX1-binding antibody fragment, and
variants and derivatives thereof), a pharmaceutically acceptable
carrier, and further comprises a labeling group or an effector
group. As used herein, a "label" refers to one or more elements,
isotopes, or chemical compounds attached to enable the detection in
a screen. Labels generally fall into three classes: (a) isotopic
labels, which may be radioactive or heavy isotopes, (b) small
molecule labels, which may include fluorescent and colorimetric
dyes, or molecules such as biotin that enable other labeling
methods, and (c) immune labels, which may be an epitope
incorporated as a fusion partner that is recognized by an antibody.
"Labeling group" refers to any detectable label. In some aspects,
the labeling group is coupled to the LOX1-binding protein via a
spacer (e.g., a peptide spacer) to reduce potential steric
hindrance. Labels may be incorporated into the compound at any
position and may be incorporated in vitro or in vivo during protein
expression. Various methods for labeling proteins are known in the
art and may be used in performing the provided methods. In
additional aspects, the labeling group is selected from the group
consisting of: isotopic labels, magnetic labels, redox active
moieties, optical dyes, biotinylated groups and polypeptide
epitopes recognized by a secondary reporter. In some aspects, the
labeling group is a fluorescent protein such as a Green Fluorescent
Protein or derivative thereof (e.g., enhanced GFP, blue fluorescent
protein or derivative thereof (e.g., EBFP (Enhanced Blue
Fluorescent Protein), EBFP2, Azurite, mKalamal, cyan fluorescent
protein or derivative thereof (e.g., ECFP (Enhanced Cyan
Fluorescent Protein), Cerulean, CyPet), yellow fluorescent protein
or derivative thereof (e.g., YFP, Citrine, Venus, YPet). In some
aspects, the polypeptide epitope is a member selected from a biotin
signaling peptide, histidine peptide (his), hemagglutinin (HA),
Flag, gold binding peptide. In additional aspects the effector
group is selected from the group consisting of a radioisotope,
radionuclide, a toxin, a therapeutic and a chemotherapeutic
agent.
[0272] In further aspects, the effector group is selected from the
group consisting of a radioisotope, radionuclide, a toxin, a
therapeutic and a chemotherapeutic agent.
[0273] The following discussion refers to diagnostic methods and
methods of treating, preventing and/or ameliorating various
diseases and conditions with a LOX1-binding protein (e.g., an
anti-LOX1 antibody such as, a full length LOX1-antibody, a
LOX1-binding antibody fragment, and variants and derivatives
thereof). In some aspects, the LOX1-binding proteins are human,
murine, or humanized antibodies.
[0274] In one aspect, the disclosure provides for the treatment,
prevention or amelioration of a disease or condition comprising
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
such as, a full length LOX1-antibody, a LOX1-binding antibody
fragment, and variants and derivatives thereof) to a subject that
has a disease or condition, or is at risk of developing a disease
or condition, associated with LOX1. In another aspect the treatment
includes the administration of a LOX1-binding protein to an
isolated tissue or cell from a subject, where the subject has a
disease or conditions, or is at risk of developing a disease or
condition, associated with LOX1.
[0275] The disclosure also provides methods for treating,
preventing and/or ameliorating a disease or condition associated
with atherosclerosis, thrombosis, CAD, ischemia (e.g., myocardial
ischemia), infarction (e.g., myocardial infarction), acute coronary
syndrome (ACS), stroke, reperfusion injury, restenosis, peripheral
vascular disease, hypertension, heart failure, inflammation (e.g.,
chronic inflammation), angiogenesis, preeclampsia and cancer in a
subject. In some aspects, the method comprises administering to a
subject in need thereof, an effective amount of a pharmaceutical
composition comprising a LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody, a LOX1-binding
antibody fragment, and variants and derivatives thereof). In
additional aspects, the LOX1-binding protein is administered alone
or as a combination therapy.
[0276] The disclosure also provides methods of reducing or
inhibiting LOX1 activity and/or stimulating or increasing
HDL-mediated signaling in a subject. In some aspects, the method
comprises administering to a subject in need thereof, (e.g. a
subject diagnosed with atherosclerosis, thrombosis, CAD, ischemia
(e.g., myocardial ischemia), infarction (e.g., myocardial
infarction), acute coronary syndrome (ACS), stroke, reperfusion
injury, restenosis, peripheral vascular disease, hypertension,
heart failure, inflammation (e.g., chronic inflammation),
angiogenesis, preeclampsia and/or cancer), an effective amount of a
LOX1-binding protein (e.g., an anti-LOX1 antibody such as, a full
length LOX1-antibody, a LOX1-binding antibody fragment, and
variants and derivatives thereof).
[0277] Additionally provided are methods of treating, preventing,
and/or ameliorating atherosclerosis. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having atherosclerosis. In other
aspects, the subject to which the LOX1-binding protein (e.g. an
anti-LOX1 antibody or fragment thereof) is administered is at risk
of developing atherosclerosis. In some aspects, the subject has a
proatherogenic condition. In further aspects, the proatherogenic
condition is systemic lupus erythematosus (SLE), diabetes,
hypertension, hyperglycemia, heart failure, vascular injury, organ
transplantation, dyslipidemia (e.g., hyperlipidemia), inflammation
(e.g., chronic inflammation and endotoxin induced inflammation)
and/or bacterial infection. Also provided are methods of decreasing
atherosclerosis. In some instances, the disclosure provides a
method of decreasing atherosclerosis in a subject that comprises
administering a LOX1-binding protein to a subject having
atherosclerosis.
[0278] Also provided are methods of treating, preventing, and/or
ameliorating thrombosis. In some instances, the method comprises
administering a LOX1-binding protein (e.g. an anti-LOX1 antibody or
fragment thereof) to a subject having thrombosis. In other aspects,
the subject to which the LOX1-binding protein is administered is at
risk of developing thrombosis. In some aspects the thrombosis is an
arterial thrombosis. In further aspects, the thrombosis is a deep
vein thrombosis.
[0279] The disclosure also provides methods of treating,
preventing, and/or ameliorating coronary artery disease (CAD) or a
condition associated with CAD. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having CAD. In other aspects, the
subject to which the LOX1-binding protein is administered is at
risk of developing CAD. In some aspects, the subject has a
proatherogenic condition. In further aspects, the proatherogenic
condition is systemic lupus erythematosus (SLE), diabetes,
hypertension, hyperglycemia, heart failure, vascular injury, organ
transplantation, dyslipidemia (e.g., hyperlipidemia), inflammation
(e.g., chronic inflammation and endotoxin induced inflammation)
and/or bacterial infection.
[0280] The disclosure also provides methods of treating,
preventing, and/or ameliorating ischemia or a condition associated
with ischemia. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject having ischemia. In other aspects, the subject
to which the LOX1-binding protein is administered is at risk of
developing ischemia. In some aspects, the subject has myocardial
ischemia. In other aspects, the subject is at risk of developing
myocardial ischemia. In additional aspects, the subject has
systemic lupus erythematosus (SLE), diabetes, hypertension,
hyperglycemia, heart failure, vascular injury, organ
transplantation, dyslipidemia (e.g., hyperlipidemia), inflammation
(e.g., chronic inflammation and endotoxin induced inflammation)
and/or a bacterial infection.
[0281] Also provided are methods of treating, preventing, and/or
ameliorating an infarction or a condition associated with an
infarction. In some instances, the method comprises administering a
LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment
thereof in a pharmaceutical composition described herein) to a
subject having an infarction. In other aspects, the subject to
which the LOX1-binding protein is administered is at risk of
developing an infarction. In some aspects, the subject has a
myocardial infarction. In other aspects, the subject is at risk of
developing a myocardial infarction. In additional aspects, the
subject has ischemia, systemic lupus erythematosus (SLE), diabetes,
hypertension, hyperglycemia, heart failure, vascular injury, organ
transplantation, dyslipidemia (e.g., hyperlipidemia), inflammation
(e.g., chronic inflammation and endotoxin induced inflammation)
and/or a bacterial infection.
[0282] The disclosure also provides methods of treating,
preventing, and/or ameliorating acute coronary syndrome (ACS) or a
condition associated with ACS. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having ACS. In other aspects, the
subject to which the LOX1-binding protein is administered is at
risk of developing ACS. In some aspects, the subject has elevated
sLOX serum levels or elevated LOX1 activity. In additional aspects,
the subject has atherosclerosis.
[0283] The disclosure also provides methods of treating,
preventing, and/or ameliorating a stroke or a condition associated
with a stroke. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject that has had a stroke. In other aspects, the
subject to which the LOX1-binding protein is administered is at
risk of having a stroke. In some aspects, the subject has elevated
sLOX serum levels or elevated LOX1 activity. In additional aspects,
the subject has atherosclerosis.
[0284] Also provided are methods of treating, preventing, and/or
ameliorating reperfusion injury or a condition associated with
reperfusion injury. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject having reperfusion injury. In other aspects,
the subject to which the LOX1-binding protein is administered is at
risk of developing reperfusion injury. In some aspects, the subject
is about to have surgery. In other aspects, the subject has had
surgery. In some aspects the surgery is transplantation or coronary
bypass surgery. In additional aspects the patient has, or is at
risk of developing, myocardial ischemia-reperfusion injury. In
further aspects the method decreases myocardial injury, decreases
collagen accumulation, reduces serum CK-MB and MDA, reduces
cardiomyocyte size, reduces leukocyte infiltration, and/or reduces
cardiac dysfunction (e.g., reduced LVP and increased LVEDP). In
further aspects, the method increases heart stroke volume,
fractional shortening, and/or injection fraction.
[0285] The disclosure also provides methods of treating,
preventing, and/or ameliorating restenosis or a condition
associated with restenosis. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof in a pharmaceutical composition described
herein) to a subject having restenosis. In other aspects, the
subject to which the LOX1-binding protein is administered is at
risk of developing restenosis. In some aspects, the subject is
about to have surgery. In other aspects, the subject has had
surgery. In some aspects the surgery is an endovascular procedure.
In further embodiments, the surgery is vascular surgery, cardiac
surgery or angioplasty. In additional aspects the procedure is
transplantation or coronary bypass surgery. In additional aspects
the treated, prevented, and/or ameliorated restenosis is in-stent
restenosis or post-angioplasty restenosis.
[0286] In additional aspects, the disclosure provides methods of
treating, preventing, and/or ameliorating peripheral vascular
disease (PVD) or a condition associated with PVD. In some
instances, the method comprises administering a LOX1-binding
protein (e.g., in a pharmaceutical composition described herein) to
a subject having PVD. In other aspects, the subject to which the
LOX1-binding protein is administered is at risk of developing PVD.
In some aspects, the subject has elevated sLOX serum levels or
elevated LOX1 activity. In additional aspects, the subject has
atherosclerosis.
[0287] The disclosure also provides methods of treating,
preventing, and/or ameliorating inflammation or a condition
associated with inflammation. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having inflammation. In further
aspects, the subject has chronic inflammation. In some aspects, the
subject has elevated oxLDLa and/or sLOX serum levels and/or
elevated LOX1 activity. In additional aspects, the subject has
atherosclerosis.
[0288] The disclosure also provides methods of treating,
preventing, and/or ameliorating preeclampsia or a condition
associated with preeclampsia. In some instances, the method
comprises administering a LOX1-binding protein (e.g., an anti-LOX1
antibody or fragment thereof in a pharmaceutical composition
described herein) to a subject having preeclampsia or eclampsia. In
further aspects, the subject has high blood pressure and large
amounts of protein in the urine. In some aspects, the subject has
elevated oxLDL and/or sLOX serum levels and/or elevated LOX1
activity. In additional aspects, the subject has swelling in the
feet, legs and/or hands.
[0289] In additional aspects, the disclosure provides methods of
stabilizing an atherosclerotic plaque in a subject. In some
instances, the method comprises administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody such as, a full length
LOX1-antibody, a LOX1-binding antibody fragment, and variants and
derivatives thereof) to a subject in need thereof. In some aspects
the method reduces the signaling of the RhoA/Racl, nitrogen
monoxide, p38MAPK, protein kinase B and C, ERK1/2, and/or
NF.kappa.B signal transduction pathway in the plaque. In other
aspects, the method decreases apoptosis in the plaque. In further
aspects, the method decreases caspase 8, caspase 9 and/or BAX
activity and/or increases BCL-2 activity in the plaque. In other
aspects, the method decreases the levels of an adhesion molecule or
cytokine produced by the plaque. In further aspects, the method
decreases E-selectin, P-selectin, ICAM-1, VCAM-1, MCP1 and/or
CD40/CD40L expression by the plaque. In additional aspects, the
method decreases atherosclerotic plaque size or formation,
macrophage accumulation and/or MMP (e.g., MMP9) expression in the
atherosclerotic plaque. In additional aspects, the method results
in decreased progression or regression of the plaque.
[0290] Also provided are methods of reducing the loss of vascular
tone in a subject. In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
such as, a full length LOX1-antibody, a LOX1-binding antibody
fragment, and variants and derivatives thereof) to a subject in
need thereof. In some aspects the method reduces the loss of
vascular tone. In further aspects, the method reduces the loss of
vascular tone in a subject through regulating HDL driven nitric
oxide (NO) production (ability of antibody to stimulate endothelial
NO production).
[0291] Additionally provided are methods of improving vascular tone
in a subject. In some instances, the method comprises administering
a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment
thereof) to a subject in need thereof.
[0292] In some aspects the disclosure provides methods of treating,
preventing, and/or ameliorating a condition associated with
hyperglycemia or hypertension. In some instances, the method
comprises administering a LOX1-binding protein to a subject having
hyperglycemia or hypertension.
[0293] Additionally provided are methods of treating, preventing,
and/or ameliorating cancer. In some instances the disclosure
provides a method of treating, preventing, and/or ameliorating
cancer in a subject that comprises administering a LOX1-binding
protein to a subject having cancer. In some aspects, the subject
has a cancer selected from the group consisting of: breast cancer,
colon cancer, ovarian cancer, melanoma, cervical cancer, lung
cancer, uterine cancer, kidney cancer, and pancreatic cancer.
[0294] Also provided are methods of inhibiting tumor cell
proliferation, migration or invasion. In some instances the
disclosure provides a method of antagonizing LOX1 activity that
comprises contacting a LOX1-binding protein with a tumor cell
expressing LOX1. In some aspects the tumor cell is from a cancer
selected from the group consisting of: breast cancer, colon cancer,
ovarian cancer, melanoma, cervical cancer, lung cancer, uterine
cancer, kidney cancer, and pancreatic cancer. In some aspects the
tumor cell is from a cancer line.
[0295] The disclosure additionally provides methods of reducing or
inhibiting angiogenesis. In some aspects the method of reducing or
inhibiting angiogenesis comprises administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof) to a
subject in need thereof. In some aspects the subject has a
condition associated with pathological angiogenesis. In additional
aspects the disclosure provides a method of inhibiting angiogenesis
that comprises contacting a LOX1-binding protein with a cell
expressing LOX1. In some aspects the cell is an endothelial cell.
In further aspects the endothelial cell is a coronary endothelial
cell. In some aspects the method is performed in vitro. In other
aspects the method is performed in vivo.
[0296] Additionally provided are methods of blocking or reducing
LOX1 activity. In some aspects the disclosure provides methods of
blocking LOX1 activity comprising administering a LOX1-binding
protein (e.g., an anti-LOX1 antibody or fragment thereof) that
reduces or inhibits the interaction between LOX1 and a LOX1 ligand
such as, oxLDL, AGEs, and/or CRP. In some aspects, LOX1 is
expressed on the surface of an endothelial cell, macrophage, smooth
muscle vascular cell and/or platelet. In some aspects the cell is
an endothelial cell such as, a coronary endothelial cell. In
additional aspects, the cell is a vascular smooth muscle cell,
macrophage, or platelet. In other aspects the cell is part of an
atherosclerotic tissue. In some aspects, the method is performed in
vivo. In other aspects, the method is performed in vitro. In some
aspects the blocked or reduced LOX1 activity is the binding and/or
taking up (e.g. internalization) of oxLDL. In additional aspects,
the blocked or reduced LOX1 activity is the induction of the p38
(MAPK), p44/42 MAPK, protein kinase C (PKC), protein kinase B
(PKB), protein tyrosine kinase (PTK), transcription factor NF-KB
and/or AP1 signaling pathway. In additional aspects the blocked or
reduced LOX1 activity is the induction of apoptosis. In further
embodiments, the induction of apoptosis is mediated by caspase-9,
caspase-3 and/or Bcl-2. In additional aspects the blocked or
reduced LOX1 activity is the expression of the A and B chains of
PDFG and/or heparin-binding EGF-like protein (HB-EGF) in
endothelial cells expressing LOX1. In some aspects, the blocked or
reduced LOX1 activity is a LOX1 activity induced by oxLDL binding
to LOX1.
