BR112017006609B1 - ISOLATED ANTIBODIES OF OXIDIZED LOW DENSITY LIPOPROTEIN RECEPTOR SIMILAR TO LECTIN-1 (LOX1), PHARMACEUTICAL COMPOSITION AND USES OF SAID PHARMACEUTICAL COMPOSITION AND ANTIBODIES - Google Patents

Abstract

LOX1-SPECIFIC BINDING PROTEINS AND THEIR USES. The present disclosure provides LOX1 (LOX1) binding proteins, such as anti-LOX1 antibodies, and compositions and methods of preparing such binding proteins. In certain aspects, the LOX1 binding proteins provided herein inhibit or antagonize the activity of LOX1. Furthermore, the disclosure provides compositions and methods for diagnosing and treating pathologies associated with atherosclerosis, thrombosis, coronary artery disease (CAD), ischemia (e.g., myocardial ischemia), infarction (e.g., myocardial infarction), coronary syndrome acute illness (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 pathologies.

Description

CROSS REFERENCE TO RELATED ORDERS

[001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/058,254, filed on October 1, 2014, which is incorporated herein in its entirety by reference.

REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING

[002] The content of the sequence listing submitted electronically in ASCII text file (Name: LOX1-100WO1_SequenceListing_ascii.txt; Size: 50,926 bytes; and Creation Date: September 30, 2015) deposited with the application is here incorporated in its entirety by reference.

BACKGROUND

[003] The present disclosure provides lectin-like oxidized low-density lipoprotein-1 (LOX1) receptor binding proteins and methods for using such binding proteins, e.g., for the treatment, prevention and/or improvement of a LOX1-associated disease or pathology including, for example, vascular dysfunction, atherosclerosis (plaque progression, rupture and/or thrombosis), coronary artery disease (CAD), ischemia (e.g. myocardial ischemia), infarction (e.g. example, myocardial infarction), stroke and acute coronary syndrome (ACS).

[004] Atherosclerosis is a complex disease that results from the accumulation of lipids, macrophages and fibrous elements in the form of lesions in the arterial wall. Lesions develop into complex plaques that narrow the arterial lumen and are a focus of chronic inflammation. Plaques are susceptible to rupture, promoting thrombosis that results in adverse cardiovascular events including stroke and myocardial infarction. Atherosclerosis is the leading cause of coronary artery disease, stroke and peripheral vascular disease, and therefore represents the most common cause of morbidity worldwide (World Health Organization 2011).

[005] Lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) is a disulfide-linked type II transmembrane protein. It was first identified as an important receptor for 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, a transmembrane domain, a neck domain, and a C-type lectin domain (CTLD), with the structure of the CTLD having been solved (Ohki et al., Structure 13:905- 917 (2005)). Furthermore, LOX1 can be proteolytically cleaved at the neck domain releasing soluble LOX1 (sLOX1). LOX1 is a class E scavenger receptor and binds several ligands including oxLDL, C-reactive protein (CRP), phosphatidylserine, advanced glycation end products (AGEs), small and 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. Several signal transduction pathways are associated with LOX1 activation including RhoA/Rac1, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2, and NFKB. See, for example, Taye et. al., Eur J Clin Invest. 43(7):740-5 (2013).

[006] Preclinical evidence implicates LOX1 in promoting vascular dysfunction, plaque progression, rupture and thrombosis, atherosclerosis and inflammatory pathologies. See, for example, 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 collagen deposition in the vessel wall (Mehta et al., Circ. Res. 100:1634-1642 (2007)), overexpression of LOX1 increased the formation of atherosclerotic plaques (Inoue et al., Circ. Res. 97:176-84 (2005); and White et al., Cardiovascular pathology 20:369-73 (2011)) with LOX1 expression observed at the margins of vulnerable plaques and associated with macrophage accumulation, apoptosis and MMP-9 expression (Li et al., Cir. Cardio. Imaging 3:464-72 (2010)). Neutralizing antibodies against LOX1 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. 69:26371 (2006)). LOX1 expression in humans is not constitutive, but dynamically inducible by pro-inflammatory stimuli. In atherosclerotic plaque, LOX1 is expressed in endothelial cells, smooth muscle cells, and macrophages. Interestingly, serum sLOX1 has been proposed to be diagnostic of plaque instability and rupture in patients with acute coronary syndrome (ACS) (Nakamura et al., J. Pharm. Biomed. Anal. 51:158-163 (2010)); is predictive for ACS recurrence or death (Kume et al., 70th Scientific Sessions of the American Heart Association Ser. 96, 1997); and is associated with an increasing number of complex injuries (Zhao et al., Clin. Cardiol. 34:172-177 (2011)).

[007] Atherosclerosis-related mortality continues to increase due to the increasing prevalence of hypertension, diabetes, dyslipidemia and lifestyle characteristics (such as smoking and obesity) that are risk factors for atherosclerosis. Intervention with standard of care treatments including: platelet inhibitors, antihypertensives, HMG-CoA reductase inhibitors (statins), thrombolytic agents, percutaneous arterial dilation, stent placement, or aorto-coronary bypass surgery had significant clinical benefit. However, despite the use of preventative and treatment strategies, there are still large numbers of patients suffering from secondary major adverse cardiovascular events (MACE). Therefore, there is a need for new therapies that can be used alone or in combination with standard care.

BRIEF SUMMARY

[008] The disclosure provides LOX1 binding proteins and their methods of use. In particular aspects, the LOX1 binding proteins disclosed herein reduce, inhibit or block the binding of LOX1 to one or more of its ligands. In some aspects, LOX1-binding proteins reduce, inhibit, or block the binding of LOX1 to 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 reduced LOX1 expression and/or 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 an associated pathology. with ACS, IM 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 , pre-eclampsia and cancer.

[009] In some aspects, the LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain variable region CDR1 (VH). region"), CDR2 of VH, CDR3 of VH, and CDR1 of light chain variable region (VL), CDR2 of VL and CDR3 of VL, where 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) substitutions , amino acid deletions and/or insertions relative to a reference set of CDRs in which: (a) the VH CDR1 has the amino acid sequence of SEQ ID NO:1; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:5; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:14; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:30; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:31; and (f) the 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 complementarity determining regions (CDRs): heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, and VH region CDR1. light chain (VL) variable, 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 substitutions, amino acid deletions and/or insertions relative to a reference set of CDRs in which: (a) the VH CDR1 has the amino acid sequence of SEQ ID NO:1; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:2; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:3; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:30; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:31; and (f) the VL CDR3 has the amino acid sequence of SEQ ID NO:32.

[0011] In some aspects, the LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain (VH) variable region CDR1, VH CDR2, VH CDR3, and VH variable region CDR1. light chain (VL), 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 substitutions, deletions and /or amino acid insertions relative to a reference set of CDRs in which: (a) the VH CDR1 has the amino acid sequence of SEQ ID NO:38; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:39; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:40; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:55; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:56; and (f) the 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) the VH CDR1 has the amino acid sequence of SEQ ID NO:1; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:2; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:3; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:30; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:31; and (f) the 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) the VH CDR1 has the amino acid sequence of SEQ ID NO:38; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:39; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:44; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:55; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:60; and (f) the 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) the VH CDR1 has the amino acid sequence of SEQ ID NO:38; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:39; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:40; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:55; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:56; and (f) the 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) that has at least 90, 95, 97, 98 or 99% sequence identity with SEQ ID NO:4 and/or a light chain variable region (VL) that has at least 90, 95, 97, 98 or 99% sequence identity with SEQ ID NO:33.

[0016] In some aspects, the LOX1 binding protein comprises a VH that has at least 90, 95, 97, 98 or 99% sequence identity with SEQ ID NO:41 and/or a VL that has at least 90, 95, 97, 98 or 99% sequence identity with SEQ ID NO:58.

[0017] In some aspects, the LOX1 binding protein comprises a heavy chain variable region (VH) that has at least 90, 95, 97, 98 or 99% sequence identity with SEQ ID NOs: 4, 19- 29, 41 or 48-54; and a light chain variable region (VL) that has at least 90, 95, 97, 98 or 99% sequence identity with 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) that has at least 90, 95, 97, 98 or 99% sequence identity and a light chain variable region (VL ) that has at least 90, 95, 97, 98 or 99% sequence identity with a VH and a VL selected from: (a) a VH that has the amino acid sequence of SEQ ID NO:4 and a VL that has 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 in relation to a sequence of reference VH of SEQ ID NO:4, 1928 or 29, and wherein the VL sequence is identical to, or has a total of 6 or less (e.g., 1, 2, 3, 4, 5 or 6) amino acid substitutions, additions and/or deletions in relation to 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 that has the amino acid sequence of SEQ ID NO:54 and a VL that has 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 that has the amino acid sequence of SEQ ID NO:2, 5-12 or 13, and a VH CDR3 that has 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 with respect to a reference VH sequence of SEQ ID NO:41, 48-53 or 54, and wherein the VL sequence is identical to, or has a total of one, two, three, four, five, six, seven, eight or fewer amino acid substitutions, additions and/or deletions relative to 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 which has the amino acid sequence of SEQ ID NO:39, 42 or 43, and a VH CDR3 which has 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 that has 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, Figure 4 or Figure 5 (e.g., a set of CDRs of Lox514, LX5140011, LX5140014, LX5140016, LX5140038, LX5140094, LX5140108, LX5140110, LX5140092, LX5140092_D, 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 a light chain variable region (VL) as described in Table 1, Figure 4 or Figure 5 (for example, a VH and VL of Lox514, LX5140011, LX5140014, LX5140016, LX5140038, LX5140094, LX5140108, LX5140110, LX5140092, LX5140092_D, LX5140093, _D, Lox696, LX6960067_ngl1, LX6960071_ngl1, LX6960073_ngl1, LX6960086_ngl1, LX6960094_ngl1, LX6960101_ngl1, LX6960102_ngl1, LX6960116_ngl1, 960073_gl or LX6960073_G82bs_gl) .

[0028] In some aspects, the isolated LOX1-binding protein comprises a set of complementarity determining regions (CDRs): heavy chain (VH) variable region CDR1, VH CDR2, VH CDR3, and variable region CDR1 light chain (VL), VL CDR2 and VL CDR3, wherein: (a) VH CDR1 comprises the amino acid sequence: E L S M H (SEQ ID NO: 1); (b) the CDR2 of VH 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) the CDR3 of VH comprises the amino acid sequence: HX7 HX8 G HX9 HX10 HX11 HX12 G V R G W D Y Y Y G M D 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) the 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) the VL CDR2 comprises the amino acid sequence: G N S N R P S (SEQ ID NO: 31); and (f) the CDR3 of VL 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 acids), 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 to the same epitope as a LOX-1-binding protein (e.g., an antibody against LOX-1 or fragment thereof) comprising a heavy chain variable region ( VH) and a light chain variable region (VL) described in Table 1, Figure 4 or Figure 5, including a LOX-1 binding protein (e.g., an antibody against LOX-1 or fragment thereof) comprising a sequence of VH 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), which compete with or present cross-competition for LOX1 binding with a LOX-1 binding protein (e.g., an antibody against LOX-1 or fragment thereof) comprising a heavy chain variable region (VH) and a light chain variable region (VL) described in Table 1, Figure 4 or Figure 5, including a LOX-1 binding protein (e.g., an antibody against LOX-1 or fragment thereof) comprising a VH sequence of SEQ ID NO:4 and a VL of SEQ ID NO:33.

[0031] In some aspects, the LOX1 binding protein binds to 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), which compete with or present cross-competition 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 the activity of LOX1. In some aspects, the LOX1-binding protein has at least one property selected from the group consisting of: (a) reduces or inhibits the binding of oxLDL, C-reactive protein (CRP), and/or advanced glycation end products (AGEs) to the LOX1 as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assays 1, 2 and/or 3 or Example 11); (b) decreases or inhibits signaling by RhoA/Rac1, nitrogen monoxide (NO), p38MAPK, protein kinases B and C, ERK1/2 and/or NFKB in an endothelial cell expressing cell surface LOX1, as determined by any assay appropriate, including an assay disclosed herein (see, for example, Example 11); (c) decreases or inhibits the activity of caspase-8, caspase-9 and/or BAX in an endothelial cell expressing cell surface LOX1, as determined by any appropriate assay, including an assay disclosed herein; (d) binds to LOX1 having the K167N single nucleotide polymorphism as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assays 1, 2 and/or 3 or Example 11 ); (e) reduces or inhibits oxLDL internalization as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assay 4, or Example 11); (f) reduces or inhibits oxLDL-induced LOX1 signaling as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assay 5, or Example 11); (g) binds 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 velocity of about 1 x 105 M-1 s-1 to about 6 x 106 M-1 s-1 (e.g., about 5 x105 M-1 s-1 ) as determined by BIACORE; and (i) binds to LOX1 with a Koff velocity of about 1 x 10-4 s-1 to about 3 x 10-4 s-1 (e.g., about 2.3 x 10-4 s- 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 bispecific antibody, a multispecific antibody, or a 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, an F(ab')2 fragment, an Fv fragment, a diabody, and an antibody molecule. single chain.

[0035] In some aspects, the LOX1 binding protein is an antibody that comprises an IgG1 heavy chain immunoglobulin constant region. In some aspects, the constant region of IgG1 comprises a mutation that decreases its effector function. In additional aspects, the IgG1 constant region comprises the L234F/L235E/P331S triple mutation, which results in an IgG1 null for effector function.

[0036] The disclosure provides an isolated nucleic acid or a set of nucleic acids that encode a LOX1-binding protein (e.g., an antibody against LOX-1 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 an NS0 murine myeloma cell, a PER.C6® human cell, or a Chinese hamster ovarian cell (CHO). Also provided are host cells and hybridomas producing LOX1 binding proteins (e.g., an antibody against LOX-1 or fragment thereof).

[0037] The disclosure also provides a method of preparing a 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., an antibody against LOX-1 or fragment thereof) under appropriate conditions and optionally provides a method for isolating the LOX1-binding protein secreted by the host cell or hybridoma. And the disclosure further provides LOX1 binding protein (e.g., an antibody against LOX-1 or fragment thereof) isolated using such methods.