[0297] Additionally provided are methods of blocking or reducing
LOX1 activity in a pathological condition associated with increased
LOX1 activity levels or LOX1 expression levels (e.g. sLOX1 serum
protein levels). In some instances, the method comprises
administering a LOX1-binding protein (e.g., an anti-LOX1 antibody
or fragment thereof) to a subject having increased LOX1 activity or
LOX1 expression levels (e.g. sLOX1 serum protein levels). In some
aspects the pathological condition is systemic lupus erythematosus
(SLE), diabetes, hypertension, hyperglycemia, heart failure,
vascular injury, transplantation, dyslipidemia (hyperlipidemia),
inflammation, (e.g., chronic inflammation and endotoxin induced
inflammation) or bacterial infection. In some aspects, the subject
has elevated serum levels of OxLDL. In some aspects, the subject
has elevated serum levels of OxLDL, 15 lipoxygenase modified LDL,
15 lipoxygenase modified HDL, glyoxidized LDL,
lysophosphatidylcholinesterase (LPC) and/or palmitic acid. In
additional aspects, the subject has elevated serum levels of TNF
alpha, ILL interferon gamma, LPS (lipopolysaccharide), CRP,
angiotensin II, endothelin I, and/or AGEs. In additional aspects,
the subject has elevated serum levels of soluble LOX1 (sLOX1). In
some aspects, the subject has a single nucleotide polymorphism
(SNP) in the LOX1 gene. In some aspects, the SNP in the LOX1 gene
is the LOX1 K167N variant.
[0298] Also provided are methods of agonizing or increasing a
high-density lipoprotein (HDL) activity. In some aspects, the
disclosure provides a method of increasing or agonizing an HDL
activity by administering a LOX1-binding protein (e.g., an
anti-LOX1 antibody or fragment thereof) to a subject in need
thereof. In some aspects the increased HDL activity is the
promotion of HDL-mediated endothelial NO production. In some
aspects, the increased HDL activity is the inhibition of the
NF.kappa.B signaling activity of the endothelial cell. In some
aspects, the increase HDL activity is the promotion of endothelial
cell repair. In some aspects, the increase HDL activity is the
reduction of inflammation.
[0299] Also provided is the use of a LOX1-binding protein such as a
LOX1-binding protein provided herein for diagnostic monitoring of
protein levels (e.g., LOX1 levels) in blood or tissue as part of a
clinical testing procedure, e.g., to determine the efficacy of a
given treatment regimen. For example, detection can be facilitated
by coupling a LOX1-binding protein (e.g., anti-LOX1 antibody) to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include nptavidin/biotin and avidin/biotin; examples of
suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin; and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S, or .sup.3H.
VIII. Pharmaceutical Compositions and Administration Methods
[0300] Methods of preparing and administering a LOX1-binding
protein provided herein to a subject in need thereof are known to
or are readily determined by those skilled in the art. The route of
administration of the LOX1-binding proteins can be, for example,
oral, parenteral, by inhalation or topical. The term parenteral as
used herein includes, e.g., intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal
administration. While all these forms of administration are clearly
contemplated as being within the scope of the disclosure, another
example of a form for administration would be a solution for
injection, in particular for intravenous or intraarterial injection
or drip. Usually, a suitable pharmaceutical composition can
comprise a buffer (e.g. acetate, phosphate or citrate buffer), a
surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g.
human albumin), etc. In other methods compatible with the teachings
herein, LOX1-binding proteins as provided herein can be delivered
directly to the organ and/or site of an atheroslerosis or tumor,
thereby increasing the exposure of the diseased tissue to the
therapeutic agent. In one aspect, the administration is directly to
the airway, e.g., by inhalation or intranasal administration.
[0301] As discussed herein, LOX1-binding proteins can be
administered in a pharmaceutically effective amount for the in vivo
treatment of LOX1-mediated diseases and conditions such as,
atherosclerosis, thrombosis, CAD, ischemia (e.g., myocardial
ischemia), infarction (e.g., myocardial infarction), acute coronary
syndrome (ACS), stroke, reperfusion injury, restenosis, peripheral
vascular disease, hypertension, heart failure, inflammation (e.g.,
chronic inflammation), angiogenesis, preeclampsia and cancer. In
this regard, it will be appreciated that the disclosed LOX1-binding
proteins can be formulated so as to facilitate administration and
promote stability of the active agent. Pharmaceutical compositions
in accordance with the disclosure can comprise a pharmaceutically
acceptable, non-toxic, sterile carrier such as physiological
saline, non-toxic buffers, preservatives and the like. For the
purposes of the instant application, a pharmaceutically effective
amount of a LOX1-binding protein, conjugated or unconjugated, means
an amount sufficient to achieve effective binding to LOX1 and to
achieve a benefit, e.g., to ameliorate symptoms of a disease or
condition or to detect a substance or a cell. Suitable formulations
for use in the therapeutic methods disclosed herein are described
in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th
ed. (1980).
[0302] Certain pharmaceutical compositions provided herein can be
orally administered in an acceptable dosage form including, e.g.,
capsules, tablets, aqueous suspensions or solutions. Certain
pharmaceutical compositions also can be administered by nasal
aerosol or inhalation. Such compositions can be prepared as
solutions in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
and/or other conventional solubilizing or dispersing agents.
[0303] The amount of a LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody and a LOX1-binding
antibody fragment, and variants and derivatives thereof) that can
be combined with carrier materials to produce a single dosage form
will vary depending upon the subject treated and the particular
mode of administration. The composition can be administered as a
single dose, multiple doses or over an established period of time
in an infusion. Dosage regimens also can be adjusted to provide the
optimum desired response (e.g., a therapeutic or prophylactic
response).
[0304] In keeping with the scope of the present disclosure, a
LOX1-binding protein can be administered to a human or other
subject in accordance with the aforementioned methods of treatment
in an amount sufficient to produce a therapeutic effect. The
LOX1-binding proteins provided herein can be administered to such
human or other animal in a conventional dosage form prepared by
combining the LOX1-binding proteins with a conventional
pharmaceutically acceptable carrier or diluent according to known
techniques. The form and character of the pharmaceutically
acceptable carrier or diluent can be dictated by the amount of
active ingredient with which it is to be combined, the route of
administration and other well-known variables. A cocktail
comprising one or more different LOX1-binding proteins can also be
used.
[0305] By "therapeutically effective dose or amount" or "effective
amount" is intended an amount of a LOX1-binding protein (e.g., an
anti-LOX1 antibody such as, a full length LOX1-antibody and a
LOX1-binding antibody fragment, and variants and derivatives
thereof) that when administered brings about a positive therapeutic
response with respect to treatment of a subject with a disease or
condition to be treated.
[0306] Therapeutically effective doses of LOX1-binding protein
compositions for treatment of LOX1-mediated diseases or conditions
such as, atherosclerosis, thrombosis, CAD, ischemia (e.g.,
myocardial ischemia), infarction (e.g., myocardial infarction),
acute coronary syndrome (ACS), stroke, reperfusion injury,
restenosis, peripheral vascular disease, hypertension, heart
failure, inflammation (e.g., chronic inflammation), angiogenesis,
preeclampsia and cancer, vary depending upon many different
factors, including means of administration, target site,
physiological state of the subject, whether the subject is human or
an animal, other medications administered, and whether treatment is
prophylactic or therapeutic. Usually, the subject is a human, but
non-human mammals including transgenic mammals can also be treated.
Treatment dosages can be titrated using routine methods known to
those of skill in the art to optimize safety and efficacy.
[0307] As used herein, to ameliorate the symptoms of a particular
disease or condition by administration of a LOX1-binding protein
refers to any lessening, whether permanent or temporary, lasting or
transient that can be attributed to or associated with
administration of the LOX1-binding protein.
[0308] The amount of at least one LOX1-binding protein, e.g.,
antibody or binding fragment to be administered is readily
determined by one of ordinary skill in the art without undue
experimentation given this disclosure. Factors influencing the mode
of administration and the respective amount of at least one
LOX1-binding protein includes, but is not limited to, the severity
of the disease, the history of the disease, and the age, height,
weight, health, and physical condition of the individual undergoing
therapy. Similarly, the amount of a LOX1-binding protein to be
administered will be dependent upon the mode of administration and
whether the subject will undergo a single dose or multiple doses of
this agent.
[0309] The disclosure also provides for the use of a LOX1-binding
protein, such as, an anti-LOX1 antibody (e.g., a full length
LOX1-antibody and a LOX1-binding antibody fragment, and a variant
and derivative thereof) in the manufacture of a medicament for
example, for improving HDL activity, and methods of treating,
preventing, and/or ameliorating atherosclerosis, thrombosis, CAD,
ischemia (e.g., myocardial ischemia), infarction (e.g., myocardial
infarction), acute coronary syndrome (ACS), stroke, reperfusion
injury, restenosis, peripheral vascular disease, inflammation
(e.g., chronic inflammation), angiogenesis, preeclampsia and
cancer.
[0310] Combination Therapies
[0311] In some aspects, a LOX1-binding protein (e.g., an anti-LOX1
antibody such as, a full length LOX1-antibody and a LOX1-binding
antibody fragment, and variants and derivatives thereof) is
administered in combination with one or more other therapies. Such
therapies include additional therapeutic agents as well as other
medical interventions. Exemplary therapeutic agents that can be
administered in combination with the LOX1-binding proteins provided
herein include, but are not limited to, platelet inhibitors,
anti-coagulants, anti-hypertensives, glycoprotein IIb/IIIa receptor
inhibitors, beta blockers, calcium channel blockers, HMG CoA
reductases inhibitors (statins), ezetimibe, fibrates (e.g.,
Gemifibrozil and Fenofibrate), Zetia, bile acid sequestrants,
nitrates, and thrombolytic agents.
[0312] In various aspects, a LOX1-binding protein is administered
to a subject before, during, and/or after a surgical procedure. In
some aspects the surgery is an endovascular procedure. In further
embodiments, the surgery is vascular surgery, cardiac surgery or
angioplasty. In additional aspects the procedure is transplantation
or coronary bypass surgery.
IX. Diagnostics
[0313] This disclosure further provides a diagnostic method useful
during diagnosis of LOX1-mediated diseases and conditions such as,
atherosclerosis, thrombosis, coronary artery disease (CAD),
ischemia (e.g., myocardial ischemia), infarction (e.g., myocardial
infarction), acute coronary syndrome (ACS), stroke, reperfusion
injury, restenosis, peripheral vascular disease, hypertension,
heart failure, inflammation, angiogenesis, preeclampsia and cancer,
which involves measuring the expression level of LOX1 (including
sLOX1) protein in tissue or body fluid such as serum from an
individual and comparing the measured expression level with a
standard LOX1 expression level in normal tissue or body fluid,
whereby an increase in the expression level compared to the
standard is indicative of a disorder treatable by a LOX1-binding
protein provided herein, such as a full-length anti-LOX1 antibody
and antigen-binding antibody fragment as provided herein.
[0314] The LOX1-binding proteins provided herein such as, anti-LOX1
antibodies (e.g., full length LOX1-antibodies and LOX1-binding
antibody fragments, and variants and derivatives thereof) can be
used to assay LOX1 protein levels in a biological sample using
classical immunohistological methods known to those of skill in the
art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985
(1985); Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Other
antibody-based methods useful for detecting LOX1 protein expression
include immunoassays, such as the enzyme linked immunosorbent assay
(ELISA), immunoprecipitation, or Western blotting.
[0315] By "assaying the expression level of LOX1 protein" is
intended qualitatively or quantitatively measuring or estimating
the level of LOX1 protein in a first biological sample either
directly (e.g., by determining or estimating absolute protein
level) or relatively (e.g., by comparing to the disease associated
polypeptide level in a second biological sample). The LOX1 protein
expression level in the first biological sample can be measured or
estimated and compared to a standard LOX1 protein level, the
standard being taken from a second biological sample obtained from
an individual not having the disorder or being determined by
averaging levels from a population of individuals not having the
disorder. As will be appreciated in the art, once the "standard"
LOX1 protein level is known, it can be used repeatedly as a
standard for comparison.
[0316] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source of cells potentially expressing LOX1. Methods for obtaining
tissue biopsies and body fluids from mammals are known in the
art.
X. Kits Comprising LOX1-Binding Proteins
[0317] This disclosure further provides kits that include a
LOX1-binding protein (e.g., an anti-LOX1 antibody such as, a full
length LOX1-antibody, a LOX1-binding antibody fragment, and
variants and derivatives thereof) in suitable packaging, and
written material and that can be used to perform the methods
described herein.
[0318] The written material can include any of the following
information: instructions for use, discussion of clinical studies,
listing of side effects, scientific literature references, package
insert materials, clinical trial results, and/or summaries of these
and the like. The written material can indicate or establish the
activities and/or advantages of the composition, and/or describe
dosing, administration, side effects, drug interactions, or other
information useful to the health care provider. Such information
can be based on the results of various studies, for example,
studies using experimental animals involving in vivo models and/or
studies based on human clinical trials. The kit can further contain
another therapy (e.g., another agent) and/or written material such
as that described above that serves to provide information
regarding the other therapy (e.g., the other agent).
[0319] In certain aspects, a kit comprises at least one purified
LOX1-binding protein in one or more containers. In some aspects,
the kits contain all of the components necessary and/or sufficient
to perform a detection assay, including all controls, directions
for performing assays, and any necessary software for analysis and
presentation of results.
XI. Immunoassays
[0320] LOX1-binding proteins (e.g., anti-LOX1 antibodies and
LOX1-binding fragments, variants, or derivatives thereof) can be
assayed for immunospecific binding by any method known in the art.
The immunoassays that can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as Western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and
known in the art (see, e.g., Ausubel et al., eds, (1994) Current
Protocols in Molecular Biology (John Wiley & Sons, Inc., NY)
Vol. 1, which is incorporated by reference herein in its
entirety).
[0321] LOX1-binding proteins (e.g., anti-LOX1 antibodies or
LOX1-binding fragments, variants, or derivatives thereof) provided
herein can be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immunological assays, for in situ
detection of LOX1 or conserved variants or peptide fragments
thereof. In situ detection can be accomplished by removing a
histological specimen from a subject, and applying thereto labeled
LOX1-binding proteins by overlaying the labeled LOX1-binding
proteins onto a biological sample. Through the use of such a
procedure, it is possible to determine not only the presence of
LOX1, or conserved variants or peptide fragments, but also its
distribution in the examined tissue. Using the present disclosure,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0322] The binding activity of a given lot of LOX1-binding protein
(e.g., an anti-LOX1 antibody such as, a full length LOX1-antibody
and a LOX1-binding antibody fragment, and variants and derivatives
thereof) can be determined according to methods known in the art.
Those skilled in the art will be able to determine operative and
optimal assay conditions for each determination by employing
routine experimentation.
[0323] Methods and reagents suitable for determination of binding
characteristics of an isolated LOX1-binding protein are known in
the art and/or are commercially available. Equipment and software
designed for such kinetic analyzes are commercially available
(e.g., BIACORE.RTM., BIAevaluation.RTM. software, GE Healthcare;
KINEXA.RTM. Software, Sapidyne Instruments).
[0324] Unless otherwise indicated, the practice of the disclosure
employs conventional techniques of cell biology, cell culture,
molecular biology, transgenic biology, microbiology, recombinant
DNA, and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature. See, for example,
Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual
(2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al.,
ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs
Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning,
Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis;
Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds.
(1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984)
Transcription And Translation; Freshney (1987) Culture Of Animal
Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL
Press) (1986); Perbal (1984) A Practical Guide To Molecular
Cloning; the treatise, Methods In Enzymology (Academic Press, Inc.,
N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For
Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds.,
Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds.
(1987) Immunochemical Methods In Cell And Molecular Biology
(Academic Press, London); Weir and Blackwell, eds., (1986) Handbook
Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in
Molecular Biology (John Wiley and Sons, Baltimore, Md.).
[0325] General principles of antibody engineering are set forth in
Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ.
Press). General principles of protein engineering are set forth in
Rickwood et al., eds. (1995) Protein Engineering, A Practical
Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General
principles of antibodies and antibody-hapten binding are set forth
in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer
Associates, Sunderland, Mass.); and Steward (1984) Antibodies,
Their Structure and Function (Chapman and Hall, New York, N.Y.).
Additionally, standard methods in immunology known in the art and
not specifically described are generally followed as in Current
Protocols in Immunology, John Wiley & Sons, New York; Stites et
al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton
& Lange, Norwalk, Conn.) and Mishell et al., (eds) (1980)
Selected Methods in Cellular Immunology (W.H. Freeman & Co.,
NY).
[0326] Standard reference works setting forth general principles of
immunology include Current Protocols in Immunology, John Wiley
& Sons, New York; Klein (1982) J., Immunology: The Science of
Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et
al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension
in Biological Analyzes (Plenum Press, NY); Campbell (1984)
"Monoclonal Antibody Technology" in Laboratory Techniques in
Biochemistry and Molecular Biology, ed. Burden et al., (Elsevere,
Amsterdam); Goldsby et al., eds. (2000) Kuby Immunnology (4th ed.;
H. Freemand & Co.); Roitt et al. (2001) Immunology (6th ed.;
London: Mosby); Abbas et al. (2005) Cellular and Molecular
Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann
and Dubel (2001) Antibody Engineering (Springer Verlan); Sambrook
(2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow and
Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor
Press); Dieffenbach et al., (2003) PCR Primer (Cold Spring Harbor
Press).
[0327] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0328] Aspects of the present disclosure can be further defined by
reference to the following non-limiting examples, which describe in
detail preparation of certain antibodies of the present disclosure
and methods for using antibodies of the present disclosure. It will
be apparent to those skilled in the art that many modifications,
both to materials and methods, can be practiced without departing
from the scope of the present disclosure.
Example 1. Isolation and Identification of Anti-LOX1 scFv
Antibodies
[0329] A large single chain Fv (scFv) human antibody library
derived from bone marrow from adult naive donors and cloned into a
phagemid vector based on filamentous phage M13 was used for
selections (Hutchings, C., Generation of Naive Human Antibody
Libraries, in Antibody Engineering, R. Kontermann and S. Dubel,
Editors. 2001, Springer Laboratory Manuals, Berlin. p. 93).
LOX1-specific scFv antibodies were isolated from the phage display
library in a series of repeated selection cycles on recombinant
mammalian expressed human LOX1 (R&D Systems) essentially as
previously described (Vaughan et al., Nat. Biotechnol. 14:309
(1996)). While several antigen-specific scFvs were obtained from
different variations of this protocol, the parental clones LOX514
(SEQ ID NO:29 (VH) and SEQ ID NO:33 (VL); also referred herein as
"LOX10514") and LOX696 (SEQ ID NO:54 (VH) and SEQ ID NO:70 (VL);
also referred herein as "LOX10696") were isolated as follows: 10
.mu.g/ml human LOX1 was immobilized on MAXISORP.TM. plates (Nunc)
and incubated with the phage display library. Unbound phage was
washed away in a series of wash cycles. The phage particles
retained on the antigen were eluted, infected into bacteria and
rescued for the next round of selection. Three rounds of selection
were performed in this way. A representative number of individual
clones from the round 2 and round 3 selection output were grown up
in 96-well plates. ScFvs were expressed in the bacterial periplasm
and screened for their inhibitory activity in a human LOX1:oxLDL
binding assay as described in Example 10, Assay 1.
[0330] Screening hits, i.e. scFv clones which showed an inhibitory
effect on LOX1:oxLDL interaction, as crude periplasmic extracts,
were subjected to DNA sequencing (Vaughan et al., Nat. Biotechnol.
14:309 (1996), and Osbourn et al., Immunotechnology 2:181 (1996)).
Unique scFvs were re-expressed in bacteria and purified by affinity
chromatography (as described in Example 3 of Intl. Appl. Publ. No.
WO01/66754), and IC.sub.50 values were determined by testing
dilution series of purified scFvs in the above assay.
[0331] The most potent scFv clones were converted to whole
immunoglobulin G1 triple mutant (IgG1-TM, human IgG1 Fc sequence
incorporating mutations L234F, L235E and P331S) antibody format
essentially as described in Persic et al., Gene 187:9 (1997) with
the following modifications. An OriP fragment was included in the
expression vectors to facilitate use with CHO cells and to allow
episomal replication. The VH domain was cloned into a vector
containing the human heavy chain constant domains and regulatory
elements to express whole IgG1-TM heavy chain in mammalian cells.
Similarly, the VL domain was cloned into a vector for the
expression of the human light chain constant domains and regulatory
elements to express whole IgG light chain in mammalian cells. To
obtain IgGs, the heavy and light chain IgG expressing vectors were
transfected into CHO mammalian cells. IgGs were expressed and
secreted into the medium. Harvests were pooled and filtered and IgG
was purified using Protein A chromatography. Culture supernatants
were loaded on a column of appropriate size of Ceramic Protein A
(BioSepra) and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl.
Bound IgG was eluted from the column using 0.1 M Sodium Citrate (pH
3.0) and neutralized by the addition of Tris-HCl (pH 9.0). The
eluted material was buffer exchanged into PBS using Nap10 columns
(Amersham, #17-0854-02) and the concentration of IgG was determined
spectrophotometrically using an extinction coefficient based on the
amino acid sequence of the IgG (Mach et al., Anal Biochem, 200:74
(1992)). The purified IgG were analyzed for aggregation and
degradation using SEC-HPLC and by SDS-PAGE.
Example 2--Anti-LOX1 Antibodies Inhibit Multi-Ligand Binding, oxLDL
Internalization and oxLDL Induced Signaling
[0332] Purified IgGs were tested for their ability to specifically
inhibit oxidized low-density lipoprotein ("oxLDL"), advanced
glycation end product of bovine serum albumin ("AGE-BSA") and
C-reactive protein ("CRP") binding to LOX1 as described in Example
10, Assays 1, 2 and 3. A number of isolated clones including
LOX10514 and LOX10696, and commercially available mouse anti-LOX1
antibody (23C11) were tested for their ability to block oxLDL,
AGE-BSA and CRP binding to LOX1. Representative plots are shown in
FIGS. 1A, 1B and 1C showing inhibition of oxLDL, AGE-BSA and CRP
binding, respectively, by LOX514 and LOX696. To confirm the
antibodies cross react and had functional activity on the LOX1 SNP
K167N the antibodies were tested for their ability to block oxLDL
binding to LOX1 SNP K167N. LOX1 SNP K167N (GenBank Accession No.
AB102861) is a naturally occurring human LOX1 variant originally
discovered in a family of patients with ischemic heart disease and
is believed to be associated with an increased risk of myocardial
infarction. See, e.g., Tatsuguchi et al., Biochem Biophys Res
Commun. 28; 303(1):247-50 (2003). Representative plots are shown in
FIG. 1D showing that both LOX514 and LOX696 block oxLDL binding to
the LOX1 SNP K167N variant. In addition to inhibition of binding, a
number of isolated clones including LOX10514 and LOX10696 were
tested for their ability to block oxLDL internalization on LOX1
expressing cells (as described in Example 10, Assay 4) and oxLDL
induced LOX1 signaling (as described in Example 10, Assay 5).
Representative plots are shown in FIGS. 2A and 2B for inhibition of
oxLDL internalization and signaling respectively. The results from
these studies are summarized in Table 2.
TABLE-US-00002 TABLE 2 Inhibition of oxLDL internalization LOX10696
23C11 LOX10514 Mean 1.31E-10 2.20E-09 6.42E-10 SD 1.14E-10 6.49E-10
8.54E-11 n 8 5 4 Mean 2.46E-10 1.31E-09 7.03E-10 SD 1.11E-10
2.82E-10 n 2 1 3 Mean 3.50E-10 2.85E-09 7.72E-10 SD 7.68E-11
8.38E-10 1.11E-10 n 2 3 3 Mean 8.73E-11 1.15E-09 2.07E-10 SD
8.23E-11 1.07E-09 1.61E-10 n 2 2 3 Mean 4.69E-09 1.11E-08 1.98E-09
SD 4.02E-09 9.52E-10 7.31E-10 n 2 2 2 Mean 6.22E-09 1.01E-08
8.28E-09 SD 2.13E-09 2.84E-09 n 6 1 8
[0333] These results demonstrate: (1) specific inhibition of LOX1
binding to oxLDL, AGE-BSA and CRP by antibodies LOX514 and LOX696;
(2) both LOX514 and LOX696 functionally cross react with the common
LOX1 SNP K167N variant; (3) LOX514 and LOX696 inhibit oxLDL
internalization; and (4) LOX514 and LOX696 inhibit oxLDL-induced
LOX1 signaling.
Example 3--Anti-LOX1 Antibodies Species Cross-Reactivity and
Specificity for Human LOX1 Over a Panel of Human Related Family
Members
[0334] Cross-reactivity of anti-human LOX1 antibodies to various
LOX1 species orthologs was assessed by a scFv binding ELISA.
Extracellular domain constructs for human (Uniprot: P78380), mouse
(Uniprot: Q9EQ09), rat (Uniprot: 070156), rabbit (Uniprot: Q9XTA8)
and cynomolgus monkey LOX1 were designed with either N or C
terminal Flag and histidine tags and cloned into Gateway
destination vectors (Invitrogen). The constructs were then
transfected into mammalian HEK293 EBNA cells for protein
expression. The proteins then underwent standard affinity and size
exclusion chromatography purification. Briefly, human
(HisFlag)-LOX1, cynomolgus LOX1-(FlagHis), mouse LOX1-(FlagHis),
rat LOX1-(FlagHis) and rabbit LOX1-(FlagHis) were coated at 10, 10,
5, 5 and 5 .mu.g/mL in PBS buffer respectively on MAXISORP.TM.
plates. Efficient antigen coating using such concentrations was
first checked by ELISA using commercial anti-human LOX1 (23C11 from
Hycult) or anti-His (Europium-labeled from Perkin Elmer)
antibodies. After blocking the wells with PBS containing 3% dried
milk, purified anti-LOX1 scFvs in PBS plus 3% dried milk were
incubated with the different coated antigens for 1 hour. Bound scFv
molecules were detected using a secondary Europium labeled anti-Myc
tag antibody (Perkin Elmer) at 100 ng/mL. Plates were read for
fluorescence using a 340 nm excitation and 615 nm emission.
Non-specific binding was determined on bovine serum albumin (New
England Biolabs) coated at 10 .mu.g/mL.
[0335] As shown in FIG. 3A, LOX514 ("LOX10514") and LOX696
("LOX10696") bind to human and cynomolgus LOX1 but not to mouse,
rat or rabbit LOX1 orthologs. The lack of antibody cross-reactivity
to mouse LOX1 is not surprising given the low homology across the C
type lectin domain between these species (.about.62% identity
between human and mouse).
[0336] The specificity of the anti-human LOX1 antibody molecules to
other human C type lectin and scavenger receptor related molecules
was assessed by an IgG binding ELISA as described in Example 10,
Assay 6. As shown in FIG. 3B, LOX514 ("LOX10514") and LOX696
("LOX10696") bind only to human LOX1 and do not bind to human
CLEC-7A, CLEC-1A, CLEC-4L, CLEC-1B, SR-A1 and SR-B3. That result
demonstrates the specificity of LOX514 and LOX696 to LOX1.
Example 4--Isolation and Identification of Potency Optimized
Anti-LOX1 scFv Antibodies by Targeted CDR Randomization and
Recombination of LOX514
[0337] LOX514 was optimized using affinity-based phage selections.
Large scFv libraries derived from the LOX514 scFv sequence were
created by oligonucleotide-directed mutagenesis of the variable
heavy (VH) complementarity determining regions 2 or 3 (VH-CDR2 or
VH-CDR3) or variable light (VL) chain complementarity determining
regions 3 (VL-CDR3) using standard molecular biology techniques as
described (Clackson, T. and Lowman, H. B. Phage Display--A
Practical Approach, 2004. Oxford University Press). The libraries
were subjected to affinity-based phage display selections in order
to select variants with a higher affinity to human LOX1. These were
expected to show an improved inhibitory activity for LOX1 binding
to oxLDL and the others LOX1 ligands. The selections were performed
essentially as described previously (Thompson et al., J Mol Biol.
256(1):77-88, (1996)). In brief, the scFv-phage particles were
incubated with recombinant biotinylated human LOX1 in solution
(biotinylated via free amines using EZ LINK.TM. Sulfo-NHS-LC-Biotin
(Thermo/Pierce, product: 21335)). ScFv bound to antigen were then
captured on streptavidin-coated paramagnetic beads (DYNABEADS.RTM.
M-280) following manufacturer's recommendations. The selected
scFv-phage particles were then rescued as described previously
(Osbourn et al., Immunotechnology, 2(3):181-96, (1996)), and the
selection process was repeated in the presence of decreasing
concentrations of biotinylated human LOX1 (a typical example would
be 50 nM to 20 pM over four rounds).
[0338] Crude scFv-containing periplasmic extracts were prepared for
a representative number of individual scFvs from the CDR-targeted
selection outputs and screened in an epitope competition HTRF.RTM.
(Homogeneous Time-Resolved Fluorescence) assay format against the
parent antibody LOX514 as described in Example 10, Assay 7. Screen
hits, i.e. scFv variants which showed a significantly improved
inhibitory effect when compared to parent LOX514, were subjected to
DNA sequencing, and unique variants from variable heavy CDR2 or
CDR3 and variable light library CDR3 outputs were produced as
purified scFv and tested. Some scFvs were then selected and
converted to IgG1-TM for further characterization. Typical examples
of optimized LOX514 antibodies obtained following this approach
include: LX5140011, LX5140014, LX5140016 and LX5140038 (as
described in Table 1 or FIG. 4).
[0339] To generate further improvement, CDR-randomized selection
outputs comprising of large numbers of scFv variants with the
ability to inhibit the binding of parent LOX514 to human LOX1 were
recombined to form libraries in which clones contained randomly
paired individually randomized VH CDR2 with VH or VL CDR3
sequences.
[0340] Crude scFv-containing periplasmic extracts were prepared of
a representative number of individual scFvs from the recombined
VH2/VH3 or VH2/VL3 libraries in the epitope competition HTRF.RTM.
assay. Screen hits, i.e. scFv variants which showed a significantly
improved inhibitory effect when compared to parent scFv and leads
generated pre-recombination, were subjected to DNA sequencing, and
unique recombined variants were produced as purified scFv and
tested. The most active scFvs were then selected and converted to
IgG1 TM for further characterization. Typical examples of optimized
LOX514 antibodies obtained from these recombined libraries were:
LX5140092, LX5140093, LX5140094, LX5140108 and LX5140110 (as
described in Table 1 or FIG. 4).
[0341] Alignments of the amino acid sequence of optimized heavy
chains for the selected antibodies from the LOX514 lineage are
shown in FIG. 4A. Alignments of the amino acid sequence of
optimized light chains for the selected antibodies from the LOX514
lineage are shown in FIG. 4B.
Example 5--Isolation and Identification of Potency Optimized
Anti-LOX1 scFv Variants Derived from LOX696
[0342] LOX696 was first optimized using affinity-based ribosome
display selection essentially as described by EP494955, U.S. Pat.
No. 5,658,754 and Hanes et al (Thompson et al., J. Mol. Biol.
256(1):77-88 (1996)).