[0038] Pharmaceutical compositions comprising a LOX1-binding protein (e.g., an antibody against LOX-1 or fragment thereof) and a pharmaceutically acceptable carrier are also provided. Methods of treating, preventing and/or ameliorating an associated pathology are also provided. with LOX1, elevated LOX1 activity and/or elevated levels of LOX1 expression, including, for example, atherosclerosis, thrombosis, coronary artery disease (CAD), ischemia (e.g., myocardial ischemia), infarction (e.g., myocardium), 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. 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, LOX1 binding protein is administered as a combination therapy with 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] Methods of treating, preventing and/or improving atherosclerosis are additionally provided. In some cases, 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 who has atherosclerosis. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing atherosclerosis. In some aspects, the subject has a pro-atherogenic pathology. In additional aspects, proatherogenic pathology 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) and/or bacterial infection. Methods for decreasing atherosclerosis are also provided. In some cases, the disclosure provides a method for decreasing atherosclerosis in a subject comprising administering a LOX1 binding protein to a subject who has atherosclerosis.

[0042] Methods of treating, preventing and/or improving thrombosis are also provided. In some cases, the method comprises administering a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) to a subject who has thrombosis. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing thrombosis. In some aspects, thrombosis is an arterial thrombosis. In further aspects, thrombosis is an arterial thrombosis.

[0043] The disclosure also provides methods of treating, preventing and/or improving coronary artery disease (CAD) or a pathology associated with CAD. In some cases, 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 who has DKA. In other aspects, the subject to whom 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 pro-atherogenic pathology. In additional aspects, proatherogenic pathology 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) and/or bacterial infection.

[0044] The disclosure also provides methods of treating, preventing and/or ameliorating ischemia or a pathology associated with ischemia. In some cases, 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 who has ischemia. In other aspects, the subject to whom 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 respects, 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 transplant, dyslipidemia (e.g., hyperlipidemia), inflammation (e.g., chronic inflammation and endotoxin-induced inflammation). ) and/or bacterial infection.

[0045] Methods of treating, preventing and/or improving a heart attack or a pathology associated with a heart attack are also provided. In some cases, 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 a heart attack. In other aspects, the subject to whom the LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) is administered is at risk of developing a heart attack. In some respects, the subject has a myocardial infarction. In other respects, 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 induced inflammation). endotoxin) and/or bacterial infection.

[0046] The disclosure also provides methods of treating, preventing and/or improving acute coronary syndrome (ACS) or a pathology associated with ACS. In some cases, 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 who has ACS. In other aspects, the subject to whom 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 serum levels of soluble LOX1 (sLOX1) or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[0047] The disclosure also provides methods of treating, preventing and/or ameliorating a stroke or a pathology associated with a stroke. In some cases, 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 who has had a stroke. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of having a stroke. In some aspects, the subject has elevated serum levels of sLOX and/or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[0048] Methods of treating, preventing and/or improving a reperfusion injury or a pathology associated with a reperfusion injury are also provided. In some cases, 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 who has reperfusion injury. In other aspects, the subject to whom the LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) is administered is at risk of developing reperfusion injury. In some respects, the subject is about to have surgery. In other respects, the guy had surgery. In some aspects, the surgery is transplant or coronary bypass surgery. In additional aspects, the patient has, or is at risk of developing, myocardial ischemia and reperfusion injury. In additional aspects, the method reduces myocardial injury, reduces serum levels of creatine kinase-isoenzyme MB (CK-MB) and serum malondialdehyde (MDA), reduces the size of cardiomyocytes, reduces leukocyte infiltration at the site of injury and/or reduces cardiac dysfunction (e.g., reduces left ventricular pressure (LVP) and increases left ventricular end-diastolic pressure (LVEDP). In additional aspects, the method increases cardiac stroke volume, fractional shortening, and/or fractional injection.

[0049] The disclosure also provides methods of treating, preventing and/or improving restenosis or a pathology associated with restenosis. In some cases, 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 who has restenosis. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing restenosis. In some respects, the subject is about to have surgery. In other respects, the guy had surgery. In some aspects the surgery is an endovascular procedure, it is vascular surgery, cardiac surgery or angioplasty. In additional aspects, restenosis, the procedure is transplantation or coronary bypass surgery. In additional aspects, the restenosis treated, prevented and/or improved 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 pathology associated with PVD. In some cases, 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 who has PVD. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing PVD. In some aspects, the subject has elevated serum levels of sLOX1 and/or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[0051] The disclosure also provides methods of treating, preventing and/or ameliorating inflammation or a pathology associated with inflammation. In some cases, 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 additional aspects, the subject has chronic inflammation. In some aspects, the subject has elevated serum levels of oxLDL and/or sLOX and/or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[0052] The disclosure also provides methods of treating, preventing and/or ameliorating pre-eclampsia or a pathology associated with pre-eclampsia. In some cases, 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 pre-eclampsia or eclampsia. In additional aspects, the subject has high blood pressure and large amounts of protein in the urine. In some aspects, the subject has elevated serum levels of oxLDL and/or sLOX 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 cases, 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 who needs it. In some aspects, the method reduces signaling from the RhoA/Rac1 signal transduction pathway, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2 and/or NFKB in the plaque. In other aspects, the method reduces apoptosis in the plaque. In additional aspects, the method decreases the activity of caspase 8, caspase 9 and/or BAX, and/or increases the activity of BCL-2 in the plaque. In other aspects, the method decreases the levels of an adhesion molecule or cytokine produced by the plaque. In additional aspects, the method decreases the expression of E-selectin, P-selectin, ICAM-1, VCAM-1, MCP1 and/or CD40/CD40L by the plaque. In additional aspects, the method decreases the size or formation of atherosclerotic plaques, the accumulation of macrophages and/or the expression of MMP (e.g., MMP9) in the atherosclerotic plaque. In additional aspects, the method results in reduced plaque progression or regression.

[0054] Methods of reducing the loss of vascular tone in a subject are also provided. In some cases, 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 who needs it. In some aspects, the method reduces the loss of vascular tone. In additional aspects, the method reduces the loss of vascular tone in a subject by regulating HDL-induced NO production (the ability of the antibody to stimulate endothelial NO production). Methods for improving vascular tone in a subject are further provided. In some cases, the method comprises administering a LOX1 binding protein to a subject in need thereof.

[0055] Methods of treating, preventing and/or improving cancer are additionally provided. In some cases, the disclosure provides a method of treating, preventing and/or ameliorating cancer in a subject comprising administering a LOX1 binding protein (e.g., an anti-LOX1 antibody or fragment thereof) to a subject who has 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] Methods of inhibiting the proliferation, migration or invasion of tumor cells are also provided. In some cases, the disclosure provides a method for antagonizing LOX1 activity comprising placing a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) in contact with a LOX1-expressing tumor cell. 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 of a cancerous lineage.

[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 pathology associated with pathological angiogenesis. In further aspects, the disclosure provides a method of inhibiting angiogenesis comprising placing a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) in contact with a LOX1-expressing cell. 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] Methods of blocking or reducing LOX1 activity are additionally provided. 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. LOX1, such as oxLDL, AGEs and/or CRP. In some aspects, LOX1 is expressed on the surface of an endothelial cell, macrophage, vascular smooth muscle cell and/or platelet. In some aspects, the cell is an endothelial cell, such as a coronary endothelial cell. In further 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 activity of LOX1 is the binding and/or uptake (e.g., internalization) of oxLDL. In additional aspects, the blocked or reduced activity of LOX1 is the induction of the signaling pathway of p38 (MAPK), p44/42 MAPK, protein kinase C (PKC), protein kinase B (PKB), protein tyrosine kinase (PTK), NF-KB and/or AP1 transcription factor. In further aspects, blocked or reduced activity of LOX1 is the induction of apoptosis. In additional embodiments, induction of apoptosis is mediated by caspase-9, caspase-3 and/or Bcl-2. In additional aspects, the blocked or reduced activity of LOX1 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 LOX1 activity induced by the binding of oxLDL to LOX1.

[0059] Methods of blocking or reducing LOX1 activity in a pathological condition associated with increased levels of LOX1 activity or increased levels of LOX1 expression (e.g., serum protein levels of sLOX1) are further provided. In some cases, the method comprises administering a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) to a subject who has increased LOX1 activity or increased levels of LOX1 expression (e.g., protein levels sLOX1 serum levels). In some aspects, the pathologic pathology 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 respects, 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, IL1, 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 K167N variant of LOX1.

[0060] Methods for agonizing or increasing the activity of high-density lipoprotein (HDL) are also provided. In some aspects, the disclosure provides a method for increasing or agonizing 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 respects, increased HDL activity is the promotion of HDL-mediated endothelial NO production. In some aspects, increased HDL activity is inhibition of endothelial cell NFKB signaling activity. In some respects, increased HDL activity is promoting endothelial cell repair. In some ways, increased HDL activity is reduced inflammation.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

[0061] Figure 1 shows the inhibition of the binding of oxLDL, AGE-BSA and CRP to human LOX1 (hLOX1) by the LOX514 and LOX696 antibodies. Binding of DyLight 649-labeled ox-LDL (Fig. 1A) or DyLight 649-labeled AGE-BSA (Fig. 1B) to cells transfected with hLOX1 or binding of biotin-labeled C-reactive protein (CRP) to hLOX1 was measured. Recombinant hLOX1 (Fig. 1C) in the presence of LOX514 ("LOX10514-IgG1-TM") (diamonds), LOX696 ("LOX10696-IgG1-TM") (circles), or 23C11 (squares), a mouse anti-LOX-1 antibody commercially available. Representative graphs are shown in Figures 1A, 1B and 1C illustrating the dose-dependent inhibition of oxLDL, AGE-BSA and CRP binding, respectively, by LOX514 and LOX696. Furthermore, LOX514 and LOX696 also block the binding of DyLight 649-labeled ox-LDL to cells transfected with hLOX1 K167N (Fig. 1D) confirming that these antibodies bind to and block the binding of oxLDL to the LOX1 SNP K167N variant. These results demonstrate a multiligand-specific inhibition of LOX1 binding to oxLDL, AGE-BSA, and CRP by the LOX514 and LOX696 antibodies; and that LOX514 and LOX696 functionally cross-react with the common LOX1 SNP K167N variant. M= molar concentration of the antibody; bars denote standard error.

[0062] Figure 2 shows the inhibition of oxLDL internalization and generation of oxLDL-dependent reactive oxygen species (ROS) by antibodies LOX514 and LOX696. Internalization of Cypher 5E-labeled ox-LDL (Fig. 2A) or oxLDL-dependent ROS generation (Fig. 2B) was measured in cells transfected with human LOX1 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 graphs are shown in Figures 2A and 2B illustrating dose-dependent inhibition of oxLDL internalization and oxLDL-dependent signaling, respectively, by LOX514 and LOX696. In the case of oxLDL-dependent ROS generation in cells transfected with hLOX1, the relative fluorescence units (RFU) of the amount of carboxy-dichlorofluorescein (DCF) generated in the assay with LOX514, LOX696 and 23C11, as an average of three replicates, (Fig. 2B). These results demonstrate that LOX514 and LOX696 antibodies inhibit oxLDL internalization and oxLDL-dependent LOX-1 signaling. M = molar concentration of the antibody; bars denote standard error.

[0063] Figure 3 shows the species cross-reactivity of the anti-LOX1, LOX514 and LOX696 antibodies. We assessed cross-reactivity of anti-human LOX1 antibodies with various LOX1 species orthologs using a scFv binding ELISA. As shown in Figure 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 for other human scavenger and C-type lectin receptors related to LOX-1 was also evaluated using an IgG binding ELISA. As shown in Figure 3B, LOX514 ("LOX10514 IgG1-TM") and LOX696 ("LOX10696 IgG1-TM") bind only to human LOX1 and do not bind to CLEC-7A, CLEC-1A, CLEC-4L, CLEC- 1B, human SR-A1 or SR-B3. As expected, CAT252 IgG1-TM, an isotype control antibody, did not bind to any of the human scavenger and C-type lectin 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 receptors types I and II (also known as MSR, from the English "macrophage scavenger receptor"); SR-B3= Platelet glycoprotein 4 (also known as CD36). Bars denote standard error.

[0064] Figure 4 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 optimized LOX514 and LOX514 antibodies are shown. Differences in relation to VH or VL of LOX514 are highlighted. FW = framework (from the English "framework"); CDR = complementarity determining regions.

[0065] Figure 5 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 optimized LOX696 and LOX696 antibodies are shown. Differences in relation to the VH or VL of LOX696 are highlighted. FW = framework; CDR = complementarity determining regions.

[0066] Figure 6 shows the specificity of anti-LOX1 antibodies for human LOX1 in comparison to various human scavenger and C-type lectin receptors related to LOX-1. The specificity of the anti-LOX1 antibodies LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D, LX6960073_gl ("LX6960073") and LX6960073_G82bS_gl ("LX6960073G82bS") was evaluated in a IgG1-TM in relation to human LOX-1 and others LOX-1-related human scavenger and C-type lectin receptors using an IgG binding ELISA. LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D, LX6960073_gl and LX6960073_G82bS_gl only bind to human LOX1 and do not bind to CLEC-7A, CLEC-1A, CLEC-4L, CLEC-1B, SR-A, 1 or human SR-B3 . NIP228, a human IgG1-TM isotype control antibody, did not bind to human LOX-1 or any of the human scavenger and C-type lectin 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 receptors types I and II (also known as MSR, from the English "macrophage scavenger receptor"); SR-B3= Platelet glycoprotein 4 (also known as CD36). Bars denote standard error.

[0067] Figure 7 shows the cross-reactivity of anti-LOX1 antibodies to various LOX1 species orthologs. We evaluated the cross-reactivity of the anti-LOX1 antibodies LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D, LX6960073_gl and LX6960073_G82bS_gl in an IgG1-TM format towards various LOX1 species orthologs using a binding ELISA of IgG. As shown in Figure 7A, the 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 LOX1 orthologs. ngo or mouse or CD86 (negative control ). Only LX5140108, LX5140110 and LX5140092_N>D also bind to rabbit LOX-1. NIP228, a human IgG1-TM isotype control antibody, did not bind to CD86 or any of the LOX-1 orthologs tested. Furthermore, the characterization of the cross-reactivity between human LOX1 (huLOX) and cynomolgus (cyLOX) was carried out for the anti-LOX1 IgG1-TM antibodies LX5140108, LX5140110 and LX6960073_G82bS_gl ("LX6960073_G82bS") using a competition ELISA. As shown in Figure 7B, LX5140108 (circles), LX5140110 (squares), and LX6960073_G82bS_gl (triangles) compete for both cynomolgus LOX1 and human LOX1, further confirming the cross-reactivity with cynomolgus of the anti-LOX1 antibodies LX5140108, LX5140110, and 6960073_G82bS_gl. Bars denote standard error.