[0343] Large scFv libraries, in ribosome display format, derived
from the LOX696 scFv sequence were created by
oligonucleotide-directed mutagenesis of the variable heavy (VH)
complementarity determining regions 2 or 3 (VH-CDR2 or VH-CDR3) or
variable light (VL) chain complementarity determining regions 3
(VL-CDR3) using standard molecular biology techniques as described
(Thompson et al., J. Mol. Biol. 256(1):77-88 (1996)). On the DNA
level, a T7 promoter was added at the 5'-end for efficient
transcription to mRNA. On the mRNA level, the construct contained a
prokaryotic ribosome-binding site (Shine-Dalgarno sequence). At the
3'-end of the single chain, the stop codon was removed and a
portion of M13 bacteriophage g111 (gene III) was added to act as a
spacer between the nascent scFv polypeptide and the ribosome
(Thompson et al., J. Mol. Biol. 256(1):77-88 (1996)).
[0344] The libraries were subjected to affinity-based ribosome
display selections in order to select variants with a higher
affinity to human LOX1. These were expected to show an improved
inhibitory activity for LOX1 binding to oxLDL and the others LOX1
ligands. The scFvs were expressed in vitro using the RIBOMAX.TM.
Large Scale RNA Production System (T7) (Promega) following the
manufacturer's protocol and a cell-free translation system. The
produced scFv antibody-ribosome-mRNA (ARM) complexes were incubated
in solution with biotinylated human LOX1 (biotinylated via free
amines using EZ LINK.TM. Sulfo-NHS-LC-Biotin (Thermo/Pierce,
product: 21335)). The specifically bound tertiary complexes
(LOX1:ARM) were captured on streptavidin-coated paramagnetic beads
(DYNABEADS.RTM. M-280) following the manufacturer's recommendations
(Dynal) whilst unbound ARMs were washed away. The mRNA encoding the
bound scFvs were then recovered by reverse transcription-PCR
(RT-PCR). The selection process was repeated on the obtained
population for further rounds of selections with decreasing
concentrations of biotinylated human LOX1 (1 .mu.M to a minimum of
2 nM over 4 rounds) in order to enrich for clones with higher
affinity for LOX1. The outputs from selection rounds 2, 3 and 4
were sub-cloned into pCantab6 (1) for bacterial expression as
scFvs, and improved clones were identified as periplasmic extracts
in an LOX696 epitope competition (see Example 10, Assay 7) as
described in Example 4.
[0345] Second-generation phage display libraries were constructed
based on the LOX696 CDR randomized ribosome display outputs or
variants. A total of four libraries were generated: (1) By
recombining VH CDR2 sequences with VL CDR3 sequences both from
round 2 outputs; (2) By performing an additional error-prone PCR on
the recombinated VH CDR2 X VL CDR3 library (see above) to
incorporate further sequence diversity using the DIVERSIFY.TM. PCR
Random Mutagenesis Kit (BD Biosciences) following the
manufacturer's recommendations; (3) By random mutagenesis on the
entire scFv sequence using error-prone PCR as described above using
as template a pool of 9 scFv DNA hits coming from the early
ribosome display libraries plus parental scFv DNA of LOX696 at
equimolar ratio; (4) By soft randomization of the entire VH CDR3 as
described in Gallop M A et al., J Med Chem, 37(9):1233-51(1994)
using as template a pool of 6 scFv DNA hits coming from the early
ribosome display VL CDR3 libraries plus parental scFv DNA of LOX696
at equimolar ratio.
[0346] Phage display selections were performed as described in
Example 4 using decreasing concentrations of biotinylated LOX1
(typically from 100 nM to 2 pM in four rounds). Crude
scFv-containing periplasmic extracts were prepared of a
representative number of individual scFvs from the different 2nd
generation libraries and tested in the epitope competition
HTRF.RTM. (as described in Example 10, Assay 7). ScFv variants
giving the greatest competition of binding were again sequenced,
and purified scFvs were prepared as in Example 1 for testing. The
most potent scFvs from those second generation libraries were
converted to IgG1 TM format for further characterization as
described in later examples.
[0347] Typical examples of optimized LOX696 antibodies obtained
from these 2nd generation libraries include: LX6960067_ngl1,
LX6960071_ngl1, LX6960073_ngl1, LX6960086_ngl1, LX6960094_ngl1,
LX6960101_ngl1, LX6960102_ngl 1 and LX6960116_ngl1 (as described in
Table 1 or FIG. 5).
[0348] Alignments of the amino acid sequence of optimized VH for
the selected antibodies from the LOX696 lineage are shown in FIG.
5A. Alignments of the amino acid sequence of optimized VL for the
selected antibodies from the LOX696 lineage are shown in FIG.
5B.
Example 6--Germlining and Stability Engineering Anti-LOX1
Antibodies as IgG1-TM
[0349] The amino acid sequence of LOX514 VH and VL were aligned to
the known human germline sequences in the VBASE database
(Tomlinson, I., VBASE. 1997, MRC Centre of Protein Engineering,
Cambridge, UK) and the closest germline was identified by sequence
similarity. For the VH domain this was VH1-24 (DP-5) and JH6, for
the VL domain it was V.lamda.1-e (DPL-8) and JL3. There is only one
difference in the two alignments located in the framework 4 of the
heavy chain. That single residue (R105--Kabat numbering) has been
reverted to the germline residue (Q) during the IgG conversion
process by standard molecular biology meaning that all optimized
IgGs from the LOX514 lineage were de-facto produced in the
germlined format. Additionally, a heavy chain N96D mutatagenesis
(Kabat numbering) was performed to remove a potential N-deamidation
motif in the CDR3 of both LX5140092 and LX5140093. Variants were
named LX5140092_N>D and LX5140093_N>D respectively (see Table
1 or FIG. 4). An alignment of the heavy and light chain amino acid
sequences for LX5140092_N>D and LX5140093_N>D is provided in
FIGS. 4A and 4B respectively.
[0350] Similarly, germline analysis of LOX696 antibody was also
performed. The closest germline was identified as VH3-09 (DP-31)
and JH3 for the heavy chain and V.lamda.2a2 (DPL-11) and JL3 for
the light chain. Excluding Vernier residues (Foote, et al., J Mol.
Biol., 224:487 (1992)), a total of five differences, all in the VH,
were detected: two in framework 1, one in framework 3 and two in
framework 4. Differences in optimized LOX696 IgG1-TM sequences were
reverted to the closest germline residues at the exception of V89
by standard molecular biology techniques: Q1E, Q6E, R105Q and
T108M. Additionally, a heavy chain G82bS mutagenesis (Kabat
numbering) was performed to remove a potential N-deamidation motif
in the framework 3 of germlined LX6960073_gl (see Table 1 or FIG.
5). The variant was named LX6960073_G82bS_gl (see Table 1 or FIG.
5). An alignment of the heavy and light chain amino acid sequences
for the non germlined LX6960073_ngl1, germlined LX6960073_g1 and
LX6960073_G82bS_gl is provided in FIGS. 5A and 5B respectively.
Example 7--Specificity and Species Cross Reactivity of Optimized
Anti-LOX1 Antibodies
[0351] The specificity of optimized anti-human LOX1 antibodies as
IgG1-TM to other human C type lectin and scavenger receptor related
molecules was assessed by an IgG binding ELISA (as described in
Example 10, Assay 7). The panel of related molecules included human
CLEC-7A (Dectin-1), human CLEC-1A, human CLEC-4L (DC-SIGN), human
CLEC-1B (CLEC-2), human SR-A1 and human SR-B3 (CD36). Human LOX1
was also coated as positive control for binding. The following
optimized antibodies were tested: LX5140108, LX5140110,
LX5140092_N>D, LX5140093_N>D, LX6960073_gl and
LX6960073_G82bS_gl. The isotype human IgG1-TM control antibody
NIP228 was included in the panel of antibodies to test for
determining nonspecific background level.
[0352] As shown in FIG. 6, all optimized anti-LOX1 antibodies bind
human LOX1 but do not bind human CLEC-7A, CLEC-1A, CLEC-4L,
CLEC-1B, SR-A1 or SR-B3, thus demonstrating the specificity of
LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D,
LX6960073_gl and LX6960073_G82bS_gl for human LOX1.
[0353] Cross-reactivity of anti-human LOX1 antibodies to various
LOX1 species orthologs was assessed by an IgG binding ELISA (as
described in Example 10, Assay 7). Briefly, human (HisFlag)-LOX1,
cynomolgus LOX1-(FlagHis), mouse LOX1-(FlagHis), rat LOX1-(FlagHis)
and rabbit LOX1-(FlagHis) were coated at 5 .mu.g/mL in PBS buffer
on MAXISORP.TM. plates. After blocking with PBS containing 3% dried
milk, purified anti-LOX1 IgG1-TM at 10 .mu.g/mL in blocking buffer
were incubated with the different coated antigens. Bound IgG
molecules were detected using a secondary Europium labeled
anti-human IgG antibody (Perkin Elmer) at 100 ng/mL. Plates were
read for fluorescence using a 340 nm excitation and 615 nm
emission. Non-specific binding was determined on CD86 FlagHis
antigen also coated at 5 .mu.g/mL. The following optimized LOX1
antibodies were tested as well as NIP228 human IgG1-TM as a
negative control: LX5140108, LX5140110, LX5140092_N>D,
LX5140093_N>D, LX6960073_gl and LX6960073_G82bS_gl. As shown in
FIG. 7A, all of the tested antibodies (LX5140108, LX5140110,
LX5140092_N>D, LX5140093_N>D, LX6960073_gl and
LX6960073_G82bS_gl) bind to human and cynomolgus LOX1 but not to
mouse or rat LOX1 proteins. Some of those antibodies (LX5140108,
LX5140110 and LX5140092_N>D) also bind to rabbit LOX1 but to a
lesser extent than to human or cynomolgus LOX1 as demonstrated by
the smaller fluorescence count.
[0354] Further cross reactivity characterization between human and
cynomolgus LOX1 were performed for the optimized anti-LOX1 IgG1-TM
antibodies LX5140108, LX5140110 and LX6960073_G82bS_gl by
competition ELISA.
[0355] Briefly, biotinylated human LOX1 was coated at 0.125
.mu.g/mL in PBS on Streptavidin plates (Abgene). After blocking
with PBS containing 3% dried milk, purified anti-LOX1 IgG1-TM at
12.5 ng/mL in blocking buffer were co-incubated with competitor
human or cynomolgus LOX1 proteins. A 1:3 titration of competitor in
blocking buffer was used starting at 400 nM for LX5140108 and
LX5140110 and at 204 for LX6960073_G82bS_gl. Bound anti-LOX1 IgG
molecules were detected using a secondary Europium labeled
anti-human IgG antibody (Perkin Elmer) at 100 ng/mL. Plates were
read for fluorescence using a 340 nm excitation and 615 nm
emission.
[0356] As shown in FIG. 7B and in Table 3 below, all three
optimized anti-LOX1 IgG1-TM (LX5140108, LX5140110 and
LX6960073_G82bS_gl) compete with cynomolgus LOX1 and exhibit
IC.sub.50 within 5-fold of the IC.sub.50 for a human LOX1
competition, demonstrating that cynomolgus cross-reactivity of
anti-LOX1 antibodies LX5140108, LX5140110 and LX6960073_G82bS_gl is
strong.
TABLE-US-00003 TABLE 3 Optimized Antibody binding to cynomolgus
LOX1 and human LOX1 IC50 for huLOX1 IC50 for cynoLOX1 Anti-LOX1
antibodies competition (M) competition (M) LX5140108 2.2 .times.
10.sup.-10 .sup. 2.5 .times. 10.sup.-10 LX5140110 2.8 .times.
10.sup.-8 4.4 .times. 10.sup.-8 LX6960073_G82bS_gl 5.5 .times.
10.sup.-10 1.8 .times. 10.sup.-9
Example 8--Affinity of Optimized Anti-LOX1 Antibodies for hLOX1 as
Determined by BIACORE.TM. and KinExA.TM.
[0357] The affinity of the anti-LOX1 antibody for recombinant Human
and Cynomolgus orthologs LOX1 extracellular domain (ECD) was
determined at 3TC using real time interaction monitoring by Biacore
(for Human and Cyno) and at equilibrium using KinExA (for Human
LOX1 affinity measurements).
[0358] In the Biacore assay recombinant protein G+the anti-LOX1
antibody captured by the protein G were immobilized via amine
linking to a CMS chip surface, and the association and dissociation
profiles of the LOX1 orthologs passed over the surface collected.
The assays were performed with the IFC temperature set at 3TC and
the sample compartment left at ambient temperature. All the
experiments were performed with the HBSEP running buffer passing
through the IFC at a constant 30 ul/min. At the end of each LOX1
application any bound antibody and antibody-complex were removed
from the protein G by two 40 seconds pulses of 10 mM glycine pH
1.5. The amount of anti-LOX1 antibody captured on the protein G
surface was tailored to firstly avoid mass transport limitations
and secondly to ensure that sufficient dissociation could be
observed for accurate modeling. The concentration range of the LOX1
interactant was sufficient to ensure that the entire binding range
from saturation of the antibody to no detectable binding was
observed. The data was analyzed using the BiaEval evaluation
software following double reference subtraction of an antibody body
only profile.
[0359] The in-solution equilibrium assay was performed at a single
concentration of anti LOX1 antibody in which a titration of
recombinant Human LOX1 ECD was made from 100 times above to 100
times below the antibody concentration. Free antibody
concentrations were determined via the capture of unoccupied
antibody that was subsequently detected by a fluorescently labeled
human-Fc specific probe. Table 4 details the observed
rate-constants and overall affinities.
TABLE-US-00004 TABLE 4 LX5140110affinity and rate constants
Ligand/LOX1 Assay On-rate (1/Ms) Off-rate (1/s) KD (pM) Human
Biacore 5.75e5 2.30e-4 401 Cynomolgus Biacore 1.09e6 1.68e-4 154
Human KinExA n/a n/a 378 to 587 pM
Example 9 Optimized Anti-LOX1 Antibodies Demonstrate Inhibition of
Multi-Ligand Binding, oxLDL Internalization and oxLDL Induced
Signaling
[0360] Purified IgGs were tested for their ability to specifically
inhibit oxLDL, AGE-BSA and CRP binding to LOX1 (see Example 10;
assays 1, 2 and 3). A number of isolated clones including LX5140110
and LX6960073_G82bS_gl were tested for their ability to block
oxLDL, AGE-BSA and CRP binding to LOX1. Representative plots are
shown in FIGS. 8A, 8B and 8C showing inhibition of oxLDL, AGE-BSA
and CRP binding, respectively, by LX5140110 and LX6960073_G82bS 1.
To confirm the antibodies cross react and had functional activity
on the LOX1 SNP K167N the antibodies were tested for their ability
to block oxLDL binding to LOX1 SNP K167N. Representative plots are
shown in FIG. 8D showing that both LX5140110 and LX6960073_G82bS g1
block oxLDL binding to the LOX1 SNP K167N variant. In addition to
inhibition of binding, a number of isolated clones including
LX5140110 and LX6960073_G82bS_gl were tested for their ability to
block oxLDL internalization on LOX1 expressing cells (see Example
10, Assay 4) and oxLDL induced LOX1 signaling (see Example 10,
Assay 5). Representative plots are shown in FIGS. 8A and 8F showing
inhibition of oxLDL internalization and oxLDL induced LOX1
signaling, respectively, by LX5140110 and LX6960073_G82bS 1. The
results from these studies are summarized in Table 5.
TABLE-US-00005 TABLE 5 Optimized anti-LOX1 antibody inhibition of
oxLDL internalization and signaling LOX514_110 LX6960073_G82bS
oxLDL/hLOX1 CHO Mean 1.04663E-10 2.00052E-11 Competition SD
1.91372E-10 6.29934E-12 n 3 3 oxLDL/K167N hLOX1 CHO Mean 4.92E-10
1.04531E-10 Competition SD 5.40E-10 2.37935E-10 n 7 5 oxLDL/hLOX1
CHO Mean 3.1223E-10 3.80346E-10 Internalisation SD 1.12532E-10
3.32497E-10 n 3 3 AGE BSA/hLOX1 CHO Mean 2.07257E-11 1.76549E-11
Competition SD 1.27853E-10 1.34571E-11 n 3 3 CRP/hLOX1 Mean
5.01529E-09 2.99815E-09 Competition SD 6.37843E-09 1.64015E-09 n 3
3 ROS ASSAY Mean 1.98E-09 2.21E-09 SD 4.47E-10 2.50E-10 n 3 3
[0361] These results demonstrate specific multi-ligand inhibition
of LOX1 binding to oxLDL, AGE-BSA and CRP by antibodies LX5140110
and LX6960073_G82bS_gl and that both LX5140110 and
LX6960073_G82bS_gl functionally cross react with the common LOX1
SNP K167N variant and inhibit oxLDL internalization and oxLDL
induced LOX1 signaling.
Example 10 Assays and Methods
[0362] The following materials and methods were used in the
experiments described in Examples 1-9.
[0363] Materials
[0364] Doxycycline hyclate (Sigma #D9891-1G); CarboxyH2DCFDA
(MolecularProbes #C400); Hoechst stain (Molecular probes #33342);
AGE-BSA (Biovision #2221-10); PBS (Gibco #14190); Recombinant
C-reactive protein (R&D #1707-CRCF); BSA 30% (SIGMA #A9576);
Lightning Link biotin conjugation kit (Innova Biosciences
#704-0010); DyLight 649 NHS ESTER (Thermo Scientific #46416);
Cypher 5E Mono NHS; Ester (GE healthcare #PA15401); HBSS (1X)
(GIBCO #14025); Corning 384 Cell; Bind black assay plate (Corning
Costar #3683); Delfia Enhancement solution (Perkin Elmer
#4001-0010); Europium Streptavidin (Perkin Elmer #1244-360); 0.5 mL
Zeba desalt columns (Pierce #89883); CHO-TREx (Invitrogen
#R718-07); oxLDL (Intracel #RP049); DiI-oXLDL (Intracel #RP-173);
Anti-LOX1 antibody (Biovision #3659); Hoechst stain (Molecular
probes #33342); PBS (Gibco #14190); Growth Medium:
Hams:F12-GlutaMax-I (Gibco #31765-027); supplemented with 10% Fetal
Bovine Serum (Invitrogen #16000-044); Blasticidin (Invitrogen
#46-1120); Zeocin (Invitrogen #R25001); Lipofectamin (Invitrogen
#11668-019); Doxycycline hyclate (Sigma #D9891-1G).
[0365] Reagent Modifications
[0366] Cypher 5E Labelling of oxLDL
[0367] The pH of ox-LDL (1 mg/mL) was adjusted to pH 8.5 using
1/10th volume of 0.5M Sodium Borate buffer and oxLDL was labeled in
accordance with the manufacturers kit instruction's at a molar
ratios of protein:dye of 40:1 (1 hour at room temperature in the
dark). Un-reacted dye was removed using Zeba spin columns according
to manufacturer's instructions (Cypher 5E Mono NHS Ester from GE
healthcare) and buffer exchanged into PBS. The labeled protein was
kept at 4.degree. C. in the dark. For calculation purposes it is
assumed that over 95% of protein was recovered, and there are no
dilution factors to consider.
[0368] DyLight 649 Labelling of oxLDL and AGE-BSA
[0369] The pH of ox-LDL or AGE-BSA (1 mg/mL) was adjusted to pH 8.5
using 1/10th volume of 0.5M Sodium Borate buffer and was labeled in
accordance with the manufacturer's instructions (DyLight 649 NHS
ester from Thermo Scientific for 1 hour at room temperature in the
dark). Un-reacted dye was removed using Zeba spin columns according
to manufacturer's instructions and buffer exchanged into PBS. For
calculation purposes it is assumed that over 95% of protein is
recovered, and there are no dilution factors to consider.
[0370] Dylight 649 Labelling of Human Anti-LOX1 Antibodies
[0371] The pH of the human anti-LOX1 antibody (1 mg/mL) was
adjusted to pH 8.5 using 1/10th volume of 0.5M sodium borate buffer
and was labeled in accordance with the manufacturer's instructions
(DyLight 649 NHS Ester from Thermo Scientific for 1 hour at room
temperature in the dark). Un-reacted dye was removed using Zeba
spin columns according to manufacturer's instructions and buffer
exchanged into PBS. For calculation purposes it is assumed that
over 95% of protein is recovered, and there are no dilution factors
to consider.
[0372] Biotin Labelling of CRP
[0373] C-reactive protein CRP (R&D systems; Carrier free) was
reconstituted in distilled water to a concentration of 4 mg/mL and
biotinylated according to the kit instructions supplied with the
Lightning-Link.TM. Biotin conjugation kit (Type A).
[0374] Human LOX1 CHO-TREX Cell Line 3A9
[0375] The human LOX1 gene (NM 002543) was used to generate the
recombinant plasmid pcDNA4/TO-LOX1 (pAM2037), which was synthesized
and delivered by Geneart. The LOX1 gene was synthesizes with
addition of Afl II restriction site and a Kozak sequence at the
5'-end, and EcoRI restriction site at the 3'-end. pAM2037 was then
transfected into Chinese hamster ovary cells expressing the
tetracycline repressor (CHO-TREx cells) using lipfectamine2000.
CHO-TREx, is a CHO-K cell line that contains pcDNA6/TR that has the
gene for the tetracycline (Tet) repressor, that will block the
expression of genes cloned into the pcDNA4/TO. When these cells are
treated with tetracycline the repression is released and the LOX1
gene will be expressed.
[0376] The transfected mixed population of cells was sorted using a
FACS machine into 96 well plates to obtain a single cell in each
well. The pcDNA6/TR was selected with 10 .mu.g/ml blasticidin, and
300 .mu.g/ml zeocin was used to select for the pcDNA4TO-LOX1
plasmid, and 200 .mu.g/ml for maintenance. The individual cells
were propagated and stable clones were selected by their ability to
bind and internalize DiI-labeled oxLDL, using a High Content
Analysis approach to quantify the fluorescence taken up by the
cells. One clone was selected for uptake of DiI-oxLDL and stability
over several growth passages. LOX1 protein surface expression was
verified by immunostaining using an anti-LOX1 specific antibody
from Biovision. Functional activity of the LOX1 protein was
demonstrated by oxLDL uptake and generation of an oxLDL mediated
ROS response in the CHO-TREx-LOX1 cells.
[0377] Assays 1 and 2--LOX1: oxLDL and LOX1:AGE-BSA Binding
Assays
[0378] Saturation Binding Curve
[0379] Both DyLight 649 labeled oxLDL and AGE-BSA (1 mg/mL) were
diluted 1:50 in HBSS and titrated across a 384 well plate using a
1+1 serial dilution over 16 points in triplicate. For the
determination of total binding 10 .mu.L of HBSS was added to all
wells, followed by the addition of 10 .mu.L of human LOX1
transfected CHO TREX cells (4000 cells per well). For the
determination of nonspecific binding, 10 .mu.L of HBSS was replaced
by 10 .mu.L of unlabeled oxLDL (1 mg/mL). The assay plate was
incubated for 1 hour at room temperature. Following incubation,
plates were read on an Applied Biosystems 8200 cellular detection
system (FMAT plate reader) using PMT1 setting of 422 with a minimum
count set to 10; Colour<0.4 and FL1<5600. Specific binding
was determined by subtracting the mean total binding signal from
the mean non-specific binding signal and plotting the resulting
binding isotherm in Prism Graphpad software using a one site
specific binding algorithm. The calculated concentration at which
KD was determined, was used for subsequent high-throughput
screening and antibody competition experiments.
[0380] Evaluation of Anti-hLOX1 Monoclonal Antibodies
[0381] The competition between anti-LOX1 antibodies and DyLight 649
labeled ligand (oxLDL; AGE-BSA) for binding to LOX1 receptor
expressed on CHO TREX cells was performed as follows.
[0382] Competitor antibodies were used without any pre-dilution and
were titrated across a 384 well plate using a 1+1 serial dilution
in assay buffer (HBSS) over 24 points in duplicate (10 .mu.L per
well). Dylight 649 labeled ligands (at their respective KD
concentrations) were added to all antibody containing wells (10
.mu.L per well) followed by the addition of human LOX1 transfected
CHO TREX cells (4000 cells per well in 10 .mu.L) or K167N SNP
transfected CHO TREX cells.
[0383] The assay plates were incubated for 1 hour at room
temperature. Following incubation, plates were read on an Applied
Biosystems 8200 cellular detection system (FMAT plate reader) using
PMT1 setting of 422 with a minimum count set to 10; Colour<0.4
and FL1<5600. The data was analyzed using a 4-parameter logistic
equation with GRAPHPAD.TM. Prism version 5 (GraphPad Inc,
California) to determine apparent IC50 values.
Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log
IC50-X)*HillSlope)) where X is the logarithm of concentration and Y
is the % specific binding. The IC50 is the sample concentration
that produces 50% inhibition of specific binding.
TABLE-US-00006 TABLE 6 Stock concentrations used in oxLDL and
AGE-BSA binding assays Assay conditions for both HTS and IC.sub.50
Assays Working Solution Con. Final Assay Con. DyLight 649 labeled
8.7 nM 2.9 nM oxLDL DyLight 649 labeled AGE 7.5 .mu.g/mL 2.5
.mu.g/mL BSA hLOX1 transfected CHO 4 .times. 10.sup.5 cells/mL 4000
cells/well TREX cells Peri-prep material for HTS 100% 30% (oxLDL
binding assay) Purified scFv/IgG samples Serial 1 + 1 dilution 30%
for IC.sub.50 analysis of purified proteins over 24 points
[0384] Assay 3--LOX1:CRP Binding Assay
[0385] Saturation Binding Curve
[0386] LOX1 (R&D systems) was immobilized on the surface of a
384 well Nunc Maxisorp plate at a concentration of 3 .mu.g/mL in
PBS, overnight at 4.degree. C. (25 .mu.L per well). The following
day the assay plate was washed 12.times. in PBS (without calcium
and magnesium). A working solution of biotinylated CRP was prepared
at 20 .mu.g/mL (769 nM) in PBS/0.1% BSA/0.01% tween 20, and
titrated across a 384 well plate using a 1+1 serial dilution over
16 points in triplicate (12.54, per well). A further 12.5 .mu.L of
assay buffer (in PBS/0.1% BSA/0.01% tween 20) was added to each
well, and the assay plate was incubated for 2 hours at room
temperature. The plate was washed 16.times. in PBS/0.01% Tween 20,
followed by the addition of 50 .mu.L of Europium labeled
streptavidin (1:1000 dilution). The assay plate was incubated for a
further 1 hour at room temperature followed by washing 16.times. in
PBS/0.01% Tween 20. The plate was developed by adding 100
.mu.L/well of DELFIA enhancement solution, and reading on a Wallac
Victor V plate reader using factory installed DELFA europium
protocol. The resulting binding isotherm was plotted in Prism
Graphpad software using a one site specific binding algorithm. The
calculated concentration, at which the apparent KD was determined,
was used for subsequent high-throughput screening and antibody
competition experiments.
[0387] Evaluation of Anti-Human LOX1 Monoclonal Antibodies
[0388] The competition between antibody and biotin labeled CRP for
binding to recombinant human LOX1 was performed as follows: LOX1
(R&D systems) was immobilized on the surface of a 384 well Nunc
Maxisorp assay plate at a concentration of 3 .mu.g/mL in PBS,
overnight at 4.degree. C. (25 .mu.L per well). The following day
the assay plate was washed 12.times. in PBS (without calcium and
magnesium). Competitor antibodies were used without any
pre-dilution and were titrated across a 384 well dilution plate
using a 1+1 serial dilution in assay buffer (PBS/0.1% BSA/0.01%
tween 20) over 24 points in duplicate (30 .mu.L per well). Biotin
labeled CRP (30 .mu.L per well at KD concentration) was then added
to all antibody containing wells. Samples (50 .mu.L) were then
transferred using a 384 MiniTrak liquid handling robot from the
dilution plate to the assay plate containing immobilized
recombinant human LOX1. Plates were incubated for 2 hours at room
temperature. The plate was washed 16.times. in PBS/0.01% Tween 20,
followed by the addition of 50 .mu.L of Europium labeled
streptavidin (1:1000 dilution). The assay plate was incubated for a
further 1 hour at room temperature followed by washing 16.times. in
PBS/0.01% Tween 20. The plate was developed by adding 100
.mu.L/well of DELFIA enhancement solution, and reading on a Wallac
Victor V plate reader using factory installed DELFA europium
protocol. The data was analyzed using a 4-parameter logistic
equation with GRAPHPAD.TM. Prism version 5 (GraphPad Inc,
California) to determine apparent IC.sub.50 values.
Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log
IC50-X)*HillSlope)) where X is the logarithm of concentration and Y
is the % specific binding. The IC.sub.50 is the sample
concentration that produces 50% inhibition of specific binding.
TABLE-US-00007 TABLE 7 Stock concentrations used in CRP binding
assays Assay conditions for both HTS and IC.sub.50 Assays Working
Solution Con. Final Assay Con. Biotin labeled CRP 784 nM 6.12 nM
Recombinant LOX1 39.2 .mu.M 0.11 .mu.M Europium Labeled 0.1 mg/mL
0.1 .mu.g/mL Streptavidin Purified scFv/IgG Serial 1 + 1 dilution
50% samples for IC.sub.50 of purified proteins analysis over 24
points
[0389] Assay 4--LOX1:oxLDL Internalization Assay
[0390] Saturation Binding Curve
[0391] Cypher 5E labeled oxLDL (40:1 Molar ratio at 1 mg/mL) was
diluted 1:50 in HBSS and titrated across a 384 well plate using a
1+1 serial dilution over 16 points in triplicate. For the
determination of total binding, 10 .mu.L of HBSS was added to all
wells, followed by the addition of 10 .mu.L of human LOX1
transfected CHO TREX cells (4000 cells per well). For the
determination of nonspecific binding, 10 .mu.L of HBSS was replaced
by 10 .mu.L of unlabeled oxLDL (1 mg/mL).
[0392] The assay plate was incubated for 1 hour at 37.degree. C. A
parallel experiment was performed where all reagents are kept at
4.degree. C. in order to demonstrate that internalization was a
metabolically active process. Following incubation, plates were
read on an Applied Biosystems 8200 cellular detection system (FMAT
plate reader) using PMT1 setting of 422 with a minimum count set to
10; Colour<0.4 and FL1<5600. Specific binding was determined
by subtracting the mean total binding signal from the mean
non-specific binding signal and plotting the resulting binding
isotherm in Prism Graphpad software using a one site specific
binding algorithm. The calculated concentration at which KD was
determined, was used for subsequent high-throughput screening and
antibody competition experiments.
[0393] Evaluation of Anti-hLOX1 Monoclonal Antibodies
[0394] The competition between anti-LOX1 antibodies and Cypher5E
labeled ox-LDL for internalization in CHO TREX cells expressing
LOX1 receptor was performed as follows.
[0395] Competitor antibodies were used without any pre-dilution and
were titrated across a 384 well plate using a 1+1 serial dilution
in assay buffer (HBSS) over 24 points in duplicate (10 .mu.L per
well). Dylight 649 labeled ligands (at their respective KD
concentrations) were added to all antibody containing wells (104,
per well) followed by the addition of human LOX1 transfected CHO
TREX cells (4000 cells per well in 10 .mu.L) or K167N SNP
transfected CHO TREX cells.
[0396] The assay plates were incubated for 1 hour at room
temperature. Following incubation, plates were read on an Applied
Biosystems 8200 cellular detection system (FMAT plate reader) using
PMT1 setting of 422 with a minimum count set to 10. Colour<0.4
and FL1<5600. The data was analyzed using a 4-parameter logistic
equation with GRAPHPAD.TM. Prism version 5 (GraphPad Inc,
California) to determine apparent IC.sub.50 values.
Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log
IC50-X)*HillSlope)) where X is the logarithm of concentration and Y
is the % specific binding. The IC.sub.50 is the sample
concentration that produces 50% inhibition of specific binding.