[0068] Figure 8 shows the inhibition of oxLDL, AGE-BSA and CRP binding to human LOX1 (hLOX1), the inhibition of oxLDL internalization and the 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 cells transfected with hLOX1 or binding of biotin-labeled C-reactive protein (CRP) to hLOX1 was measured. Recombinant hLOX1 (Fig. 8C) in the presence of LX5140110-IgG1-TM ("LOX514_110"; 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 graphs are shown in Figures 8A, 8B and 8C illustrating the dose-dependent inhibition of oxLDL, AGE-BSA and CRP binding to hLOX1, respectively, by LX5140110 and LX6960073_G82bS_gl. Furthermore, LX5140110 and LX6960073_G82bS_gl also block the binding of DyLight 649-labeled ox-LDL to cells transfected with hLOX1 K167N (Fig. 8D) confirming that these antibodies bind to and block the binding of oxLDL 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, we measured the internalization of cypher 5E-labeled ox-LDL (Fig. 8E) or the 5E-dependent ROS generation. OXLDL (Fig. 8F) in Human Lox1 Cells in the presence of LX5140110-IGG1-TM ("514_110", Circles Fig. 8E; and "LOX10514_110", Losagos Fig. 8F), or LX6960073_G82BS_GL-IGG1-TM (" 0073_g82bs ", squares Fig. 8E; and "LOX10696_73", squares Fig. 8F). Representative graphs are shown in Figures 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 inhibition by multiple ligands of LOX1 binding to oxLDL, AGE-BSA, and CRP by antibodies LX5140110 and LX6960073_G82bS_gl; (2) that LX5140110 and LX6960073_G82bS_gl functionally cross-react with the common variant LOX1 SNP K167N; and (3) LX5140110 and LX6960073_G82bS_gl inhibit oxLDL internalization and oxLDL-dependent LOX-1 signaling. M= molar concentration of the antibody; bars denote standard error.

[0069] Figure 9A shows the inhibition of OxLDL uptake in human aortic endothelial cells (HAECs) by LX5140110. Uptake of OxLDL-AlexaFluor568 by HAECs incubated with 0, 0.5, 1, 5, or 10 nM of the 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 the binding and internalization of oxidized low-density lipoprotein (OxLDL) by HAECs. Approximately 12 images for each set were analyzed and the average number of red fluorescent vesicles containing Alexafluor-568-conjugated OxLDL in each cell was determined after a 1-hour incubation. The number of vesicles per cell is reported with standard derivation 1. These results demonstrate that LX5140110 5 nM or 10 nM significantly inhibits OxLDL uptake by HAECs (p=0.0003 or p=0.0002 for 5 nM and 10 nM, respectively). * indicates P<0.05 compared to untreated controls (cells incubated with Alexa-Fluor-labeled OxLD alone and no antibody); bars denote standard deviation.

[0070] Figure 9B shows the reduction of OxLDL-dependent NFkB signaling in human aortic endothelial cells (HAECs) by LX5140110. HAECs coexpressing NFKB-luciferase and GFP were serum starved for 24 hours and incubated for 8 hours with: vehicle (control); OxLDL alone (50 μg/ml); OxLDL (50 μg/ml) + LX5140110 ("514") (10 nM); or OxLDL (50 μg/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 to vehicle control only); # indicates P<0.05 (compared to OxLDL alone); bars denote standard error.

[0071] Figure 9C shows the inhibition of the OxLDL-dependent increase in 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 μg/ml); OxLDL (50 μg/ml) + LX5140110 ("514") (1 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (3 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (10 nM); or OxLDL (50 μg/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 P<0.05 compared to vehicle controls only; # indicates P<0.05 compared to OxLDL (50 μg/ml) control (no antibody); bars denote standard deviation.

[0072] Figure 9D shows that LX5140110 blocks the OxLDL-dependent reduction of nitric oxide production by HAECs. HAECs were serum starved (1% serum) overnight and incubated for 24 hours with: vehicle (control); OxLDL (50 μg/ml); OxLDL (50 μg/ml) + LX5140110 ("514") (0.5 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (1 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (5 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (10 nM); or OxLDL (50 μg/ml) + NIP (10 nM) before adding DAF-FM DA (5 μM) into fresh medium and measuring total DAF-FM DA fluorescence. These results demonstrate that the addition of 5 nM or 10 nM LX5140110, but not 10 nM NIP (control antibody), significantly inhibits the OxLDL-dependent reduction of nitric oxide production in 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 P<0.05 (compared to vehicle control only); bars denote standard error.

[0073] Figure 9E shows that LX5140110 blocks OxLDL-dependent increased production of reactive oxygen species (ROS) by HAECs. HAECs were serum starved (1% serum) overnight and incubated for 24 hours with: vehicle (control); OxLDL (50 μg/ml); OxLDL (50 μg/ml) + LX5140110 ("514") (0.5 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (1 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (5 nM); OxLDL (50 μg/ml) + LX5140110 ("514") (10 nM); or OxLDL (50 μg/ml) + NIP (10 nM) before incubating cells with fresh phenol-free medium containing 400 μM of the luminol analog L-012. Relative light units (RLU) quantified from the luminescence of the luminol analogue L-012 indicate changes in ROS production. 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 compared to vehicle controls only; # indicates P<0.05 compared to OxLDL (50 μg/ml) control (no antibody); bars denote standard error.

[0074] Figure 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 thus confirming the specificity of L-012 in measuring superoxide (ROS) in Figure 9E. * indicates that P<0.05 (compared to vehicle); bars denote standard error.

[0075] Figures 9G and 9H show that LX5140110 blocks OxLDL-mediated phosphorylation of focal adhesion kinase (FAK) on 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 the anti-LOX1 antibody LX5140110 (514) or 10 nM of the control antibody (NIP), before incubating the cells for 1 hour in fresh medium with 50 μg/ml of OxLDL and run 10 μg of protein lysates on polyacrylamide gels with a gradient of 4 to 15%. The protein samples subjected to SDS-PAGE were transferred to nitrocellulose membranes for Western blot. A representative blot showing changes in FAK phosphorylation at Tyr397 and GAPDH expression is shown in Figure 9G, while quantification of the percentage increase in FAK phosphorylation at Tyr397 (pY397-FAK) normalized to GAPDH expression is shown in Figure 9am. These results demonstrate that addition of 5 nM or 10 nM LX5140110, but not 10 nM NIP (control antibody), inhibits OxLDL-mediated phosphorylation of FAK at Tyr397 in HAECs. * indicates that P<0.05 (compared to the untreated control); # indicates that P<0.05 compared to LX5140110 (514) 0 nM; bars denote standard error.

[0076] Figure 9I shows that LOX1 is necessary for OxLDL signaling in HAECs. Human aortic endothelial cells (HAECs) were cotransduced with adenovirus encoding NFKB-LUC and a nontargeting shRNA (Ad-NTsh) or shRNA for Lox-1 (Ad-Lox-1sh) to specifically inhibit LOX1 expression. 24 hours after transduction, cells were treated with or without OxLDL (50 μg/ml) and incubated at 37°C for 24 hours. Firefly luciferase activity was measured in cell lysates using chemiluminescence. Ad-LOX-1sh significantly inhibited OxLDL-mediated NFkB signaling in HAECs compared to cells incubated with a control virus with non-targeting shRNA (Ad-NTsh). * indicates P<0.05 (compared to untreated control); # indicates P<0.05 compared to OxLDL + Ad-NTsh.

[0077] Figure 9J shows that a viral vector expressing interfering small hairpin RNAs (shRNA) targeted to the 5' UTR region of the human LOX1 gene (LOX1-shRNA) reduces protein expression of LOX1. LOX1 protein expression was monitored by performing an immunoblot using anti-Lox1 (IB:LOX-1) and anti-GAPDH (IB:GAPDH, used here as protein loading control) antibodies of the following samples: lysates of HAECs transduced with Non-targeting shRNA (NT shRNA) (lanes 1 and 2) or lysates from HAECs transduced with shRNA for Lox-1 (lane 3). Lysates in lanes 2 and 3 were treated with OxLDL (50 μg/ml), whereas 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 nontargeting shRNA (lanes 1 and 2). These data confirm that LOX1-shRNA effectively inhibits LOX1 expression in HAECs.

[0078] Figure 9K shows that a viral vector expressing small interfering hairpin RNAs (shRNA) targeting the 5' UTR region of the human LOX1 gene (Ad-LOX-1shRNA) reduces LOX1 protein expression in a dose-dependent manner . HAECs transduced with increasing concentrations of shRNA for Lox-1 (Ad-LOX-1shRNA) (0, 10, 20, 30, and 100 MOI for lanes 1 to 5, respectively) were stimulated with OxLDL (50 μg/ml) (with exception of the unstimulated control sample shown in lane 1). Cell lysates were obtained and immunoblotted 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] Figure 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. Also provided are related nucleic acids, compositions comprising LOX1-binding proteins, and methods of preparing LOX1-binding proteins. Further provided are methods of using LOX1-binding proteins to, for example, ameliorate diseases or conditions associated with LOX1-binding proteins, such as: atherosclerosis, thrombosis, coronary artery disease (CAD), ischemia (e.g., ischemia of the myocardium), 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, pre- eclampsia or cancer in a subject and diagnostic uses.

[0081] So that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are presented throughout the detailed description.

I. Definitions

[0082] As used in the present specification and the attached claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "one" (or "an"), as well as the terms "one or more", and "at least one", may be used interchangeably here.

[0083] The term “about” is used herein to designate 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 limits above and below the established numerical values or ranges. In general, the term "about" is used herein to modify a numerical value or range above and below the value indicated by a 10% variance.

[0084] Furthermore, "and/or" is to be considered as specific disclosure of each of the two specified characteristics or components with or without the other. Thus, the term "and/or", when 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 otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this disclosure relates. 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 a person skilled in the art with a general dictionary of many of the terms used in the present disclosure.

[0086] Units, prefixes, and symbols are denoted in their form accepted by the Système International de Unites (SI). Numeric ranges include the numbers that define the range. Unless otherwise noted, amino acid sequences are written from left to right in the amino to carboxy orientation. The headings provided herein are not limitations on the various aspects of the disclosure, which may be taken 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] Whenever aspects are described with the language "comprising", other analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[0088] Amino acids are referred to here by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides are referred to by their commonly accepted one-letter codes.

[0089] The terms "LOX1" and "lectin-1-like oxidized low-density lipoprotein receptor" are used interchangeably here and refer to LOX1 and/or biologically active fragments of LOX1. The cDNA and amino acid sequences of the three isoforms of hLOXI are provided in GenBank Accession 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 here and refer to any statistically significant decrease in biological activity, including complete blockage of activity. For example, "inhibition" may refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in the biological activity of LOX1. Thus, when the terms "inhibition" or "suppression" have been used 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, 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 decreasing a LOX1-mediated induced signal transduction in the cell to 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/Rac1, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2, and/or NFKB. In further aspects, the inhibited or blocked LOX1-mediated biological activity is programmed cell death (i.e., apoptosis). In additional embodiments, the decreased, inhibited, or blocked LOX1-mediated biological activity is the increased LOX1-mediated activity of caspase 8, caspase 9, and/or decreased BAX. The cell expressing cell surface LOX1 may be a naturally occurring cell (e.g., human endothelial cells, smooth muscle cells, and macrophages), a cell of a cell lineage, or a recombinant cell produced by introducing a nucleic acid encoding LOX1. in the host cell.

[0091] The terms "antibody" or "immunoglobulin", as used interchangeably herein, include entire antibodies and any antigen-binding fragments 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, C1. The VH and VL regions can be further subdivided into regions of hypervariability, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called framework regions (FW). Each VH and VL is composed of three CDRs and four FWs, arranged from the amino terminus to the 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. Antibody constant regions can mediate immunoglobulin binding 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 in which, for example, an scFv is covalently linked (e.g., via peptide bonds or via a chemical linker) to the N-terminus of the heavy chain and/or the light chain. of a typical antibody, or interspersed in the heavy chain and/or light chain of a typical antibody.

[0092] The term "antibody" may 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, bispecific antibodies, multispecific 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 mutants (scFv), multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant portion of an antibody, and any other modified immunoglobulin molecule comprising a recognition site antigenic as long as the antibodies exhibit the desired biological activity. An antibody can belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of its heavy chain constant domains called 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 with other molecules such as toxins, radioisotopes, etc.

[0094] The term "germlining" means that amino acids in specific positions in an antibody are mutated back to amino acids that occur in the same position as found in the germline.

[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 fragments of antibody. The disclosure further provides LOX1-binding antibody fragments, wherein the antibody fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, or a chain antibody molecule. only.

[0096] A "monoclonal antibody" refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies which 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 mutants (scFv), fusion proteins comprising an antibody moiety, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies prepared 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 complementarity 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 specificity, affinity, and ability desired (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 cases, Fv framework region (FW) residues from a human immunoglobulin are replaced by corresponding residues in an antibody from a non-human species that has the desired specificity, affinity and capacity.

[0098] Humanized antibodies can be further modified by replacing additional residues in the Fv framework region and/or within the substituted non-human residues to refine and optimize the specificity, affinity and/or capacity of the antibodies. 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 corresponding to the non-human immunoglobulin, whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may 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. Patent Nos. 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, alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FW) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs on each chain are held together in close proximity by the FW regions and, with the CDRs on 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 interspecies 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)). Additionally, combinations of these two approaches are sometimes used in the art to determine CDRs.

[00100] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 to 107 of the light chain and residues 1 to 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) incorporated herein by reference). The numbering of amino acid positions as in Kabat refers to the numbering system used for heavy chain variable domains or light chain variable domains of antibody assembly 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 may contain fewer or more amino acids corresponding to a shortening of, or insertion into, an FW or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (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 residue 82 of the heavy chain FW.