TABLE-US-00008 TABLE 8 Stock concentrations used in oxLDL
Internalization assays Assay conditions for both HTS and IC.sub.50
Assays Working Solution Con. Final assay Con. Cypher5E labeled
oxLDL 9.6 nM 3.2 nM Human LOX1 transfected 4 .times. 10.sup.5
cells/mL 4000 cells CHO TREX cells per well Purified scFv/IgG
Serial 1 + 1 dilution 30% samples for IC.sub.50 analysis of
purified proteins over 24 points
[0397] Assay 5--ROS Induction in Recombinant CHO-LOX1 Cells
[0398] Preparation of Cells
[0399] Human LOX1 transfected CHO TREX cells were induced 24 hours
prior to conducting experiments with a 1 .mu.g/mL (final
concentration) of doxycycline. On the day of the experiment, cells
were scraped and centrifuged for 5 minutes at 1200 rpm. Supernatant
was discarded and the cell pellet was re-suspended in 50 mLs of PBS
and centrifuged at 1200 rpm for 5 minutes. This procedure was
repeated twice. Supernatant was discarded and the cell pellet was
re-suspended in 5 mLs of HBSS. Cells were counted on a
haemocytometer using the Trypan Blue exclusion method. The number
of cells was adjusted to give 4.times.10.sup.5 cells total. Cells
were kept on ice until use.
[0400] Saturation Binding Curve
[0401] Cypher 5E labeled oxLDL (40:1 Molar ratio at lmg/mL) was
diluted 1:50 in HBSS and titrated across a 384 well plate using a
1+1 serial dilution over 16 points in triplicate. For the
determination of total binding, 10 .mu.L of HBSS was added to all
wells, followed by the addition of 10 .mu.L of human LOX1
transfected CHO TREX cells (4000 cells per well). For the
determination of nonspecific binding, 10 .mu.L of HBSS was replaced
by 10 .mu.L of unlabeled oxLDL (1 mg/mL).
[0402] The assay plate was incubated for 1 hour at 37.degree. C. A
parallel experiment was performed where all reagents are kept at
4.degree. C. in order to demonstrate that internalization was a
metabolically active process.
[0403] Following incubation, plates were read on an Applied
Biosystems 8200 cellular detection system (FMAT plate reader) using
PMT1 setting of 422 with a minimum count set to 10; Colour<0.4
and FL1<5600.
[0404] Specific binding was determined by subtracting the mean
total binding signal from the mean non-specific binding signal and
plotting the resulting binding isotherm in Prism Graphpad software
using a one site specific binding algorithm. The calculated
concentration at which KD was determined, was used for subsequent
high-throughput screening and antibody competition experiments.
[0405] CHO-TREx-LOX1 transfected cells were seeded in assay plates
at 7000 cells/well (100 .mu.l/well in media+10% FCS (tetracycline
free)), and incubated overnight 37.degree. C., 5% CO.sub.2. The
following day doxycycline was added at a final concentration of 50
ng/ml per well in order to induce LOX expression and plates were
incubated overnight at 37.degree. C. in a 95% 0215% CO.sub.2
atmosphere. For each screening experiment three separate plates
were prepared, and the anti-LOX antibodies were run in triplicates
with one replicate per plate. The following day, the anti LOX1
antibodies were prepared by serially diluting the antibodies in
warm cell culture media as 2.times. concentrated solutions in a
separate dilution plate. All media was removed from the assay
plates, and 25 .mu.L aliquots of a dilution series of the anti-LOX1
antibodies (2.times. concentrated solution) were added to each well
of the assay plate and incubated for 20 min at 37.degree. C., in a
95% O.sub.2/5% CO.sub.2 atmosphere. This was followed by the
addition of 254 per well of oxLDL (at a fixed final concentration
of 25 .mu.g/ml) and plates were incubated for 60 min at 37.degree.
C. in 95% O.sub.2/5% CO.sub.2.
[0406] Wells were then washed carefully with 1.times.100 .mu.l warm
HBSS/Ca/Mg and 50 .mu.l/well of carboxy-H2DCFDA (1.5 .mu.M in warm
HBSS/Ca/Mg) was thereafter added, and assay plates were incubated
for 30 minutes. During the last 5 min of incubation with
carboxy-H.sub.2DCFDA, cells were counter stained with Hoechst stain
(final concentration of 8.3 .mu.g/ml) in warm HBSS/Ca/Mg, by adding
10 .mu.l 50 .mu.g/ml Hoechst in warm HBSS/Ca/Mg to the
carboxy-H.sub.2DCFDA already present in the wells.
[0407] Assay plates were then washed 2.times. with warm HBSS+Ca/Mg
and read immediately on an Arrayscan high content plate reader
(ThernoFischerScientific, Cellomics) using XF53-Hoechst (Ch1) and
XF53-FITC (Ch2) settings. For image analysis, the Compartemental
BioApplication Algorithm was used, and the fluorescence generated
by the ROS probe was quantified by the CircSpotAvgIntensity
parameter. The competitive data, and resulting IC.sub.50s were
plotted and calculated in ExcelFit v.5.1 using a sigmoidal
dose-response one-site-zero model using a 4 parameter logistic
model (Model 903).
[0408] Assay 6--LOX1 Specificity ELISAs
[0409] The IgG specificity ELISAs were performed essentially as
follows on the LOX1 and LOX1-related molecules listed in Table 9
below. MAXISORB.TM. (NUNC) plates were coated with antigen at 5
.mu.g/mL in PBS and incubated over night at 4.degree. C., except 10
.mu.g/mL was used for human SR-B3. Plates were washed 3.times. with
PBS and blocked with 200 4/well blocking buffer (PBS+3% dried milk)
for 1 hour. After plates were washed 3.times. with PBS, IgGs were
diluted to 0.2 .mu.g/mL in blocking buffer, added at 50 4/well, and
1 incubated for 1 hour. Plates were then washed 3.times. with
PBS-Tween, the detection reagent (anti-human IgG lambda light chain
peroxidase conjugate (Sigma A5175, diluted at 0.23 .mu.g/mL) or
kappa light chain (Sigma A7164, diluted at 0.8 .mu.g/mL)) was added
(50 4/well in blocking buffer), and plates were incubated for 1
hour. Plates were washed 3.times. with PBS-Tween, 50 4/well of TMB
was added, and the plates were left to develop for 5-15 minutes.
The reaction was quenched with 50 4/well 0.1M H2504, and plates
were read on an ENVISION.TM. plate reader, or similar equipment, at
450 nm.
TABLE-US-00009 TABLE 9 Reagents used in LOX1 specificity ELISAs
Reagent Supplier Catalogue Human HisFlag tagged LOX1 MedImmune N/A
Human CLEC-7A (Dectin-1) R&D Systems 1859-DC Human CLEC-1A
R&D Systems 1704-CL Human CLEC-4L (DC-SIGN) R&D Systems
161-DC Human CLEC-1B (CLEC-2) R&D Systems 1718-CL Human SR-A1
(MSR) R&D Systems 2708-MS Human SR-B3 (CD36) R&D Systems
1955-CD
[0410] Assay 7--Epitope Competition
[0411] LOX514 and LOX696 were labeled with DyLight 649 as described
above in the protein modifications section and used as competitor
molecules for both high throughput screening and profiling
alongside ligand binding assays. Labeled antibodies were used at
several concentrations above their respective KD concentrations in
order to make it more difficult for higher affinity unlabeled
antibodies to compete against.
[0412] Determining the K.sub.D of DyLight 649 Labeled LOX514 and
LOX696
[0413] Dylight 649 labeled human anti-LOX1 antibodies were diluted
1:50 in HBSS and titrated across a 384 well plate using a 1+1
serial dilution over 16 points in triplicate. For the determination
of total binding, 10 .mu.L of HBSS was added to all wells, followed
by the addition of 10 .mu.L of human LOX1 transfected CHO TREX
cells (4000 cells per well). For the determination of nonspecific
binding, 10 .mu.L of HBSS was replaced by 10 .mu.L of unlabeled
human anti-LOX1 antibody (lmg/mL).
[0414] The assay plate was incubated for 1 hour at room
temperature.
[0415] Following incubation, plates were read on an Applied
Biosystems 8200 cellular detection system (FMAT plate reader) using
PMT1 setting of 422 with a minimum count set to 10; Colour<0.4
and FL1<5600.
[0416] Specific binding was determined by subtracting the mean
total binding signal from the mean non-specific binding signal and
plotting the resulting binding isotherm in Prism Graphpad software
using a one site specific binding algorithm. Antibodies were used
at multiples over and above the calculated KD concentration.
TABLE-US-00010 TABLE 10 Stock concentrations tor LOX514 and LOX696
epitope competition assays Assay conditions for both HTS and
IC.sub.50 Assays Final assay concentration K.sub.D (.times.KD)
DyLight 649 labeled 0.8 nM 6.5 nM (.times.8) LOX10514 DyLight 649
labeled 3.3 nM 20.8 nM (.times.6.3) LOX10696 Human LOX1 transfected
4 .times. 10.sup.5 cells/mL 4000 cells CHO TREX cells per well
Peri-prep material for HTS 100% 30% (oxLDL binding assay) Purified
scFv/IgG samples Serial 1 + 1 dilution 30% for IC.sub.50 analysis
of purified proteins over 24 points
Example 11--Effects of Anti-LOX1 Antibodies on Endothelial Lipid
Uptake, Cell Signaling and Nitric Oxide Homeostasis in Tissue
Culture and in Human Blood Vessels
[0417] The LOX1 receptor acts as one of the proximate causes of the
atherogenic process by specific ligation and internalization of
OxLDL with subsequent activation of multiple intracellular signal
transduction cascades. See, e.g., Twigg et al., Cardiol Res Pract.
2012:632408 (2012). Activation of the LOX1 receptor in endothelial
cells contributes to endothelial dysfunction including increased
ROS production and impaired nitric oxide (NO) signaling induced, in
part, by arginase 2 upregulation. (See, e.g., Ryoo et al.,
Atherosclerosis 214(2):279-287 (2011), Ryoo et al., Circ Res.
102(8):923-932 (2008); and Ryoo et al., Circ Res. 99(9):951-60
(2006). Additionally, LOX1 receptor activation leads to initiation
and perpetuation of an inflammatory process in the vascular wall
with increased cytokine production and adhesion molecule
expression. The development of a biological inhibitor of the LOX1
receptor would therefore likely be an effective way of attenuating
the development of the atherogenic process. Here, we demonstrate
that LX5140110, blocks uptake of OxLDL by human aortic endothelial
cells (HAECs) in a dose-dependent manner. Additionally, LX5140110
prevents arginase 2 activation, a process previously demonstrated
to be LOX1 dependent. Furthermore, LX5140110 prevents an
Ox-LDL-dependent decrease in NO production and increase in
superoxide (ROS) production. Additionally, using an NFkB luciferase
reporter construct, we demonstrate that LX5140110 significantly
attenuates the activation of NFkB--a master inflammatory regulator
in atherogenesis. See, e.g., Pamukcu et al., Thrombosis Res.
128(2):117-23 (2011). Finally, the LX5140110 antibody prevents the
phosphorylation and activation of Focal Adhesion Kinase (FAK), a
process critical in barrier function in endothelial cells. Thus, we
show that LX5140110 blocks a number of LOX1-dependent processes
that lead to activation of the endothelium and initiate and promote
atherogenesis. The results of these experiments are discussed in
turn.
[0418] A. LX5140110 Blocks the Uptake of OxLDL in HAECs (FIG.
9A)
[0419] Methods:
[0420] OxLDL Conjugation with Alexa Fluor-568
[0421] 500 microliters of human oxidized LDL (1 mg/ml, Intracel,
Frederick, Md.) was labeled with Alexafluor-568 using the
Alexafluor-568 protein labeling kit (Molecular probes, Eugene,
Oreg.) following the manufacturer's protocol. Briefly, OxLDL was
incubated with Alexafluor-568 in 0.1 M bicarbonate (pH 8.3) at room
temperature for one hour. Alexafluor-568-conjugated-OxLDL was then
purified with purification resin column.
[0422] Alexa Fluor-568-Conjugated-OxLDL Uptake by HAEC
[0423] HAEC were seeded onto fibronectin-coated (10 .mu.g/ml)
coverslips for eight hours in serum-containing media before serum
starvation for 18 hours.
[0424] Cells were then incubated with 0, 0.5, 1, 5, or 10 nM of
LX5140110 (("514") or 10 nM of control antibody NIP for one hour in
serum free media. Antibody was washed off completely with fresh
serum-free media before approximately 50 ng/ml of
AlexaFluor568-OxLDL was added to the media for uptake for one hour.
Cells were then permeabilized for 2 min with 0.5% Triton X-100
(Fisher Scientific) in 3% paraformaldehyde (Sigma) followed by
fixation with 3% paraformaldehyde for 20 min. Fixed cells were
labeled with fluorescein phalloidin (Life Technologies, Grand
Island, N.Y.) and DAPI (Life Technologies) and were observed on an
epifluorescence Nikon TE-200 microscope. Images were captured with
a Rolera EMCCD camera (QImaging, Vancouver, Canada) with Volocity
software (PerkinElmer, Lexington, Mass.). Images were further
analyzed with Volocity software by counting the
Alexafluor-568-conjugated-OxLDL red fluorescent particles
(exclusion of particles with size smaller than 0.1 .mu.m2) inside
HAEC cells (images not shown). Approximately 12 images for each set
were acquired and analyzed for the average number of
Alexafluor-568-conjugated-OxLDL red fluorescent particles in each
cell. Dose response data are shown in FIG. 9A (number of vesicles
per cell with standard derivation). As shown in FIG. 9A, 5 nM or 10
nM LX5140110 significantly inhibited OxLDL uptake by HAECs
(p=0.0003 or p=0.0002, respectively).
[0425] B. LX5140110 Attenuates NFkB Signaling in HAECs (FIG.
9B)
[0426] Methods: Confluent 6 well plates of HAECs that were
co-expressing NF.kappa.B-LUCIFERASE and GFP were serum starved for
24 hours before they were subjected to following conditions
(numbers indicate the bars in FIG. 9B from left to right): 1.
Control; 2. OxLDL alone (50 .mu.g/ml); 3. OxLDL+LX5140110 ("514")
(10 nM); 4. OxLDL+NIP (control antibody) (10 nM).
[0427] Antibodies were added 1 hour before OxLDL (50 .mu.g/ml). The
incubation with OxLDL was for an additional 8 hours. Cells were
lysed and subjected to a Luciferase activity assay (Promega), and
luminescence was determined with a FlexStation 3 microplate reader
(Molecular Devices). Briefly, HAECs were lysed with Promega
5.times. lysis buffer and supernatants were plated in white plate
containing 50 .mu.l of Promega substrate in each well. 48 hours
after transfection, cells were lysed in ice-cold modified lysis
buffer consisting of 20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% NP40, 1% sodium deoxycholate, 1 mM
Na.sub.3VO.sub.4, 2.5 mM sodium pyrophosphate, 1 mM
.beta.-glycerophosphate, 1 .mu.g/mL leupeptin, and 1:1000 diluted
protease inhibitor cocktail (Sigma). Loading buffer was added to a
final concentration of 2x, boiled for 5 min, spun for 3 minutes and
loaded in a gel. GFP fluorescence was used as normalization
control. N=5 for each group; * indicates P<0.05 (compared with
untreated control); # indicates P<0.05 (compared with OxLDL+0 nM
ab 514 (LX5140110). As shown in FIG. 9B, addition of 10 mM
LX5140110, but not NIP (control antibody), significantly reduced
OxLDL-dependent NFkB signaling in HAECs. This suggests that
LX5140110 is capable of inhibiting LOX1-dependent NFkB activation,
a signaling pathway that is well established to be downstream of
the LOX-1 receptor. See, e.g., Zhao W, et al., "Lipopolysaccharide
induced LOX-1 expression via TLR4/MyD88/ROS activated
p38MAPK--NF-KB pathway." Vascul Pharmacol. (2014).