[00101] Kabat residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence. Chothia refers instead to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). The end of Chothia's 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 inserts at H35A and H35B; if neither 35A nor 35B are present, the loop ends in 32; if only 35A is present, the loop ends in 33; if both 35A and 35B are present, the loop ends in 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

[00102] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, M. P. et al., Dev. Comp. Immunol. 27: 55-77 (2003), which is incorporated herein 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 easy comparison of variable regions and CDR for all species. According to IMGT numbering schemes, VH CDR1 is in positions 26 to 35, VH CDR2 is in positions 51 to 57, VH CDR3 is in positions 93 to 102, VL CDR1 is in positions 27 to 32, VL CDR2 is in positions 50 to 52 and VL CDR3 is in positions 89 to 97.

[00103] As used in the specification, the described VH CDR sequences correspond to the classical Kabat numbering positions, namely Kabat VH CDR1 is in positions 31 to 35, VH CDR2 is in positions 50 to 65 and CDR3 of VH is in positions 95 to 102. CDR1 of VL, CDR2 of VL and CDR3 of VL also correspond to the classical Kabat numbering positions, namely positions 24 to 34, 50 to 56 and 89 to 97, respectively.

[00104] 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 prepared 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 light chain polypeptides. heavy human.

[00105] The term "chimeric antibodies" refers to antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies derived from a mammalian species (e.g., mouse, rat, etc.) with the desired specificity, affinity, and capacity while the constant regions are homologous to sequences in antibodies derived from another (usually human) to avoid elicitation of an immunological response in that species.

[00106] The terms "TM" or "TM mutant" refer to a mutation in the constant region of IgG1 that results in a decreased effector function (e.g., ADCC) of an antibody that has the mutation. A TM mutant comprises a combination of three mutations, L234F/L235E/P331S, resulting in a null human IgG1 effector (EU numbering in 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.

[00107] The terms "YTE" or "YTE mutant" refer to a mutation in IgG1 Fc that results in increased binding to human FcRn and improves the serum half-life of the antibody that has the mutation. A YTE mutant comprises a combination of three mutations, M252Y/S254T/T256E (EU numbering in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.), introduced in the heavy chain of an IgG1. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. Mutant YTE has been shown to increase the serum half-life of antibodies by about fourfold compared to wild-type versions of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514- 24 (2006)). See also U.S. Patent No. 7,083,784, which is incorporated herein in its entirety by reference.

[00108] The terms "LOX1-binding protein", "anti-LOX1 antibody" or "an antibody that specifically binds to LOX1" refer to a LOX1-binding protein, such as an anti-LOX1 antibody that is capable of binding to LOX1 with sufficient affinity so that the antibody is useful as a therapeutic agent or diagnostic reagent when LOX1 is the target. The extent of binding of an anti-LOX1 antibody to an unrelated non-LOX1 protein is less than about 10% of the antibody binding to LOX1 measured, for example, by a radioimmunoassay (RIA), BIACORE® (using recombinant LOX1 as analyte and antibody as ligand, or vice versa), the kinetic exclusion assay (KINEXA®) 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 <1 nM, <0.5 nM, <0.1 nM , <10 pM or <1 pM, or, in some cases, 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 <1 μM, <100 nM, <10 nM, <1 nM , <0.1 nM, <10 pM, <1 pM, or <0.1 pM, or, in some cases, a KD of about 150 pM to about 600 pM.

[00109] An "antagonistic" 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, LOX1-binding protein inhibits the ability of LOX1 to bind to oxHDL, HSP60, activated leukocytes and/or platelets. In certain aspects, a LOX1-binding protein substantially or completely inhibits the biological activity of LOX1. Desirably, the biological activity of LOX1 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%.

[00110] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen ). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that 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 to the antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind to the antigen more quickly 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 the purposes of the present disclosure.

[00111] "Potency" is typically expressed as an IC50 value, in nM or pM, unless otherwise stated. IC50 is the median inhibitory concentration of an antibody molecule. In functional assays, the IC50 is the concentration that reduces a biological response by 50% of its maximum. In ligand binding studies, IC50 is the concentration that reduces receptor binding by 50% of the maximum level of specific binding. The IC50 can be calculated by means known in the art.

[00112] The number-fold potency improvement for the LOX1-binding protein disclosed herein (e.g., an antibody, such as a full-length LOX1 antibody, a LOX1-binding antibody fragment, and variants and derivatives thereof) compared to a reference antibody may 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 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, or at least about 180 times or more.

[00113] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer cells ( NK, "Natural Killer"), neutrophils and macrophages) allows these cytotoxic effector cells to specifically bind to a target cell carrying an antigen 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 necessary for such killing. Target cell lysis 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 ability to specifically bind to a target cell carrying an antigen will be capable of effecting cell-mediated cytotoxicity. For simplicity, the cell-mediated cytotoxicity that results from the activity of an Fc fusion protein is also referred to here as ADCC activity.

[00114] A LOX1-binding protein (e.g., an antibody against LOX1, 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. Polypeptides (e.g., anti-LOX1 antibodies including full-length antibodies and LOX1-binding antibody fragments), a polynucleotide, a vector, isolated polynucleotides, vectors, cells or compositions include those that have been purified to a degree that no longer are 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.

[00115] By "subject" or "individual" or "animal" or "patient" or "mammal" is intended to mean any subject, particularly a mammalian subject, for which diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sporting animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses , cattle, bears, and so on.

[00116] The term "pharmaceutical composition" refers to a preparation that is in such a form as to allow the biological activity of the active ingredient (e.g., a LOX1-binding protein disclosed herein) to be effective, and which does not contain additional components that are unacceptably toxic to a subject to which the composition would be administered. Such a composition may be sterile.

[00117] An "effective amount" or a "pharmaceutically effective amount" of a LOX1-binding protein, such as an anti-LOX1 antibody, or other therapeutic agent, is an amount sufficient to accomplish a specifically indicated objective. An "effective amount" can be determined empirically and routinely in relation to the stated objective. The disclosure provides therapeutics for the treatment, prevention or amelioration of diseases and conditions associated with LOX1 and/or diminished HDL-mediated signaling. Such diseases and pathologies 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, pre-eclampsia and cancer.

[00118] The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody in order to generate a "labeled" antibody. The tag may be detectable by itself (e.g., radioisotope tags or fluorescent tags) or, in the case of an enzymatic tag, may catalyze the chemical change of a compound or substrate composition that is detectable.

[00119] Terms such as "treating" or "treatment" or "treating" or "improving" or "improving" refer to (1) therapeutic measures that cure, delay, attenuate pathologies associated with, and/or halt the progression of a diagnosed disease or pathology and (2) prophylactic or preventive measures that prevent and/or delay the development of a targeted disease or pathology. Thus, those in need of treatment include those already with the disease or pathology; those at risk of developing the disease or pathology; and those in which the disease or pathology is to be prevented. In certain aspects, the subject is successfully "treated" in accordance with the methods provided herein when the subject exhibits, for example, a total, partial or transient improvement or elimination of a symptom associated with the disease or condition. Such diseases or pathologies 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.

[00120] A "nucleic acid" or "polynucleotide", as used herein, may 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. Nucleic acids can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogues thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase. A "nucleic acid" or "polynucleotide" may comprise modified nucleotides, such as methylated nucleotides and analogues thereof. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA (genomic or cDNA).

[00121] 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 condensation agents, DNA expression vectors or RNA encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[00122] The terms "polypeptide", "peptide", and "protein" are used interchangeably here to refer to amino acid polymers of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. 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 analogues of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It is understood that because the polypeptides of the present disclosure are based on antibodies, in certain aspects, the polypeptides may occur as single chains or associated chains.

[00123] The terms "identical" or percentage "identity" of sequences, 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 residues. amino acid that are the same when compared and aligned (introducing gaps where necessary) for maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using a sequence comparison program or algorithm or by visual inspection. Various algorithms and programs are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[00124] One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Academic. Sci., 87:2264-2268 (1990), as modified in Karlin et al., Proc. Natl. Academic. 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, Southern San Francisco, California), or Megalign (DNASTAR) are additional publicly available 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 program package (e.g., using an 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 program package, which incorporates the Needleman and Wunsch algorithm (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percentage of identity between two amino acid sequences (e.g., using 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 percentage of identity between nucleotide or amino acid sequences is determined using the Myers and Miller algorithm (CABIOS, 4:11-17 (1989)). For example, percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residual table, a gap length penalty of 12, and a gap length penalty of 4. One skilled in the art can determine the appropriate parameters for maximum alignment using a particular alignment program. In certain aspects, the predefined parameters of the alignment program are used.

[00125] In certain aspects, the percent sequence identity "X" of a first amino acid sequence with a second amino acid sequence is calculated as 100 x (Y/Z), where Y is the number of classified amino acid residues as identical matches in the alignment of the first and second sequences (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 greater than that of the second sequence, the percentage identity of the first sequence with the second sequence will be higher than the percentage identity of the second sequence with the first sequence.

[00126] A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by another amino acid residue with 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., e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar 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, the substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain aspects, conservative substitutions in the LOX1-binding protein sequences of the disclosure do not abrogate binding of the protein containing the substituted amino acid sequence to LOX1. Methods of identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. ., Protein Eng. 12(10):879-884 (1999); and Burks et al., Natl. Sci. USA 94:412-417 (1997)).

[00127] The term "epitope", as used herein, refers to a protein determinant of LOX1, for example human LOX1 (hLOX1) or monkey LOX1 (e.g. cynomolgus monkey), capable of binding to a LOX1 binding protein (e.g., an antibody) of the disclosure. Epitopes usually consist of chemically active surface clusters 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 nonconformational epitopes are distinguished by the fact that 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., competitively inhibit the binding of, in a statistically significant manner) 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.

[00128] A LOX1-binding protein (e.g., an antibody) is said to "compete" with a reference molecule for binding to LOX1 when it binds to LOX1 in such a way that it blocks, to some degree, binding of the LOX1 reference molecule. 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, a LOX1-binding protein can be said to competitively inhibit the binding of the reference molecule to LOX1, for example, by at least 90%, at least 80%, at least 70%, at least least 60% or at least 50%.

II. LOX1-binding proteins

[00129] The present disclosure provides LOX1 binding proteins that specifically bind to 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 bispecific antibody, a multispecific antibody, or an antibody fragment thereof. In certain aspects, the antibody against LOX1 is a full-length antibody.

[00130] In some aspects, the antibody against LOX1 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 further aspects, the antibody against LOX1 is an Fd, a single-chain Fv (scFv), a disulfide-linked Fv, a V-NAR domain, an IgNar, an intrabody, an IgGΔCH2, a minibody, an F(ab' )3, a tetrabody, a triabody, a diabody, a single domain antibody, a DVD-Ig, an Fcab, a mAb2, a (scFv)2 or a scFv-Fc.

[00131] 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., an antibody against LOX1 or fragment thereof) additionally 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 a fragment thereof; (b) a human IgD constant region or a fragment thereof; (c) a human IgE constant region or a fragment thereof; (d) a human IgG1 constant region or a fragment thereof; (e) a human IgG2 constant region or a fragment thereof; (f) a human IgG3 constant region or a fragment thereof; (g) a human IgG4 constant region or a fragment thereof; and (h) a human IgM constant region or a fragment thereof. In further aspects, the LOX1-binding protein (e.g., an antibody against LOX1 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., an antibody against LOX1 or fragment thereof) comprises an IgG1 heavy chain constant region containing a mutation that decreases its 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 additional aspects, the constant region of IgG1 comprises the triple mutations L234F/L235E/P331S (TM), where the position numbering is according to the EU index as in Kabat, resulting in a null effector human IgG1. In some aspects, the IgG1 constant region comprises the YTE triple mutation mutant, as disclosed above in the Definitions section. In further aspects, the LOX1 binding protein is an antibody containing an IgG1 constant region comprising both the TM triple mutation and the YTE triple mutation.

[00132] In certain aspects, a heavy chain constant region or fragment thereof, for example, a human IgG constant region or a fragment thereof, may include one or more amino acid substitutions relative to an IgG constant domain. wild-type, where the modified IgG has an increased half-life compared to the half-life of an IgG that has the constant domain of wild-type IgG. For example, the IgG constant domain may contain one or more amino acid substitutions of amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389 and 428 to 436, wherein the position numbering is according to the EU index as presented in Kabat. In certain aspects, the IgG constant domain may contain one or more of an amino acid substitution at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W) or Threonine (T), an amino acid substitution 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 ), an amino acid substitution at Kabat position 257 with Leucine (L), an amino acid substitution at Kabat position 309 with Proline (P), an amino acid substitution at Kabat position 311 with Serine (S), a substitution of 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 an amino acid substitution at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F) or Tyrosine. More specifically, the IgG constant domain may contain amino acid substitutions relative to a wild-type human IgG constant domain, including an amino acid substitution at Kabat position 252 with Tyrosine (Y), an amino acid substitution at Kabat position 254 with Threonine (T) and an amino acid substitution at Kabat position 256 with Glutamic acid (E).

[00133] In further aspects, the LOX1 binding protein (e.g., an antibody against LOX1 or fragment thereof) comprises a light chain immunoglobulin constant domain selected from the group consisting of: (a) a cap constant domain human Ig; and (b) a human Ig lambda constant domain. In further aspects, the LOX1 binding protein (e.g., an antibody against LOX1 or fragment thereof) comprises a human IgG1 heavy chain constant domain containing the triple mutations L234F/L235E/P331S ("IgG-TM") resulting in a null effector human IgG1 and a human lambda light chain constant domain.

[00134] 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 less substitutions, deletions and/or amino acid insertions relative to a reference VH or VL disclosed herein. In some aspects, the LOX1 binding protein has a VH and/or a VL as shown in Table 1.

[00135] Exemplary LOX-1 binding proteins are provided in Table 1. Table 1: Exemplary LOX-1 binding proteins

[00136] 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 relative to a reference VH sequence of SEQ ID NO:4. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00137] 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00138] 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 relative to a reference VL sequence of SEQ ID NO:33. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00139] 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00140] 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 that has the amino acid sequence of SEQ ID NO:30, a VL CDR2 that has the amino acid sequence of SEQ ID NO:31, and a VL CDR3 that has 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 such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparation and use of LOX1 binding protein.

[00141] The disclosure also provides an isolated LOX1 binding protein comprising a VH and VL sequence, which has a total of one, two, three, four, five, six, seven, eight or less substitutions, deletions and/or or amino acid insertions relative to LOX1 binding proteins with reference VH and VL disclosed herein. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00142] 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 such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1-binding protein.

[00143] In some aspects, the isolated LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain (VH) variable region CDR1, VH CDR2, VH CDR3, and variable region CDR1 light chain (VL), 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 relative to a reference set of CDRs in which: (a) the CDR1 of VH has the amino acid sequence of SEQ ID NO:1; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:5; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:14; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:30; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:31; and (f) the VL CDR3 has the amino acid sequence of SEQ ID NO:32. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00144] 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) CDR1 of VH has the amino acid sequence of SEQ ID NO:1; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:2; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:3; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:30; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:31; and (f) the VL CDR3 has the amino acid sequence of SEQ ID NO:32. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00145] The disclosure also provides an isolated LOX1 binding protein comprising a heavy chain variable region (VH) that has at least 90, 95, 97, 98 or 99% sequence identity with a disclosed reference VH sequence herein and/or a light chain variable region (VL) that has at least 90, 95, 97, 98 or 99% sequence identity with a reference VL sequence disclosed herein (e.g., the sequences of VH and VL revealed in Table 1, Figure 4 or Figure 5). In further aspects, the disclosure provides an isolated LOX1 binding protein comprising a VH that has at least 90, 95, 97, 98 or 99% sequence identity to SEQ ID NO:4 and/or a VL that has at least 90, 95, 97, 98 or 99% sequence identity with SEQ ID NO:33. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00146] In some aspects, the LOX1 binding protein comprises a heavy chain variable region (VH) that has at least 90, 95, 97, 98 or 99% sequence identity and a light chain variable region (VL ) which has at least 90, 95, 97, 98 or 99% sequence identity with 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00147] In some aspects, the LOX1 binding protein comprises a VH that has at least 90, 95, 97, 98 or 99% sequence identity and a VL that has at least 90, 95, 97, 98 or 99% sequence identity. % sequence identity with 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 that has the amino acid sequence of SEQ ID NO:27 and a VL that has 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; (l) 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00148] 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 In additional aspects, the LOX1 binding protein comprises a VH CDR3 having the amino acid sequence of SEQ ID NO:3, 1417 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 that has the amino acid sequence of SEQ ID NO:2, 5-12 or 13 and a VH CDR1 that has the amino acid sequence of SEQ ID NO:1. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00149] In further aspects, the disclosure provides an isolated LOX1 binding protein comprising an 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 of SEQ ID NO:31, and a VL CDR1 having has 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 of 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 of 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00150] In further aspects, the LOX1 binding protein comprises one, two or three VH CDRs, such as a VH CDR1 identical to, or having a total of one, two or less amino acid substitutions, deletions or insertions with respect to SEQ ID NO:1, a VH CDR2 identical to, or having a total of one, two or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO:2, 5-12 or 13, or a VH CDR3 identical to, or having a total of one, two, three, four or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO:3, 14-17 or 18.

[00151] In further aspects, the LOX1 binding protein comprises one, two or three VL CDRs, such as a VL CDR1 identical to, or having a total of one, two or less amino acid substitutions, deletions or insertions with respect to SEQ ID NO:30, a VL CDR2 identical to, or having a total of one, two or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO:31, or a VH CDR3 identical with , or which has a total of one, two, three, four or fewer amino acid substitutions, deletions or insertions in relation to SEQ ID NO:32, 34 or 35.

[00152] 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 relative to a reference VH sequence of SEQ ID NO:41. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00153] 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 LOX1 binding proteins, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding proteins.

[00154] 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 relative to a reference VL sequence of SEQ ID NO:58. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00155] 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00156] 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.

[00157] 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, 6569 or 70. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00158] In some aspects, the isolated LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain (VH) variable region CDR1, VH CDR2, VH CDR3, and variable region CDR1 light chain (VL), 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 substitutions, deletions and/or amino acid insertions relative to a reference set of CDRs in which: (a) the VH CDR1 has the amino acid sequence of SEQ ID NO:38; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:39; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:44; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:55; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:60; and (f) the VL CDR3 has the amino acid sequence of SEQ ID NO:61. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00159] In some aspects, the isolated LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain (VH) variable region CDR1, VH CDR2, VH CDR3, VH variable region CDR1. light chain (VL), 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 substitutions, deletions and /or amino acid insertions relative to a reference set of CDRs in which: (a) the VH CDR1 has the amino acid sequence of SEQ ID NO:38; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:39; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:40; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:55; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:56; and (f) the VL CDR3 has the amino acid sequence of SEQ ID NO:57. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00160] 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) CDR1 of VH has the amino acid sequence of SEQ ID NO:38; (b) the VH CDR2 has the amino acid sequence of SEQ ID NO:39; (c) the VH CDR3 has the amino acid sequence of SEQ ID NO:40; (d) the VL CDR1 has the amino acid sequence of SEQ ID NO:55; (e) the VL CDR2 has the amino acid sequence of SEQ ID NO:56; and (f) the VL CDR3 has the amino acid sequence of SEQ ID NO:57. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00161] 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 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. 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 that has the amino acid sequence of SEQ ID NO: 39, 42 or 43, and a VH CDR1 that has the amino acid sequence of SEQ ID NO:38. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00162] In further aspects, the disclosure provides an isolated LOX1 binding protein comprising an 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 of SEQ ID NO:56, and a VL CDR3 having has 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 of SEQ ID NO:56 or 60, and a CDR1 of VL 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 such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparation and use of LOX1 binding protein.

[00163] In further aspects, the LOX1 binding protein comprises one, two or three VH CDRs, such as a VH CDR1 identical to, or having a total of one, two or less amino acid substitutions, deletions or insertions with respect to SEQ ID NO:38, a VH CDR2 identical to, or having a total of one, two or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO:39, 42 or 43, or a CDR3 of VH identical to, or having a total of one, two, three, four or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO:40, 44-46 or 47. The disclosure also provides nucleic acids encoding the LOX1-binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1-binding protein.

[00164] In further aspects, the LOX1 binding protein comprises one, two or three VL CDRs, such as a VL CDR1 identical to, or having a total of one, two or less amino acid substitutions, deletions or insertions with respect to SEQ ID NO: 55 or 59, a VL CDR2 identical to, or having a total of one, two or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO: 56 or 60, or a CDR3 of VH identical to, or having a total of one, two, three, four or fewer amino acid substitutions, deletions or insertions with respect to SEQ ID NO: 57, 61-63 or 64. The disclosure also provides nucleic acids encoding the LOX1-binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1-binding protein.

[00165] 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, Figure 4 or Figure 5. The disclosure also provides nucleic acids encoding LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00166] In some additional aspects, the LOX1 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL) as described in Table 1, Figure 4 or Figure 5. The disclosure also provides nucleic acids encoding the LOX1-binding protein, vectors containing such nucleic acids and host cells transformed with these nucleic acids and vectors, and methods of preparing and using the LOX1-binding protein.

[00167] In some aspects, the isolated LOX1 binding protein comprises a VH CDR2 having the amino acid sequence of G F D P E D HX1 HX2 HX3 HX4 HX5 HX6 Q K F Q G (Gly Phe Asp Pro Glu Asp HX1 HX2 HX3 HX4 HX5 HX6 Gln Lys Phe Gln Gly), where HX1 is Gly, Trp, Tyr or Phe; HX2 is Glu, Thr, Gln, Ser, Lys or Ala; HX3 is Thr, Tyr, Ile or Asn; HX4 is Ile, Ala, Arg or His; HX5 is Tyr, Val, Thr, Leu or Gln, and HX6 is Ala, Asp, Gly, Ser or His (SEQ ID NO:71).

[00168] In further aspects, the isolated LOX1 binding protein comprises a VH CDR3 having the amino acid sequence of HX7 HX8 G HX9 HX10 HX11 HX12 G V R G W D Y Y Y G M D V (HX7 HX8 Gly HX9 HX10 HX11 HX12 Gly Val Arg Gly Trp Asp Tyr Tyr Tyr Gly Met Asp Val Trp) where HX7 is Pro, Ser or Val; HX8 is Asn, Thr, Trp or Asp; HX9 is Gln, Arg or Thr; HX10 is Gln or His; HX11 is Gly or Gln; HX12 is Lys or Gly (SEQ ID NO:72).

[00169] In other aspects, the isolated LOX1 binding protein comprises a VL CDR3 having the amino acid sequence of Q S Y D S LX1 LX2 LX3 LX4 LX5 LX6 (Gln Ser Tyr Asp Ser LX1 LX2 LX3 LX4 LX5 LX6), wherein LX1 is Ser or Met; LX2 is Leu, Tyr or His; LX3 is Ser or Arg; LX4 is Gly, Ala or no amino acid; LX5 is Trp or Phe; and LX6 is Val, Gly or Ala (SEQ ID NO:73).

[00170] In some aspects, the isolated LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, and variable region CDR1 light chain (VL), VL CDR2 and VL CDR3, wherein: (a) VH CDR1 comprises the amino acid sequence: E L S M H (SEQ ID NO: 1); (b) the CDR2 of VH 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) the CDR3 of VH comprises the amino acid sequence: HX7 HX8 G HX9 HX10 HX11 HX12 G V R G W D Y Y Y G M D 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) the 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) the VL CDR2 comprises the amino acid sequence: G N S N R P S (SEQ ID NO: 31); and (f) the CDR3 of VL 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 acids), 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).

[00171] In further aspects, the isolated LOX1 binding protein comprises a VH CDR2 having the amino acid sequence of G HX1 S HX2 HX3 HX4 HX5 HX6 HX7 HX8 HX9 HX10 D S V K G (Gly HX1 Ser HX2 HX3 HX4 HX5 HX6 HX7 HX8 HX9 HX10 Asp Ser Val Lys Gly), where HX1 is Ile or Val; HX2 is Trp or Leu; HX3 is Asn or Gln; HX4 is Ser or Glu; HX5 is Gly, Leu or Pro; HX6 is Ser, Tyr or Asp; HX7 is Ile, Thr or Arg; HX8 is Gly or Tyr; HX9 is Tyr or Met; and HX10 is Ala or Asp (SEQ ID NO:74).

[00172] In further aspects, the isolated LOX1 binding protein comprises a VH CDR3 having the amino acid sequence of E G HX11 W N Y D A HX12 D HX13 (Glu Gly HX11 Trp Asn Tyr Asp Ala HX12 Asp HX13), where HX11 is Asn or Being; HX12 is Phe or Leu; and HX13 is Ile or Val (SEQ ID NO:75).

[00173] In further aspects, the isolated LOX1 binding protein comprises a VL CDR1 having the amino acid sequence of T G T S LX1 D V G G Y N Y V S (Thr Gly Thr Ser LX1 Asp Val Gly Gly Tyr Asn Tyr Val Ser), wherein LX1 is Ser or Asn (SEQ ID NO:76).

[00174] In further aspects, the isolated LOX1 binding protein comprises a VL CDR2 having the amino acid sequence of D V S LX2 R P S (Asp Val Ser X4 Arg Pro Ser), where LX2 is Asn or Lys (SEQ ID NO :77).

[00175] In further aspects, the isolated LOX1 binding protein comprises a VL CDR3 having the amino acid sequence of LX3 LX4 LX5 LX6 LX7 LX8 S T N W V (LX3 LX4 LX5 LX6 LX7 LX8 Ser Thr Asn Trp Val), wherein LX3 is Ser, Leu, Met or Ala; LX4 is Ser, Gly or Gln; LX5 is Tyr, Arg, Ser or Gly; LX6 is Thr or Met; LX7 is Ser, Trp, Gly or Val; and LX8 is Ser or Arg (SEQ ID NO:78).

[00176] In some aspects, the isolated LOX1 binding protein comprises a set of complementarity determining regions (CDRs): heavy chain (VH) variable region CDR1, VH CDR2, VH CDR3, and variable region CDR1 light chain (VL), VL CDR2 and VL CDR3, wherein: (a) VH CDR1 comprises the amino acid sequence: D Y A M H (SEQ ID NO: 38); (b) the CDR2 of VH comprises the amino acid sequence: , HX3 is selected from the group consisting of N and Q, HX4 is selected from the group consisting of S and E, HX5 is selected from the group consisting of G, L and P, HX6 is selected from the group consisting of S, Y and D, HX7 is selected from the group consisting of I, T and R, HX8 is selected from the group consisting of G and Y, HX9 is selected from the group consisting of M and Y, and

[00177] HX10 is selected from the group consisting of A and D (SEQ ID NO:74); (c) the CDR3 of VH comprises the amino acid sequence: consisting of I and V (SEQ ID NO:75); (d) VL-CDR1 comprises the amino acid sequence: T G T S LX1 D V G G Y N Y V S, wherein LX1 is selected from the group consisting of N and S (SEQ ID NO:76); (e) the CDR2 of VL comprises the amino acid sequence: D V S LX2 R P S, wherein LX2 is selected from the group consisting of N and K (SEQ ID NO:77); and (f) the CDR3 of VL comprises the amino acid sequence: LX3 LX4 LX5 LX6 LX7 LX8 S T N W V, wherein LX3 is selected from the group consisting of L, M, A and S, LX4 is selected from the group consisting of S, Q and G, LX5 is selected from the group consisting of Y, S, G and R, LX6 is selected from the group consisting of T and M, LX7 is selected from the group consisting of S, W, G and V, and LX8 is selected from the group consisting of in S and R (SEQ ID NO:78).

[00178] In some aspects, the set of CDRs of a LOX1-binding protein disclosed herein is provided within antibody framework regions or other protein supports known in the art. Exemplary antibody framework regions include: germline framework regions such as VH1-24 (DP-5), JH6, VH3-09 (DP-31) and JH3 for the heavy chain antibody framework region and VÀ1e (DPL-8), VÀ2a2 (DPL-11) and JL3 for the light chain antibody framework region and/or any appropriate framework regions well known to the person skilled in the art.