[0428] C. LX5140110 Inhibits the Activation of Arginase in HAEC
(FIG. 9C)
[0429] Methods: Confluent 6 well plates of HAEC were serum-starved
for 24 hours before they were subjected to following conditions
(numbers indicate the bars in FIG. 9C from left to right): 1.
Control; 2. OxLDL (50 .mu.g/ml); 3. OxLDL+LX5140110 ("514") (1 nM);
4. OxLDL+LX5140110 ("514") (3 nM); 5. OxLDL+LX5140110 ("514") (10
nM); 6. OxLDL+NIP (10 nM). Antibodies were added 1 hour before HAEC
were incubated in OxLDL (50 .mu.g/ml) for another 3 hours. Cells
were lysed and arginase activity was determined using the urea
assay using .alpha.-isonitrosopropiophenone. Briefly, supernatants
of extracted cell lysates were prepared following incubation with
lysis buffer (50 mM Tris-HCl, pH7.5, 0.1 mM EDTA and protease
inhibitors) for 30 min at 4.degree. C. and centrifugation for 20
minutes at 14,000.times.g at 4.degree. C. Supernatants were then
incubated with 150 mM L-arginine for 1 hour at 37.degree. C. After
1 hour the reaction was stopped using 400 .mu.l acid solution
mixture (H2SO:H3PO4:H2O 1:3:7) and then 25 .mu.l 9%
.alpha.-isonitrosopropiophenone (in 100% EtOH) was added and heated
for 30 min 95.degree. C., and read after 10 min at 540 nm. N=3 for
each group; * indicates that P<0.05 as compared with untreated
controls; # indicates that P<0.05 as compared with 0 nM ab 514
(LX5140110). As shown in FIG. 9C, addition of 3 nM or 10 nM
LX5140110, but not 10 nM NIP (control antibody), significantly
reduced OxLDL-dependent arginase activity in HAECs in a
dose-dependent manner. This further suggests that LX514110 inhibits
LOX1-dependent activation of arginase 2, a downstream signaling
pathway that has recently been shown to be coupled to LOX-1 in
vascular endothelium. See, e.g., Ryoo et al., Atherosclerosis
214:279-87 (2011).
[0430] D. LX5140110 Blocks OxLDL-Dependent Reduction in Nitric
Oxide Production (FIG. 9D)
[0431] Methods: NO production was determined using DAF
fluorescence. Briefly, to measure NO, HAEC cells were plated into
white 96-well plates (ThermoLabsystems) at a density of
approximately 5.times.104 cells per well and serum-starved (1%
serum) overnight. Cells investigated in 7 experimental groups
(numbers indicate the bars in FIG. 9D from left to right): 1.
Control; 2. OxLDL (50 .mu.g/ml); 3. OxLDL+LX5140110 ("514") (0.5
nM); 4. OxLDL+LX5140110 ("514") (1 nM); 5. OxLDL+LX5140110 ("514")
(5 nM); 6. OxLDL+LX5140110 ("514") (10 nM); 7. OxLDL+NIP (10 nM).
Antibodies were added 1 hour before HAEC were incubated in OxLDL
(50 .mu.g/ml) for 24 hours. Medium was then removed and the cells
were placed at 37.degree. C. in EBM2 media containing DAF-FM DA (5
.mu.M) for 30 min at 37.degree. C. Medium was then replaced with
fresh media and the cells were incubated for another 20 min prior
to measuring total fluorescence with a FlexStation 3 microplate
reader (Molecular Devices) at excitation 495 nm and emission 515
nm. To confirm that NO was produced by eNOS, the NOS inhibitor
L-NAME was used as a control (data not shown). N=5 for each group;
* indicates that P<0.05 (compared with untreated control). As
shown in FIG. 9D, addition of 5 nM or 10 nM LX5140110, but not 10
nM NIP (control antibody), significantly inhibited in a
dose-dependent manner OxLDL-dependent reduction in nitric oxide
production of HAECs.
[0432] E. LX5140110 Blocks OxLDL-Dependent Increases in ROS
Production (FIGS. 9E and 9F)
[0433] Methods: Superoxide production was determined using the
Luminol analog L-012. To measure superoxide (ROS), HAEC cells were
plated into white TC-treated 96-well plates (ThermoLabsystems) at a
density of approximately 5.times.10.sup.4 cells per well and serum
starved (1% serum) for overnight. The 7 study populations were as
follows (numbers indicate the bars in FIG. 9E from left to right):
1. Control; 2. OxLDL (50 .mu.g/ml); 3. OxLDL+LX5140110 ("514") (0.5
nM); 4. OxLDL+LX5140110 ("514") (1 nM); 5. OxLDL+LX5140110 ("514")
(5 nM); 6. OxLDL+LX5140110 ("514") (10 nM); 7. OxLDL+NIP (10 nM).
Antibodies were added 1 hour before HAEC were incubated in OxLDL
(50 .mu.g/ml) for 24 hours. Medium was then removed and the cells
were further incubated at 37.degree. C. in phenol free DMEM (Sigma)
containing 400 .mu.M of the luminol analogue L-012 (Wako) for a
minimum of 20 minutes prior to the addition of agonists.
Luminescence was quantified over time using a FlexStation 3
microplate reader (Molecular Devices). The specificity of L-012 for
reactive oxygen species was confirmed by co-incubation with the
superoxide scavenger SOD (5 mM), and this yielded virtually
undetectable levels of luminescence under control or
OxLDL-stimulated conditions (see FIG. 9F). Relative light units
(RLU) quantified from the luminescence of L-012 therefore indicate
changes in production of superoxide. To confirm that superoxide was
produced by eNOS, the NOS inhibitor L-NAME was used as a control.
N=5 for each group. As shown in FIGS. 9E and 9F, addition of 0.5
nM, 1 nM, 5 nM or 10 nM LX5140110, but not 10 nM NIP (control
antibody), inhibited OxLDL-dependent ROS production in HAECs. Thus,
LX5140110 prevents OxLDL-mediated eNOS uncoupling and subsequent
increase in ROS production by preventing LOX-1 activation and
downstream activation of arginase 2. See, e.g., Ryoo et al.,
Atherosclerosis 214:279-87 (2011).
[0434] F. LX5140110 Blocks OxDL Mediated Phosphorylation of Focal
Adhesion Kinase (FAK), Y397 (FIGS. 9G and 9H).
[0435] Methods: HAECs were cultured with ECM media (ScienCell,
Carlsbad, Calif.) with 5% serum for one day before serum-starvation
for 18 hours. LX5140110 or the control antibody NIP was then added
to cells at the concentration indicated in FIG. 9G and incubated
for one hour. Cells were washed with fresh media to remove
antibodies before they were incubated with 50 .mu.g/ml of OxLDL for
one hour. Cells were washed with ice-cold PBS buffer and then
protein was extracted using modified RIPA buffer (0.1% DOC, 0.1%
Trition X-100, 2 mM EDTA, 1 mM PMSF, 2 mM sodium vanadate, 20 mg/ml
leupeptin, and 20 mg/ml aprotinin in PBS). Cell lysates were
clarified by centrifugation at 15,000.times.g at 4.degree. C. for
10 minutes. Protein concentration was then quantified by the
bicinchoninic assay (Pierce, Rockford, Ill.). 10 .mu.g of protein
lysates were mixed with 2.times. Laemmli sample buffer, boiled, and
then subjected to SDS-PAGE using 4-15% gradient polyacrylamide
gels, followed by transfer to nitrocellulose membranes for Western
blotting.
[0436] Primary antibodies used in the experiments described in
FIGS. 9G and 9H include anti-pY397-FAK (rabbit polyclonal, Life
Technologies, Grand Island, N.Y.) and anti-GAPDH (mouse monoclonal,
Novus Biologicals, Littleton, Colo.). Horseradish
peroxidase-conjugated anti-mouse and anti-rabbit secondary
antibodies were obtained from ICN Biochemicals, Inc. (Costa, Mesa,
Calif.).
[0437] A Student t-test was used to analyze the differences in FAK
phosphorylation on Tyr397 in control, OxLDL-treated, and
antibody-preincubated cells. P values are supplied in FIG. 9H and
significance (*) was adjudged to be present at p values less than
0.05 for all data. The figures include standard error bars. A
representative blot showing changes in FAK phosphorylation at
Tyr397 and expression of GAPDH in HAECs is shown in FIG. 9G, while
quantification of the percentage increase of FAK phosphorylation at
Tyr397 (pY397-FAK) normalized to GAPDH expression is shown in FIG.
9H. These results demonstrate that addition of 5 nM or 10 nM
LX5140110, but not 10 nM NIP (control antibody), significantly
inhibited OxLDL-dependent mediated FAK phosphorylation at Tyr397 in
HAECs. * indicates that P<0.05 (compared with untreated
control); # indicates that P<0.05 as compared with 0 nM
LX5140110 (514). This data suggests that LX5140110 inhibits
OxLDL-mediated induction of LOX-1 dependent FAK phosphorylation, a
process that regulates endothelial cytoskeletal functions including
cell-cell and cell-matrix adhesion with subsequent effects on
endothelial barrier function.
[0438] G. LOX1 is the Primary Receptor Responsible for OxLDL
Signaling in HAECs (FIGS. 91, 9J and 9K).
[0439] Methods: Construction of LOX1 shRNA Adenoviruses:
[0440] Ad-shNontargeted- and Ad shLOX1-encoded viruses were
generated using a pAdBLOCK-iT kit (Life Sciences). Briefly,
oligonucleotides that were nontargeted, and other targeting 5'UTR
region of Human LOX1 were designed with proprietary software from
Life Sciences and cloned into pU6-ENTR. Sequences used were as
follows. Non targeted: Top, 5'-CAC CGA TGG ATT GCA CGC AGG TTC TCG
AAA GAA CCT GCG TGC AAT CCA TC-3' (SEQ ID NO:79); Bottom, 5'-AAA
AGA TGG ATT GCA CGC AGG TTC TTT CGA GAA CCT GCG TGC AAT CCA TC-3'
(SEQ ID NO:80). LOX1sh: Top, 5'-CAC CGC TTC ACT CTC TCA TTC TTA GCG
AAC TAA GAA TGA GAG AGT GAA GC-3' (SEQ ID NO:81); Bottom, 5'-AAA
AGC TTC ACT CTC TCA TTC TTA GTT CGC TAA GAA TGA GAG AGT GAA GC-3'
(SEQ ID NO:82).
[0441] The resulting pU6-sh-Nontargeted and pU6-LOX1shRNA plasmids
were tested for function in transient transfection experiments with
HAEC cells. The constructs showing the greatest inhibition were
recombined with pAD/BLOCK-iTDEST (Invitrogen) to generate
pAd-Nontargeted and Ad-shLOX1. Viruses were amplified, purified,
and concentrated using a Millipore Kit.
[0442] To confirm that LOX1 is necessary for OxLDL signaling in
HAECs, LOX1 RNA expression was inhibited using a viral vector
expressing interfering short hairpin RNAs (shRNA) directed to the
5'UTR region of the human LOX1 gene (LOX1shRNA). Briefly, 60
percent confluent HAECs were transduced with 25 MOI (multiplicity
of infection, a measure of virulence) of adenovirus expressing
LOX1shRNA ("LOX1-shRNA" or "Ad-LOX-1 shRNA"). 24 hours later, media
was replaced with low serum media (1%) containing NF.kappa.B-LUC
encoding viruses. The day after viral transduction, cells were
treated with 50 .mu.g/mL of OxLDL and incubated for an additional 8
hours, followed by luciferase activity measurement by
chemiluminescence. Cell lysates were then subjected to
immunoblotting with anti-Lox-1 or anti-GAPDH antibodies. As shown
in FIGS. 9J and 9K, addition of LOX1shRNA significantly reduced
LOX1 protein expression (see, e.g., lane 3 of FIG. 9J as compared
to lanes 1 and 2) confirming that LOX1shRNA effectively inhibited
LOX1 expression in HAECs. In addition, as shown in FIG. 9I,
AdshLOX1 significantly inhibited OxLDL-mediated NFkB signaling in
HAECs compared to cells incubated with OxLDL and a control,
non-targeted shRNA (Ad-NTsh) construct. * indicates P<0.05
(compared with untreated control); # indicates P<0.05 as
compared with OxLDL+Ad-NTsh.
[0443] A summary of signaling pathways involved in LOX1 receptor
signaling and blocked by the anti-LOX1 antibodies disclosed herein
(including e.g. LX5140110; "514 Ab") is shown in FIG. 10.
[0444] The foregoing description of the specific aspects will so
fully reveal the general nature of the disclosure that others can,
by applying knowledge within the skill of the art, readily modify
and/or adapt for various applications such specific aspects,
without undue experimentation, without departing from the general
concept of the present disclosure. Therefore, such adaptations and
modifications are intended to be within the meaning and range of
equivalents of the disclosed aspects, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance.
[0445] All publications, patents, patent applications, and/or other
documents cited herein are incorporated by reference in their
entirety for all purposes to the same extent as if each individual
publication, patent, patent application, and/or other document were
individually indicated to be incorporated by reference for all
purposes.