[00179] In some aspects, the LOX1 binding protein contains one or more framework regions of a heavy chain variable region (VH) and/or a light chain variable region (VL) that are a germline framework. The framework regions of the heavy chain domain may be selected from frameworks VH1-24 (DP-5), JH6, VH3-09 (DP-31), JH3 and/or any appropriate framework regions or protein scaffolds well known in the art. . The light chain framework regions may be selected from frameworks VÀ1e (DPL-8), VÀ2a2 (DPL-11) and JL3 and/or any appropriate framework regions or protein scaffolds well known in the art. One or more CDRs can be taken from the LOX1-binding proteins disclosed herein (e.g., a LOX1-binding protein described in Table 1, Figure 4 or Figure 5) and incorporated into an appropriate protein scaffold and/or support.

[00180] In further 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.

[00181] 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), which compete with or present cross-competition 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 capable of competitively inhibiting the binding to LOX1 of an antibody comprising a VH of SEQ ID NO:4 and a VL of SEQ ID NO:33.

[00182] Also provided is a LOX1-binding protein, such as an antibody against LOX1 (e.g., a full-length anti-LOX1 antibody, a LOX1-binding antibody fragment, and variants and derivatives thereof) that can bind to LOX1 with greater affinity than a LOX1-binding protein (e.g., an antibody against LOX1 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00183] In further 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.

[00184] 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), which compete with or present cross-competition 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 capable of competitively inhibiting the binding to LOX1 of an antibody comprising a VH of SEQ ID NO:41 and a VL of SEQ ID NO:58.

[00185] Also provided is a LOX1-binding protein, such as an antibody against LOX1 (e.g., a full-length anti-LOX1 antibody, a LOX1-binding antibody fragment, and variants and derivatives thereof) that can bind to LOX1 with 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 LOX1 binding protein, vectors containing such nucleic acids and host cells transformed with such nucleic acids and vectors, and methods of preparing and using the LOX1 binding protein.

[00186] 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 bispecific antibody, a multispecific antibody or any combination thereof. In some aspects, the anti-LOX1 antibody is an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, a dsFv fragment, a scFv fragment, or a sc(Fv)2 fragment.

[00187] 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) which can bind to LOX1 molecules in different 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 additional 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 than: CLEC-7A, CLEC-1A, CLEC-4L, CLEC-1B, SR-A1 and/or SR-B3. See, for example, Figures 3, 6 and 7.

[00188] In some aspects, the LOX1 binding protein (e.g., an isolated anti-LOX1 antibody or the same) further comprises a human IgG1 TM mutant heavy chain, wherein the heavy chain variable region (VH) comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence reference SEQ ID NO:4, 19-28 or 29.

[00189] In some aspects, the LOX1 binding protein (e.g., an isolated anti-LOX1 antibody or the same) further comprises a human IgG1 TM mutant heavy chain, wherein the heavy chain variable region (VH) comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence reference SEQ ID NO:41, 48-53 or 54.

[00190] 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) additionally includes a variable region of heavy chain (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 constant region of the kappa and lambda light chains, for example, a human kappa constant region or a human lambda constant region. In a specific aspect, the light chain constant region is a human cap constant region.

[00191] In further aspects, the LOX1-binding protein (e.g., a full-length LOX1 antibody, a LOX1-binding antibody fragment, and a variant and derivative thereof) contains a light chain variable region ( VL) that contains a human cap constant region, for example, the VL comprises an amino acid sequence of VL that has at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity to sequences with 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) additionally comprising a heavy chain human IgG1 TM mutant 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.

[00192] In further 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) which further comprises a human cap constant region, for example, the light chain may comprise a light chain amino acid sequence which is 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 mutant human IgG1 TM 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.

[00193] 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 the binding of oxLDL, C-reactive protein (CRP) and/or advanced glycation end products ( AGEs) to LOX1 as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assays 1, 2 and/or 3 or Example 11); (b) decreases or inhibits signaling by RhoA/Rac1, nitrogen monoxide (NO), p38MAPK, protein kinases B and C, ERK1/2 and/or NFKB in an endothelial cell expressing cell surface LOX1, as determined by any assay appropriate, including an assay disclosed herein (see, for example, Example 11); (c) decreases or inhibits the activity of caspase-8, caspase-9 and/or BAX in an endothelial cell expressing cell surface LOX1, as determined by any appropriate assay, including an assay disclosed herein; (d) binds to LOX1 having the K167N single nucleotide polymorphism as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assays 1, 2 and/or 3 or Example 11 ); (e) reduces or inhibits oxLDL internalization as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assay 4, or Example 11); (f) reduces or inhibits oxLDL-induced LOX1 signaling as determined by any appropriate assay, including an assay disclosed herein (see, for example, 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 velocity of about 1 x 105 M-1 s-1 to about 6 x 106 M-1 s-1 (e.g., about 5 x105 M-1 s-1 ) as determined by BIACORE; and (i) binds to LOX1 with a Koff velocity of about 1 x 10-4 s-1 to about 3 x 10-4 s-1 (e.g., about 2.3 x 10-4 s- 1) as determined by BIACORE.

[00194] In certain aspects, blocking the biological activity of LOX1 by a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) described herein reduces atherosclerosis and/or decreases one or more pathologies associated with atherosclerosis. In particular aspects, the LOX1 antagonist (e.g., an anti-LOX1 antibody or fragment thereof) inhibits or diminishes the 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.

[00195] 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 10-6 M, or less than 10-7 M, or less than 10-8 M, or less than 10-9 M, or less than 10-10 M, or less than 10-11 M, less than 10-12 M, less than 10-13 M, less than 10-14 M, or less than 10-15 M as measured, for example, by KINEXA® or BIACORE®. In one aspect, the anti-LOX1 antibody can bind to hLOX1 and cynoLOX1 with a KD of less than about 1 x 10-8 M to about 1 x 10-10 M as measured by BIACORE®.

[00196] 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 less than 1*10-3 s-1 or less than 2*10—3 s-1. In other aspects, the LOX1 binding protein binds to LOX1 with a koff of less than 10-3 s-1, less than 5*10-3 s-1, less than 10-4 s-1, less than 5* 10-4 s-1, less than 10-5 s-1, less than 5*10-5 s-1, less than 10-6 s-1, less than 5*10-6 s-1, less than 5 *10-7 s-1, less than 10-8 s-1, less than 5*10-8 s-1, less than 10-9 s-1, less than 5*10-9 s-1, or less at 10-10 s-1 as measured, for example, by KINEXA® or BIACORE®. In one aspect, the anti-LOX1 antibody can bind to hLOX1 and cynoLOX1 with a Koff of 1 to 10 *10-4 s-1 as measured by BIACORE®.

[00197] In another aspect, the 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 (hLOX1) and/or cynomolgus LOX1, with an association rate constant or kon rate of at least 105 M-1 s-1, at least 5x105 M-1 s- 1, at least 106 M-1 s-1, at least 5x106 M-1 s-1, at least 107 M-1 s-1, at least 5x07 M-1 s-1, or at least 108 M-1 s -1, or at least 109 M-1 s-1 as measured, for example, by KINEXA® or BIACORE®. In one aspect, the anti-LOX1 antibody can bind to hLOX1 and cynoLOX1 with a kon rate of about 1 x105 M-1 s-1 to about 20 x105 M-1 s-1 as measured by BIACORE®.

[00198] 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 amino acid sequence and /or VL that binds to LOX1 may have at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a sequence set forth herein (see, for example, the Table 1, Figure 4 or Figure 5). In some aspects, the VH and/or VL amino acid sequence(s) that bind to LOX1 comprise(s) 15 or less (e.g., 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14 or 15) additions, substitutions (e.g., conservative substitutions) or deletions of amino acids with respect to a sequence presented herein. In some aspects, the VH and/or VL amino acid sequence(s) that bind to LOX1 comprise(s) 8 or less (e.g., 1, 2, 3, 4, 5, 6 , 7 or 8) additions, substitutions (e.g., conservative substitutions) or deletions of amino acids in relation to a sequence presented herein. In further aspects, the VH and/or VL amino acid sequence that binds to LOX1 comprises 5 or less (e.g., 1, 2, 3, 4, or 5) additions, substitutions (e.g., conservative substitutions) or deletions of amino acids in relation to a sequence presented in this document. A LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) containing VH and VL regions having a certain percentage 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 the VH and/or VL regions described herein, followed by antibody evaluation altered coded for binding to LOX1 and optionally assessing retained function using the functional assays described herein or an assay known in the art that can be routinely modified to test retained function.

[00199] 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 ) by LOX1 can be determined experimentally using any appropriate method known in the art, for example, flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA). "), or kinetics (e.g. analysis with KINEXA® or BIACORE®). 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 may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other LOX1 binding parameters (e.g., KD or Kd, kon, koff) are performed with standardized solutions of LOX1 and LOX1 binding proteins, and a standardized buffer as known in the art, such as the buffer described in this document.

[00200] 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, for example, Table 1, Figure 4 or Figure 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.

[00201] In certain aspects, the LOX1 binding protein is not an anti-LOX1 antibody. A variety of methods of identifying and producing non-antibody polypeptides that bind with high affinity to a target protein are known in the art. See, for example, Skerra, Curr. Opinion. Biotech. 18:295-304 (2007), Hosse et al., Protein Science 15:14-27 (2006), Gill et al., Curr. Opinion. 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 herein in its entirety by reference . In certain aspects, phage display technology can be 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 repeat consensus domain, and thioredoxin.

Proteins that bind to the same epitope as a LOX1-binding protein

[00202] 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, for example, Table 1, Figure 4 or Figure 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 clusters 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 nonconformational epitopes are distinguished by the fact that 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., competitively inhibit the binding of, in a statistically significant manner) 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.

[00203] 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 to the same epitope than an isolated LOX1-binding protein disclosed herein (see, for example, Table 1, Figure 4 or Figure 5).

[00204] The ability of a test LOX1 binding protein to inhibit the binding of, for example, 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, demonstrate that the test LOX1 binding protein can compete with this antibody for binding to LOX1; Such a LOX1-binding protein may, according to a non-limiting theory, bind to the same or a related epitope (e.g., structurally similar or spatially close) on LOX1 as LOX1-binding proteins (e.g., antibodies anti-LOX1, 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.

[00205] 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), which 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), which 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

[00206] 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 the binding of oxLDL, C-reactive protein (CRP) and/or advanced glycation end products (AGEs) to LOX1 as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, runs 1, 2 and/or 3 or Example 11); (b) decreases or inhibits signaling by RhoA/Rac1, nitrogen monoxide (NO), p38MAPK, protein kinases B and C, ERK1/2 and/or NFKB in an endothelial cell expressing cell surface LOX1, as determined by any assay appropriate, including an assay disclosed herein (see, for example, Example 11); (c) decreases or inhibits the activity of caspase-8, caspase-9 and/or BAX in an endothelial cell expressing cell surface LOX1, as determined by any appropriate assay, including an assay disclosed herein; (d) binds to LOX1 having the K167N single nucleotide polymorphism as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assays 1, 2 and/or 3 or Example 11 ); (e) reduces or inhibits oxLDL internalization as determined by any appropriate assay, including an assay disclosed herein (see, for example, Example 10, assay 4, or Example 11); (f) reduces or inhibits oxLDL-induced LOX1 signaling as determined by any appropriate assay, including an assay disclosed herein (see, for example, 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 velocity of about 1 x 105 M-1 s-1 to about 6 x 106 M-1 s-1 (e.g., about 5 x105 M-1 s-1 ) as determined by BIACORE; and (i) binds to LOX1 with a Koff velocity of about 1 x 10-4 s-1 to about 3 x 10-4 s-1 (e.g., about 2.3 x 10-4 s- 1) as determined by BIACORE.

[00207] 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) may 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 signal transduction pathway of RhoA/Rac1, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2, and/or NFKB as measured using, for example, a cell-based assay as described herein with an IC50 of less than about 500 pM, less than about 450 pM, less than about 450 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, less than about 150 pM, less than about 100 pM, less than about 75 pM, less than about 100 nM, less than about 75 nM, less to about 50 nM, less than about 30 nM, less than about 20 pM, less than about 10 nM, or less than about 5 nM.

[00208] In certain aspects, a LOX1-binding protein (e.g., an anti-LOX1 antibody, such as a full-length anti-LOX1 antibody, a LOX1-binding antibody fragment, and variants and derivatives thereof) can suppress, inhibit or reduce cynomolgus LOX1-mediated activation of the RhoA/Rac1, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2 and/or NFKB signaling pathway in endothelial cells, smooth muscle cells and /or cynomolgus macrophages expressing LOX1 with an IC50 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 IC50 of about 1 nM to about 500pM.

[00209] In further aspects, an antagonistic LOX1-binding protein (e.g., an anti-LOX1 antibody, such as a full-length anti-LOX1 antibody, a LOX1-binding antibody fragment, and variants and derivatives thereof ) can suppress, inhibit or reduce human LOX1-mediated activation of the signal transduction pathway of RhoA/Rac1, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2 and/or NFkB as measured using e.g. a cell-based assay as described herein in endothelial cells, smooth muscle cells and/or macrophages expressing LOX1 with an IC50 of about 300 pM, about 250 pM, about 100 pM, about 55 pM, about of 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.

[00210] 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 the binding of LOX1 to oxLDL. In some aspects, the LOX1-binding protein inhibits or reduces the binding of LOX1 to various LOX1 ligands. In some aspects, the LOX1-binding protein inhibits or reduces the binding of LOX1 to oxLDL and AGEs. In some aspects, the LOX1-binding protein inhibits or reduces the binding of LOX1 to oxLDL and CRP. In some aspects, the LOX1-binding protein inhibits or reduces the binding of LOX1 to oxLDL, AGEs, and CRP. In additional aspects, LOX1-binding protein inhibits or reduces the binding of LOX1 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 for measuring the inhibition or reduction of binding of LOX-1 to one or more ligands include those described herein in the Examples and any other appropriate method known in the art.

V. Preparation of anti-LOX1 antibodies

[00211] In certain aspects, LOX1-binding proteins are anti-LOX1 antibodies, such as a full-length anti-LOX1 antibody, LOX1-binding antibody fragments, and variants and derivatives thereof.

[00212] 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 induce the production, by lymphocytes, of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells which can then be selected and separated from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against LOX1, such as hLOX1, as determined by immunoprecipitation, immunoblot, or by an in vitro binding assay (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated in culture in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo in the form of ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described above for polyclonal antibodies.

[00213] Alternatively, anti-LOX1 monoclonal antibodies can also be prepared using recombinant DNA methods as described in U.S. Patent No. 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B cells or hybridoma cells, 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 or myeloma cells (e.g., NS0 cells) that do not otherwise produce an immunoglobulin protein, monoclonal antibodies are generated by the host cells. Furthermore, 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.

[00214] 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, may (m) be further modified in several different ways 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 replaced (1) by the regions of, for example, a human antibody to generate a chimeric antibody or (2) by a polypeptide which is not an immunoglobulin to generate a fusion antibody. In some aspects, 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.

[00215] In certain aspects, the anti-LOX1 LOX1-binding protein binds human LOX1, such as a full-length anti-hLOX1 antibody and a human hLOX1-binding 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 produces 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);

[00216] Furthermore, the human anti-LOX1 antibody (e.g., a LOX1-binding human antibody fragment) can be selected from a phage library, wherein the 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. Academic. 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 generating and using antibody phage libraries are also described in U.S. Patent 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 herein in its entirety by reference).

[00217] Affinity maturation strategies and chain rearrangement strategies (Marks et al., BioTechnology 10:779-783 (1992), which is incorporated in its entirety by reference) are known in the art and can be employed to generate antibodies high affinity human anti-LOX1.

[00218] In some aspects, an antibody against LOX1 (such as a full-length anti-LOX1 antibody and a LOX1-binding antibody fragment, and variants and derivatives thereof) may be a humanized antibody. Methods for manipulating, humanizing or modifying surface residues ("resurfacing") of non-human or human antibodies can also be used and are known in the art. A humanized, resurfaced, or similarly engineered antibody may have one or more amino acid residues from a source other than human, e.g., but not limited to, mouse, rat, rabbit, primate. non-human or other mammal. These non-human amino acid residues are replaced by residues that are often called "import" residues, which are typically taken from a variable, constant or other "import" domain of a known human sequence. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. In general, CDR residues are directly and very substantially involved in influencing binding to LOX1. Accordingly, part or all of the non-human or human CDR sequences are retained while the non-human sequences of the variable and constant regions may be replaced with human or other amino acids.

[00219] Anti-LOX1 antibodies can also be optionally humanized, subjected to modification of surface residues ("resurfaced"), engineered or engineered human antibodies with high affinity retention for the LOX1 antigen and other favorable biological properties. To achieve this purpose, the antibody against humanized (or human) or engineered LOX1, such as a full-length anti-LOX1 antibody and a LOX1-binding antibody fragment, and variants and derivatives thereof, such as antibodies with surface residues modified ("resurfaced"), may optionally be prepared by a process of analyzing the parent sequences and various humanized and manipulated conceptual products using three-dimensional models of the parent, manipulated and humanized sequences. Three-dimensional models of immunoglobulins are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these results allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, that is, analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen, such as LOX1. In this way, framework residues (FW) can be selected and combined from the consensus sequences and imported in order to achieve the desired characteristic of the antibody, such as greater affinity for the target antigen(s).

[00220] Humanization, modification of surface residues ("resurfacing") or manipulation of the anti-LOX1 antibodies of the disclosure can be carried out 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. Academic. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Patent 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; international applications 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; international application publications Nos. WO90/14443; WO90/14424; WO90/14430; and EP patent publication No. EP 229246; each of which is incorporated herein in its entirety by reference, including the references cited therein.

[00221] Antagonistic human anti-LOX1 antibodies can also be prepared in transgenic mice containing human immunoglobulin locus that are capable, after immunization, of producing the complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425 and 5,661,016.

[00222] In certain aspects, the anti-LOX1 antibody is a LOX1-binding antibody fragment. Various techniques for producing antibody fragments are known. Traditionally, these fragments are derived by proteolytic digestion of intact antibodies (e.g. Morimoto et al., J. Biochem. Biophys. Meth. 24:107117 (1993); Brennan et al., Science 229:81 (1985)). In certain aspects, LOX1-binding antibody fragments are produced recombinantly. All Fab, Fv and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, thereby allowing the production of large quantities of these fragments. Such LOX1-binding antibody fragments can also be isolated from the antibody phage libraries discussed above. LOX1-binding antibody fragments can also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments are known in the art.

[00223] In accordance with the present disclosure, techniques known in the art can be adapted for the production of single-chain antibodies specific for LOX1 (see, for example, U.S. Patent No. 4,946,778). Furthermore, 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 efficient identification of monoclonal Fab fragments with the desired specificity for LOX1. LOX1-binding anti-LOX1 antibody fragments can be produced by techniques known in the art including, but not limited to: (a) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (b) a Fab fragment generated by reducing the disulfide bonds of an F(ab')2 fragment, (c) a Fab fragment generated by treating the anti-LOX1 antibody with papain and a reducing agent, and (d) Fv fragments.

[00224] In certain aspects, a LOX1-binding protein (e.g., an antibody against LOX1, such as a full-length anti-LOX1 antibody and a LOX1-binding antibody fragment, and variants and derivatives thereof) may be modified to increase its half-life in serum. This can be achieved, for example, by incorporating a salvage receptor-binding epitope into the LOX1-binding protein by mutating the appropriate region on the LOX1-binding protein or by incorporating the epitope into a peptide tag which 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 for increasing the serum half-life of a LOX1-binding protein (e.g., an antibody against LOX1, such as a full-length anti-LOX1 antibody and a LOX1-binding antibody fragment, and variants and derivatives thereof), for example, conjugation with a heterologous molecule, such as PEG.

[00225] Heteroconjugate 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) also fall within the scope of the disclosure. Heteroconjugate LOX1-binding proteins are composed of two covalently linked proteins. It has been proposed, for example, that such proteins target immune cells to unwanted cells (see, e.g., U.S. Patent No. 4,676,980). It is contemplated that heteroconjugated LOX1-binding proteins may be prepared in vitro using methods known in protein synthesis chemistry, including those involving cross-linking 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.

[00226] Modified anti-LOX1 antibodies, such as full-length anti-LOX1 antibodies and LOX1-binding antibody fragments, and variants and derivatives thereof, as provided herein, may comprise any type of variable region that promotes association of the antibody with LOX1. In this sense, the variable region can comprise or be derived from any type of mammal that can be induced to establish a humoral response and generate immunoglobulins against the LOX1 antigen. Thus, the variable region of an anti-LOX1 antibody can be, for example, of human, murine, non-human primate (e.g., cynomolgus monkeys, Macaca, 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 (typically derived from a non-human source) can be manipulated or specifically designed to improve the binding properties or reduce the immunogenicity of the molecule. In this regard, variable regions useful in accordance with the disclosure may be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[00227] 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 same) are altered by at least partial replacement of one or more CDRs and/or by alteration of sequences and partial replacement of the framework region. Although the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisioned that the CDRs will be derived from an antibody of a different class and, in certain aspects, from a antibody from a different species. It is not necessary to replace all CDRs with the complete CDRs of the donor variable region to transfer antigen-binding capacity from one variable domain to another. Instead, 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. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, it will be well within the competence of those skilled in the art, whether by performing routine experimentation or by trial and error testing to obtain a functional antibody. with reduced immunogenicity.

[00228] Notwithstanding changes to the variable region, those skilled in the art will recognize that modified anti-LOX1 antibodies (e.g., full-length anti-LOX1 antibodies and LOX1-binding antibody fragments, and variants and derivatives thereof) of the disclosure will comprise antibodies in which at least a portion 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 localization in tumors or reduced half-life in serum compared to a antibody of approximately the same immunogenicity comprising a native or unchanged 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 may 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 may comprise changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or the light chain constant domain (CL). In some aspects, modified anti-LOX1 antibodies will comprise constant regions in which one or more domains are contemplated to be partially or entirely deleted. In some aspects, modified anti-LOX1 antibodies will comprise domain-deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some aspects, the omitted constant region domain can be replaced with a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically conferred by the missing constant region.

[00229] In addition to its configuration, it is known in the art that the constant region mediates several effector functions. For example, binding of complement component C1 to antibodies activates the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Additionally, antibodies bind to cells via the Fc region, with an Fc receptor site in the Fc region of the antibody binding to an Fc receptor (FcR) on a cell. There are several Fc receptors that are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Antibody binding to Fc receptors on cell surfaces triggers several important and diverse biological responses including engulfment and destruction of antibody-coated particles, elimination of immune complexes, lysis of antibody-coated target cells by killer cells (called mediated cytotoxicity). by antibody-dependent cells or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.

[00230] 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 which, in turn, time, it affects the biological profile of the administered anti-LOX1 antibody. For example, deletion or inactivation (by point mutations or other means) of a constant region domain can reduce binding to the Fc receptor of the circulating modified antibody. In other cases, constant region modifications may moderate complement binding and thus reduce serum half-life and nonspecific association of a conjugated cytotoxin. Still other modifications of the constant region can be used to eliminate disulfide bonds or oligosaccharide chemical moieties that allow for improved localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in accordance with the present disclosure can be easily effected using well-known biochemical or molecular manipulation techniques well within the reach of the skilled artisan.

[00231] In some aspects, a LOX1-binding protein provided herein is an antibody against LOX1 (e.g., a full-length anti-LOX1 antibody and a LOX1-binding antibody fragment, and variants and derivatives thereof ) that lacks one or more effector functions. For example, in some aspects, the anti-LOX1 antibody has no antibody-dependent cellular cytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity (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.

[00232] 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 spacer of 5 to 20 amino acids. Such a spacer may be added, for example, to ensure that the constant domain regulatory elements remain free and accessible or that the hinge region remains flexible. Amino acid spacers may, in some cases, be shown to be immunogenic and induce an unwanted immune response against the construct. Thus, in certain aspects, any spacer added to the construct may be relatively non-immunogenic, or even omitted entirely, in order to maintain the desired biochemical qualities of the modified anti-LOX1.

[00233] In addition to the deletion of entire 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 deletion or partial replacement of some or even a single amino acid in a constant region. For example, mutation of a single amino acid in selected areas of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby. Similarly, one or more constant region domains that control effector function (e.g., complement C1Q binding) can be completely or partially deleted. Such partial deletions of the constant regions can improve selected characteristics of the antagonistic anti-LOX1 antibody (e.g., serum half-life) while leaving other desirable functions associated with the corresponding constant region domain intact. Furthermore, the constant regions of the anti-LOX1 antibodies provided herein can be modified through mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this regard it is possible to destroy 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 in the constant region to enhance desirable characteristics, such as decreasing or increasing effector function or providing binding sites for more cytotoxin, tag or carbohydrate moieties. In such aspects, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

[00234] The disclosure is also directed to variants and equivalents that are substantially homologous in terms of structure and/or similar in one or more functions to the anti-LOX1 antibodies disclosed herein (e.g., murine LOX1-binding proteins, chimeric , humanized and human). These variants may contain, for example, conservative substitution mutations of amino acid residues. As is generally understood in the art, a conservative substitution of amino acid residues refers to the replacement of one amino acid residue with another of the same general class such as, for example, an acidic amino acid for another acidic amino acid, a basic amino acid for another amino acid. basic or a neutral amino acid for another neutral amino acid. It is known in the art what is intended by a conservative substitution of amino acids.

[00235] LOX1 binding proteins, such as anti-LOX1 antibodies, provided herein can be derivatized 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 otherwise improve the stability, formulation and/or therapeutic properties of the LOX1 binding protein. A non-exhaustive review of such fractions can be found, for example, in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

SAW. Nucleic acids encoding LOX1-binding proteins and their expression

[00236] The present disclosure provides nucleic acid molecules that encode LOX1 binding proteins (see, for example, those described in Table 1, Figure 4 or Figure 5) including anti-LOX1 antibodies, such as anti-LOX1 antibodies of length full-length, LOX1-binding antibody fragments, and variants and derivatives thereof. The nucleic acid molecules disclosed herein may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and it can be double-stranded or single-stranded, and if it is single-stranded, it can be the coding strand or non-coding (antisense) strand. In certain aspects, the nucleic acid molecule is isolated. In further aspects, the 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 produced recombinantly.

[00237] In some aspects, the nucleic acid molecule comprises a LOX1 binding protein coding sequence operably linked to a control sequence that controls 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 expression of the coding sequence in a host cell or in vitro.

[00238] 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 secretion of the expressed protein from the host cell transformed with the nucleic acid molecule encoding LOX1. The protein containing a leader sequence is called a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. Such leader peptide sequences and their use to facilitate the secretion of recombinant proteins into host cells is generally known in the art. In further aspects, the heterologous polynucleotide sequence encodes additional 5' and/or 3' amino acid residues that may function, for example, to facilitate purification, add to or improve protein stability and/or therapeutic or diagnostic properties of the binding protein. to recombinantly expressed LOX1.

[00239] 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.

[00240] 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 which have a total of one, two, three, four, five, six, seven, eight or less amino acid substitutions in one or more of the VL CDRs in relation 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 which have a total of one, two, three, four, five, six, seven, eight or less amino acid substitutions in one or more of the VL CDRs with respect to: SEQ ID NO:55 or 59; SEQ ID NO:56 or 60 or SEQ ID NO:57, 61-63 or 64, respectively.

[00241] The disclosure further provides an isolated nucleic acid molecule encoding a heavy chain (VH) variable region, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 amino acid sequences identical to, or which have a total of one, two, three, four, five, six, seven, eight or less amino acid substitutions in one or more of the VH CDRs with respect 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 (VH) variable region, wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 amino acid sequences identical to, or which have a total of one, two, three, four, five, six, seven, eight or less amino acid substitutions in one or more of the VH CDRs with respect to: SEQ ID NO:38; SEQ ID NO:39, 42 or 43; and SEQ ID NO:40, 44-46 or 47, respectively.

[00242] The disclosure further provides an isolated nucleic acid, such as a cDNA, that encodes a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least 70%, 75%, 80% , 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with 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 that has at least 90%, 95%, 97%, 98%, 99% identity. of sequences with the reference amino acid sequence.

[00243] Furthermore, the disclosure provides an isolated nucleic acid molecule that encodes a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with 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 that has at least 90%, 95%, 97%, 98%, 99% identity. of sequences with the reference amino acid sequence.

[00244] 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, Figure 4 or Figure 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.

[00245] In certain aspects, the heavy chain variable region (VH) and the light chain variable region (VL) are encoded by nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with 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.