Sequence CWU 1
1
8215PRTArtificial SequenceLox514 CDR-1 Heavy Chain sequence 1Glu
Leu Ser Met His1 5217PRTArtificial SequenceLX5140110 CDR-2 Heavy
Chain sequence 2Gly Phe Asp Pro Glu Asp Phe Lys Tyr His Thr His Gln
Lys Phe Gln1 5 10 15Gly320PRTArtificial SequenceLX5140110 CDR-3
Heavy Chain sequence 3Val Trp Gly Thr Gln Gly Lys Gly Val Arg Gly
Trp Asp Tyr Tyr Tyr1 5 10 15Gly Met Asp Val 204129PRTArtificial
SequenceLX5140110 Heavy Chain sequence 4Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu 20 25 30Ser Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Gly Phe Asp
Pro Glu Asp Phe Lys Tyr His Thr His Gln Lys Phe 50 55 60Gln Gly Arg
Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Leu Val Trp Gly Thr Gln Gly Lys Gly Val Arg Gly Trp Asp Tyr
100 105 110Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser 115 120 125Ser517PRTArtificial SequenceLox514 CDR-2 Heavy
Chain sequence 5Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln
Lys Phe Gln1 5 10 15Gly617PRTArtificial SequenceLX5140011 CDR-2
Heavy Chain sequence 6Gly Phe Asp Pro Glu Asp Trp Glu Tyr Ala Tyr
Asp Gln Lys Phe Gln1 5 10 15Gly717PRTArtificial SequenceLX5140014
CDR-2 Heavy Chain sequence 7Gly Phe Asp Pro Glu Asp Tyr Thr Ile Arg
Val Gly Gln Lys Phe Gln1 5 10 15Gly817PRTArtificial
SequenceLX5140016 CDR-2 Heavy Chain sequence 8Gly Phe Asp Pro Glu
Asp Trp Gln Thr His Thr Ala Gln Lys Phe Gln1 5 10
15Gly917PRTArtificial SequenceLX5140038 CDR-2 Heavy Chain sequence
9Gly Phe Asp Pro Glu Asp Trp Thr Ile His Val Asp Gln Lys Phe Gln1 5
10 15Gly1017PRTArtificial SequenceLX5140094 CDR-2 Heavy Chain
sequence 10Gly Phe Asp Pro Glu Asp Trp Gln Tyr His Val Ser Gln Lys
Phe Gln1 5 10 15Gly1117PRTArtificial SequenceLX5140108 CDR-2 Heavy
Chain sequence 11Gly Phe Asp Pro Glu Asp Trp Ser Asn His Val Ser
Gln Lys Phe Gln1 5 10 15Gly1217PRTArtificial SequenceLX5140092
CDR-2 Heavy Chain sequence 12Gly Phe Asp Pro Glu Asp Trp Lys Tyr
His Leu Ser Gln Lys Phe Gln1 5 10 15Gly1317PRTArtificial
SequenceLX5140093 CDR-2 Heavy Chain sequence 13Gly Phe Asp Pro Glu
Asp Trp Ala Tyr His Gln Ala Gln Lys Phe Gln1 5 10
15Gly1420PRTArtificial SequenceLX5140011 CDR-3 Heavy Chain sequence
14Pro Asn Gly Gln Gln Gly Lys Gly Val Arg Gly Trp Asp Tyr Tyr Tyr1
5 10 15Gly Met Asp Val 201520PRTArtificial SequenceLX5140108 CDR-3
Heavy Chain sequence 15Ser Thr Gly Arg Gln Gly Lys Gly Val Arg Gly
Trp Asp Tyr Tyr Tyr1 5 10 15Gly Met Asp Val 201620PRTArtificial
SequenceLX5140092_D CDR-3 Heavy Chain sequence 16Pro Asp Gly Thr
His Gln Gly Gly Val Arg Gly Trp Asp Tyr Tyr Tyr1 5 10 15Gly Met Asp
Val 201720PRTArtificial SequenceLX5140092_N CDR-3 Heavy Chain
sequence 17Pro Asn Gly Thr His Gln Gly Gly Val Arg Gly Trp Asp Tyr
Tyr Tyr1 5 10 15Gly Met Asp Val 201820PRTArtificial
SequenceLX5140093_D CDR-3 Heavy Chain sequence 18Pro Asp Gly Gln
Gln Gly Lys Gly Val Arg Gly Trp Asp Tyr Tyr Tyr1 5 10 15Gly Met Asp
Val 2019129PRTArtificial SequenceLX5140011 Heavy Chain sequence
19Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Glu Tyr Ala Tyr Asp
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser20129PRTArtificial SequenceLX5140014 Heavy Chain sequence
20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Tyr Thr Ile Arg Val Gly
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser21129PRTArtificial SequenceLX5140016 Heavy Chain sequence
21Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Gln Thr His Thr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser22129PRTArtificial SequenceLX5140038 Heavy Chain sequence
22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Thr Ile His Val Asp
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser23129PRTArtificial SequenceLX5140094 Heavy Chain sequence
23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Gln Tyr His Val Ser
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser24129PRTArtificial SequenceLX5140108 Heavy Chain sequence
24Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Ser Asn His Val Ser
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Leu Thr Ser Thr Gly Arg Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser25129PRTArtificial SequenceLX5140092_D Heavy Chain sequence
25Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Lys Tyr His Leu Ser
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asp Gly Thr His Gln Gly
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser26129PRTArtificial SequenceLX5140092_N Heavy Chain sequence
26Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Lys Tyr His Leu Ser
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Thr His Gln Gly
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser27129PRTArtificial SequenceLX5140093_D Heavy Chain sequence
27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Ala Tyr His Gln Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asp Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser28129PRTArtificial SequenceLX5140093_N Heavy Chain sequence
28Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu
Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Trp Ala Tyr His Gln Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys
Gly Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser29129PRTArtificial SequenceLox514 Heavy Chain sequence 29Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu
20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln
Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Thr Pro Asn Gly Gln Gln Gly Lys Gly
Val Arg Gly Trp Asp Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp Gly
Arg Gly Thr Thr Val Thr Val Ser 115 120 125Ser3014PRTArtificial
SequenceLox514 CDR-1 Light Chain sequence 30Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Asp Val His1 5 10317PRTArtificial
SequenceLox514 CDR-2 Light Chain sequence 31Gly Asn Ser Asn Arg Pro
Ser1 53211PRTArtificial SequenceLox514 CDR-3 Light Chain sequence
32Gln Ser Tyr Asp Ser Ser Leu Ser Gly Trp Val1 5
1033111PRTArtificial SequenceLX5140110 Light Chain sequence 33Gln
Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu 35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Ser Ser 85 90 95Leu Ser Gly Trp Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 100 105 1103410PRTArtificial SequenceLX5140094
CDR-3 Light Chain sequence 34Gln Ser Tyr Asp Ser Met Tyr Arg Phe
Gly1 5 103511PRTArtificial SequenceLX5140093 CDR-3 Light Chain
sequence 35Gln Ser Tyr Asp Ser Ser His Arg Ala Trp Ala1 5
1036110PRTArtificial SequenceLX5140094 Light Chain sequence 36Gln
Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu 35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Ser Met 85 90 95Tyr Arg Phe Gly Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 11037111PRTArtificial SequenceLX5140093
Light Chain sequence 37Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser
Ser Asn Ile Gly Ala Gly 20 25
30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg
Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr
Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Tyr Asp Ser Ser 85 90 95His Arg Ala Trp Ala Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110385PRTArtificial SequenceLox696 CDR-1
Heavy Chain sequence 38Asp Tyr Ala Met His1 53917PRTArtificial
SequenceLox696 CDR-2 Heavy Chain sequence 39Gly Ile Ser Trp Asn Ser
Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys1 5 10
15Gly4011PRTArtificial SequenceLX6960073_G82bS_gl CDR-3 Heavy Chain
sequence 40Glu Gly Ser Trp Asn Tyr Asp Ala Leu Asp Ile1 5
1041120PRTArtificial SequenceLX6960073_G82bS_gl Heavy Chain
sequence 41Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser
Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Ser Trp Asn
Tyr Asp Ala Leu Asp Ile Trp Gly Gln 100 105 110Gly Thr Met Val Thr
Val Ser Ser 115 1204217PRTArtificial SequenceLX6960067_ngl1 CDR-2
Heavy Chain sequence 42Gly Val Ser Leu Gln Glu Leu Tyr Thr Gly Tyr
Ala Asp Ser Val Lys1 5 10 15Gly4317PRTArtificial
SequenceLX6960086_ngl1 CDR-2 Heavy Chain sequence 43Gly Ile Ser Trp
Asn Ser Pro Asp Arg Tyr Met Asp Asp Ser Val Lys1 5 10
15Gly4411PRTArtificial SequenceLox696 CDR-3 Heavy Chain sequence
44Glu Gly Asn Trp Asn Tyr Asp Ala Phe Asp Ile1 5
104511PRTArtificial SequenceLX6960067_ngl1 CDR-3 Heavy Chain
sequence 45Glu Gly Ser Trp Asn Tyr Asp Ala Phe Asp Ile1 5
104611PRTArtificial SequenceLX6960073_ngl1 CDR-3 Heavy Chain
sequence 46Glu Gly Ser Trp Asn Tyr Asp Ala Leu Asp Ile1 5
104711PRTArtificial SequenceLX6960101_ngl1 CDR-3 Heavy Chain
sequence 47Glu Gly Asn Trp Asn Tyr Asp Ala Phe Asp Val1 5
1048120PRTArtificial SequenceLX6960067_ngl1 Heavy Chain sequence
48Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Gly Val Ser Leu Gln Glu Leu Tyr Thr Gly Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Gly Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Ser Trp Asn Tyr Asp
Ala Phe Asp Ile Trp Gly Arg 100 105 110Gly Thr Thr Val Thr Val Ser
Ser 115 12049120PRTArtificial SequenceLX6960071_ngl1 Heavy Chain
sequence 49Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asp Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Asn Trp Asn
Tyr Asp Ala Phe Asp Ile Trp Gly Arg 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 12050120PRTArtificial SequenceLX6960073_ngl1 Heavy
Chain sequence 50Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val
Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Ser Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser
Ile Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Gly Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Ser
Trp Asn Tyr Asp Ala Leu Asp Ile Trp Gly Arg 100 105 110Gly Thr Thr
Val Thr Val Ser Ser 115 12051120PRTArtificial
SequenceLX6960086_ngl1 Heavy Chain sequence 51Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly
Ile Ser Trp Asn Ser Pro Asp Arg Tyr Met Asp Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Gly Asn Trp Asn Tyr Asp Ala Phe Asp Ile Trp Gly
Arg 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12052120PRTArtificial SequenceLX6960101_ngl1 Heavy Chain sequence
52Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Gly Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Asn Trp Asn Tyr Asp
Ala Phe Asp Val Trp Gly Arg 100 105 110Gly Thr Thr Val Thr Val Ser
Ser 115 12053120PRTArtificial SequenceLX6960073_gl Heavy Chain
sequence 53Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser
Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Gly Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Ser Trp Asn
Tyr Asp Ala Leu Asp Ile Trp Gly Gln 100 105 110Gly Thr Met Val Thr
Val Ser Ser 115 12054120PRTArtificial SequenceLox696 Heavy Chain
sequence 54Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Asn Trp Asn
Tyr Asp Ala Phe Asp Ile Trp Gly Arg 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 1205514PRTArtificial SequenceLX6960073_G82bS_gl
CDR-1 Light Chain sequence 55Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr Asn Tyr Val Ser1 5 10567PRTArtificial
SequenceLX6960073_G82bS_gl CDR-2 Light Chain sequence 56Asp Val Ser
Lys Arg Pro Ser1 55711PRTArtificial SequenceLX6960073_G82bS_gl
CDR-3 Light Chain sequence 57Met Gly Gly Met Gly Arg Ser Thr Asn
Trp Val1 5 1058111PRTArtificial SequenceLX6960073_G82bS_gl Light
Chain sequence 58Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly
Ser Pro Gly Gln1 5 10 15Pro Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser
Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Lys Arg Pro
Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr
Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Met Gly Gly Met Gly Arg 85 90 95Ser Thr Asn Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
1105914PRTArtificial SequenceLX6960116_ngl1 CDR-1 Light Chain
sequence 59Thr Gly Thr Ser Asn Asp Val Gly Gly Tyr Asn Tyr Val Ser1
5 10607PRTArtificial SequenceLox696 CDR-2 Light Chain sequence
60Asp Val Ser Asn Arg Pro Ser1 56111PRTArtificial SequenceLox696
CDR-3 Light Chain sequence 61Ser Ser Tyr Thr Ser Ser Ser Thr Asn
Trp Val1 5 106211PRTArtificial SequenceLX6960067_ngl1 CDR-3 Light
Chain sequence 62Leu Gly Arg Thr Trp Ser Ser Thr Asn Trp Val1 5
106311PRTArtificial SequenceLX6960071_ngl1 CDR-3 Light Chain
sequence 63Met Gly Ser Met Gly Arg Ser Thr Asn Trp Val1 5
106411PRTArtificial SequenceLX6960094_ngl1 CDR-3 Light Chain
sequence 64Ala Gln Arg Thr Val Ser Ser Thr Asn Trp Val1 5
1065111PRTArtificial SequenceLX6960067_ngl1 Light Chain sequence
65Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Leu Gly Arg Thr Trp Ser 85 90 95Ser Thr Asn Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 11066111PRTArtificial
SequenceLX6960071_ngl1 Light Chain sequence 66Gln Ser Ala Leu Thr
Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Pro Ile Thr Ile
Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile
Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Met Gly Ser Met Gly Arg
85 90 95Ser Thr Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 11067111PRTArtificial SequenceLX6960073_ngl1 Light Chain
sequence 67Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln1 5 10 15Pro Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val
Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Met Gly Gly Met Gly Arg 85 90 95Ser Thr Asn Trp Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 11068111PRTArtificial
SequenceLX6960094_ngl1 Light Chain sequence 68Gln Ser Ala Leu Thr
Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile
Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile
Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Gln Arg Thr Val Ser
85 90 95Ser Thr Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 11069111PRTArtificial SequenceLX6960116_ngl1 Light Chain
sequence 69Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Asn Asp Val
Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Met Gly Ser Met Gly Arg 85 90 95Ser Thr Asn Trp Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 11070111PRTArtificial
SequenceLox696 Light Chain sequence 70Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp
Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp
Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90
95Ser Thr Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 1107117PRTArtificial SequenceLox514 VH
CDR2misc_feature(7)..(7)Xaa is Gly, Trp, Tyr, or
Phemisc_feature(8)..(8)Xaa is Glu, Thr, Gln, Ser, Lys, or
Alamisc_feature(9)..(9)Xaa is Thr, Tyr, Ile, or
Asnmisc_feature(10)..(10)Xaa is Ile, Ala, Arg, or
Hismisc_feature(11)..(11)Xaa is Tyr, Val, Thr, Leu, or
Glnmisc_feature(12)..(12)Xaa is Ala, Asp, Gly, Ser, or His 71Gly
Phe Asp Pro Glu Asp Xaa Xaa Xaa Xaa Xaa Xaa Gln Lys Phe Gln1 5 10
15Gly7220PRTArtificial SequenceLox514 VH
CDR3misc_feature(1)..(1)Xaa is Pro, Ser, or
Valmisc_feature(2)..(2)Xaa is Asn, Thr, Trp, or
Aspmisc_feature(4)..(4)Xaa is Gln, Arg, or
Thrmisc_feature(5)..(5)Xaa is Gln or Hismisc_feature(6)..(6)Xaa is
Gly or Glnmisc_feature(7)..(7)Xaa is Lys or Gly 72Xaa Xaa Gly Xaa
Xaa Xaa Xaa Gly Val Arg Gly Trp Asp Tyr Tyr Tyr1 5 10 15Gly Met Asp
Val 207311PRTArtificial
SequenceLox514 VL CDR3misc_feature(6)..(6)Xaa is Ser or
Metmisc_feature(7)..(7)Xaa is Leu, Tyr, or
Hismisc_feature(8)..(8)Xaa is Ser or Argmisc_feature(9)..(9)Xaa is
Gly, Ala, or no amino acidmisc_feature(10)..(10)Xaa is Trp or
Phemisc_feature(11)..(11)Xaa is Val, Gly, or Ala 73Gln Ser Tyr Asp
Ser Xaa Xaa Xaa Xaa Xaa Xaa1 5 107417PRTArtificial SequenceLox696
VH CDR2misc_feature(2)..(2)Xaa is Ile or Valmisc_feature(4)..(4)Xaa
is Trp or Leumisc_feature(5)..(5)Xaa is Asn or
Glnmisc_feature(6)..(6)Xaa is Ser or Glumisc_feature(7)..(7)Xaa is
Gly, Leu, or Promisc_feature(8)..(8)Xaa is Ser, Tyr, or
Aspmisc_feature(9)..(9)Xaa is Ile, Thr, or
Argmisc_feature(10)..(10)Xaa is Gly or Tyrmisc_feature(11)..(11)Xaa
is Tyr or Metmisc_feature(12)..(12)Xaa is Ala or Asp 74Gly Xaa Ser
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ser Val Lys1 5 10
15Gly7511PRTArtificial SequenceLox696 VH
CDR3misc_feature(3)..(3)Xaa is Asn or Sermisc_feature(9)..(9)Xaa is
Phe or Leumisc_feature(11)..(11)Xaa is Ile or Val 75Glu Gly Xaa Trp
Asn Tyr Asp Ala Xaa Asp Xaa1 5 107614PRTArtificial SequenceLox696
VL CDR1misc_feature(5)..(5)Xaa is Ser or Asn 76Thr Gly Thr Ser Xaa
Asp Val Gly Gly Tyr Asn Tyr Val Ser1 5 10777PRTArtificial
SequenceLox696 VL CDR2misc_feature(4)..(4)Xaa is Asn or Lys 77Asp
Val Ser Xaa Arg Pro Ser1 57811PRTArtificial SequenceLox696 VL
CDR3misc_feature(1)..(1)Xaa is Ser, Leu, Met, or
Alamisc_feature(2)..(2)Xaa is Ser, Gly, or
Glnmisc_feature(3)..(3)Xaa is Tyr, Arg, Ser, or
Glymisc_feature(4)..(4)Xaa is Thr or Metmisc_feature(5)..(5)Xaa is
Ser, Trp, Gly, or Valmisc_feature(6)..(6)Xaa is Ser or Arg 78Xaa
Xaa Xaa Xaa Xaa Xaa Ser Thr Asn Trp Val1 5 107950DNAArtificial
SequenceTop Ad-shNontargeted synthetic primer 79caccgatgga
ttgcacgcag gttctcgaaa gaacctgcgt gcaatccatc 508050DNAArtificial
SequenceBottom Ad-shNontargeted synthetic primer 80aaaagatgga
ttgcacgcag gttctttcga gaacctgcgt gcaatccatc 508150DNAArtificial
SequenceTop LOX1sh synthetic primer 81caccgcttca ctctctcatt
cttagcgaac taagaatgag agagtgaagc 508250DNAArtificial SequenceBottom
LOX1sh synthetic primer 82aaaagcttca ctctctcatt cttagttcgc
taagaatgag agagtgaagc 50
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