[00246] The disclosure further provides an isolated nucleic acid, such as a cDNA, that encodes a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least 70%, 75%, 80% , 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with 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 that is at least 90%, 95%, 97%, 98%, 99% sequence identity with the reference amino acid sequence.

[00247] The disclosure also provides an isolated nucleic acid molecule that encodes a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with 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 that has at least 90%, 95%, 97%, 98%, 99% sequence identity. with the reference amino acid sequence.

[00248] In certain aspects, the heavy chain variable region (VH) and the light chain variable region (VL) are encoded by nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with 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.

[00249] 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, Figure 4 or Figure 5. In some further aspects, the isolated nucleic acid encodes a LOX1 binding protein comprising a heavy chain variable region (VH) and a light chain variable region (VL) as described in Table 1 , Figure 4 or Figure 5.

[00250] In a composition of nucleic acid molecules as described above, the nucleic acid encoding a VH and the nucleic acid encoding a VL may reside in a single vector, or may be in separate vectors. Thus, the disclosure provides one or more vectors comprising the composition of nucleic acid molecules or combination of nucleic acid molecules described above.

[00251] In some cases, a polynucleotide composition encoding a VH and VL as described above may 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 further 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 a light chain variable region (VL) described in Table 1 , Figure 4 or Figure 5.

[00252] In further aspects, the disclosure provides a host cell comprising a nucleic acid or nucleic acids or vector as provided above, wherein the host cell may, in some cases, express a LOX1-binding protein (e.g., an antibody anti-LOX1, 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 used in a method of preparing a LOX1-binding protein as provided herein, wherein the method includes (a) culturing the host cell and (b) isolating the LOX1-binding proteins expressed from of the host cell.

[00253] The disclosure also provides a method for preparing a LOX1-binding protein comprising culturing a host cell or hybridoma capable of expressing the LOX1-binding protein under appropriate conditions, and optionally provides a method for isolating the LOX1-binding protein secreted from the host cell or hybridoma. Furthermore, the disclosure further provides 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.

[00254] In certain aspects, the polynucleotides comprise the coding sequence(s) for the mature LOX1-binding protein(s) (e.g., an antibody against LOX1, such as a full-length antibody and a LOX1-binding antibody fragment) fused in the same reading frame to a tag sequence that allows, for example, purification of the encoded polypeptide. For example, the tag sequence may be a hexahistidine tag provided by a pQE-9 vector to provide purification of the mature polypeptide fused to the tag in the case of a bacterial host, or the tag sequence may be a hemagglutinin tag. (HA) derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.

[00255] 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 may contain changes in coding regions, non-coding regions, or both. In some aspects, nucleic acid variants contain changes that produce silent substitutions, additions, or deletions but do not alter the properties or activities of the encoded polypeptide. In some aspects, nucleic acid variants are produced by silent substitutions due to degeneration of the genetic code. Nucleic acid variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (changing codons in 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.

[00256] 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 synthesis chemistry using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and codon optimization based on host cell preferences. Standard methods can be routinely applied to synthesize isolated polynucleotide sequences encoding LOX1-binding proteins.

[00257] Once assembled (by synthesis, site-directed mutagenesis or other method), nucleic acid sequences encoding LOX1 binding proteins can be routinely operably linked to an appropriate control sequence for expression of the binding proteins to LOX1 in a desired host. In some aspects, nucleic acid sequences encoding LOX1 binding proteins are inserted into an expression vector and operably linked to an appropriate control sequence 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.

[00258] 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 that 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, e.g., promoters or transcription enhancers, (2) a structural or coding sequence that 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 may 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 be additionally incorporated. Regions of DNA are operationally 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 that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls 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 allowing extracellular secretion of the translated protein from a host cell. Alternatively, when a recombinant protein is expressed without a leader or shuttle sequence, the protein may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final protein. In certain aspects, the disclosure provides a composition, for example a pharmaceutical composition, comprising a nucleic acid or vector as described above or elsewhere herein, optionally further comprising one or more carriers, diluents, excipients or other additives.

[00259] 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 operatively 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 an NS0 murine myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell. In other respects, the host cell is a hybridoma.

[00260] Additionally, host cells expressing nucleic acids encoding the LOX1-binding proteins disclosed herein are hybridomas that produce a LOX1-binding protein.

[00261] In further aspects, the disclosure provides a method of preparing 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 hybridoma disclosed herein under conditions appropriate for producing the LOX1 binding protein. The disclosure optionally provides for the isolation of the LOX1 binding protein secreted from the host cell or hybridoma. The disclosure also optionally provides LOX1-binding protein produced using such a method and pharmaceutical compositions comprising LOX1-binding protein and a pharmaceutically acceptable carrier.

[00262] The choice of expression control sequence and expression vector will depend on the choice of host. A wide variety of host/expression vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papillomavirus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as E. coli plasmids, including pCR 1, pBR322, pMB9 and their derivatives, and also plasmids for a broader range of hosts, such as M13 and E. coli phages. filamentous single-stranded DNA.

[00263] Host cells suitable 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 same) 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 lineages of mammalian origin as described below. Cell-free translation systems can also be employed. Additional information on protein production methods, including antibody production, can be found, for example, in U.S. Publication No. 2008/0187954, and in U.S. Patent Nos. 6,413,746 and 6,660,501, and in International Application Publication No. WO 04009823, each of which is hereby incorporated herein by reference in its entirety.

[00264] Various mammalian or insect cell culture systems can also be advantageously employed to express a recombinant LOX1-binding protein (e.g., an anti-LOX1 antibody, such as an antibody against full-length LOX1 and a fragment of LOX1-binding antibody, and variants and derivatives thereof). Expression of recombinant LOX1-binding proteins in mammalian cells can be accomplished since these proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 monkey kidney cell lines described by Gluzman (Cell 23:175, (1981)), and other cell lines, including , for example, L, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors may comprise non-transcribed 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 non-transcribed sequences, and 5' or 3' untranslated sequences. ', such as necessary ribosome binding sites, a polyadenylation site, splicing donor and acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are reviewed by Luckow and Summers, BioTechnology 6:47 (1988).

[00265] 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 appropriate method. Such standard methods include chromatography (e.g., ion exchange, affinity, and size column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be linked to the protein to allow easy purification by passage over an appropriate affinity column. Isolated LOX1-binding proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[00266] For example, supernatants from systems that secrete recombinant LOX1-binding proteins into the culture medium can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Pellicon ultrafiltration unit from Millipore. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin may 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 used 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 methyl or other pendant aliphatic groups, may be employed to further purify a LOX1 binding protein. Some or all of the above purification steps, in various combinations, can also be routinely employed to provide homogeneous recombinant LOX1-binding proteins.

[00267] 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 it can be isolated, for example, by initial extraction from cell pellets, followed by one or more steps of concentration, desalination, aqueous ion exchange or size exclusion chromatography. High performance liquid chromatography (HPLC) can be employed for the final purification steps. Microbial cells used in expressing a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

[00268] Methods known in the art for the purification of target binding proteins, such as full-length antibodies and antigen-binding antibody fragments also include, for example, those described in U.S. application publications Nos. 2008/0312425, 2008 /0177048 and 2009/0187005, each of which is hereby incorporated herein by reference in its entirety.

VII. Treatment methods using therapeutic LOX1-binding proteins

[00269] Methods are provided for using 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). same) to treat subjects who have 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.

[00270] In further aspects, the disclosure provides a pharmaceutical composition containing a LOX1 binding protein provided herein (e.g., an anti-LOX1 antibody or a 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 the treatment, prevention and/or amelioration of a disease or pathology 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.

[00271] 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 marker group or an effector group. As used herein, a "tag" refers to one or more elements, isotopes, or chemical compounds linked to enable detection in a trace. Tags generally fall into three classes: (a) isotopic tags, which can be radioactive or heavy isotopes, (b) small molecular tags, which can include fluorescent and colorimetric dyes, or molecules such as biotin that allow for other tagging methods, and (c) immunological tags, which may be an epitope incorporated as a fusion partner that is recognized by an antibody. “Tagger group” refers to any detectable tag. In some aspects, the tag group is coupled to the LOX1-binding protein through a spacer (e.g., a peptide spacer) to reduce potential steric hindrance. Tags can be incorporated in the compound at any position and can be incorporated in vitro or in vivo during protein expression. Various methods for labeling proteins are known in the art and can be used in carrying out the methods provided. 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 label 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, mKalama1, 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 selected member of a biotin signaling peptide, histidine (his) peptide, hemagglutinin (HA), Flag, gold-binding peptide. In further aspects, the effector group is selected from the group consisting of a radioisotope, radionuclide, a toxin, a therapeutic agent and a chemotherapeutic agent.

[00272] In other aspects, the effector group is selected from the group consisting of a radioisotope, radionuclide, a toxin, a therapeutic agent and a chemotherapeutic agent.

[00273] The following discussion relates to methods of diagnosis 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 antibody against full-length LOX1, a LOX1-binding antibody fragment, and variants and derivatives thereof). In some aspects, the LOX1 binding proteins are human, murine or humanized antibodies.

[00274] 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 an antibody against full-length LOX1 , a LOX1-binding antibody fragment, and variants and derivatives thereof) to a subject who has a disease or condition, or who is at risk of developing a disease or condition, associated with LOX1. In another aspect, the treatment includes administering a LOX1-binding protein to a tissue or cell isolated from a subject, wherein the subject has a disease or condition, or is at risk of developing a disease or condition, associated with the LOX1.

[00275] The disclosure also provides methods of treating, preventing and/or ameliorating a disease or pathology associated with atherosclerosis, thrombosis, CAD, ischemia (e.g., myocardial ischemia), infarction (e.g., myocardial infarction), syndrome acute coronary artery disease (ACS), stroke, reperfusion injury, restenosis, peripheral vascular disease, hypertension, heart failure, inflammation (e.g., chronic inflammation), angiogenesis, pre-eclampsia, and cancer in one subject. In some cases, 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.

[00276] The disclosure also provides methods for 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, DKA, ischemia (e.g., myocardial ischemia), infarction (e.g., myocardial infarction), acute coronary artery disease (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 binding to LOX1 (e.g., an anti-LOX1 antibody, such as a full-length LOX1 antibody, a LOX1-binding antibody fragment, and variants and derivatives thereof).

[00277] Methods of treating, preventing and/or improving atherosclerosis are additionally provided. In some cases, 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 who has atherosclerosis. In other aspects, the subject to whom 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 pro-atherogenic pathology. In additional 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) and/or bacterial infection. Methods for decreasing atherosclerosis are also provided. In some cases, the disclosure provides a method for decreasing atherosclerosis in a subject comprising administering a LOX1 binding protein to a subject who has atherosclerosis.

[00278] Methods of treating, preventing and/or improving thrombosis are also provided. In some cases, the method comprises administering a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) to a subject who has thrombosis. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing thrombosis. In some aspects, thrombosis is an arterial thrombosis. In further aspects, thrombosis is a deep vein thrombosis.

[00279] The disclosure also provides methods of treating, preventing and/or improving coronary artery disease (CAD) or a pathology associated with CAD. In some cases, 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 who has DKA. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing DKA. In some aspects, the subject has a pro-atherogenic pathology. In additional aspects, proatherogenic pathology 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) and/or bacterial infection.

[00280] The disclosure also provides methods of treating, preventing and/or ameliorating ischemia or a pathology associated with ischemia. In some cases, 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 who has ischemia. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing ischemia. In some aspects, the subject has myocardial ischemia. In other respects, 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 transplant, dyslipidemia (e.g., hyperlipidemia), inflammation (e.g., chronic inflammation and endotoxin-induced inflammation). ) and/or bacterial infection.

[00281] Methods of treating, preventing and/or improving a heart attack or a pathology associated with a heart attack are also provided. In some cases, 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 a heart attack. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing a heart attack. In some respects, the subject has a myocardial infarction. In other respects, 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 induced inflammation). endotoxin) and/or bacterial infection.

[00282] The disclosure also provides methods of treating, preventing and/or improving acute coronary syndrome (ACS) or a pathology associated with ACS. In some cases, 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 who has ACS. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing ACS. In some aspects, the subject has elevated serum levels of sLOX or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[00283] The disclosure also provides methods of treating, preventing and/or ameliorating a stroke or a pathology associated with a stroke. In some cases, 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 who has had a stroke. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of having a stroke. In some aspects, the subject has elevated serum levels of sLOX or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[00284] Methods of treating, preventing and/or improving a reperfusion injury or a pathology associated with a reperfusion injury are also provided. In some cases, 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 who has reperfusion injury. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing reperfusion injury. In some respects, the subject is about to have surgery. In other respects, the guy had surgery. In some aspects, the surgery is transplant or coronary bypass surgery. In additional aspects, the patient has, or is at risk of developing, myocardial ischemia and reperfusion injury. In additional 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., LVP reduced and LVEDP increased). In additional aspects, the method increases cardiac stroke volume, fractional shortening and/or injection fraction.

[00285] The disclosure also provides methods of treating, preventing and/or improving restenosis or a pathology associated with restenosis. In some cases, 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 who has restenosis. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing restenosis. In some respects, the subject is about to have surgery. In other respects, the guy had surgery. In some respects, the surgery is an endovascular procedure. In additional aspects, the surgery is vascular surgery, cardiac surgery or angioplasty. In additional aspects, the procedure is transplant or coronary bypass surgery. In additional aspects, the restenosis treated, prevented and/or improved is in-stent restenosis or post-angioplasty restenosis.

[00286] In additional aspects, the disclosure provides methods of treating, preventing and/or ameliorating peripheral vascular disease (PVD) or a pathology associated with PVD. In some cases, the method comprises administering a LOX1-binding protein (e.g., in a pharmaceutical composition described herein) to a subject who has PVD. In other aspects, the subject to whom the LOX1-binding protein is administered is at risk of developing PVD. In some aspects, the subject has elevated serum levels of sLOX or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[00287] The disclosure also provides methods of treating, preventing and/or ameliorating inflammation or a pathology associated with inflammation. In some cases, 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 who has inflammation. In additional aspects, the subject has chronic inflammation. In some aspects, the subject has elevated serum levels of oxLDLa and/or sLOX and/or elevated LOX1 activity. In further aspects, the subject has atherosclerosis.

[00288] The disclosure also provides methods of treating, preventing and/or ameliorating pre-eclampsia or a condition associated with pre-eclampsia. In some cases, the method comprises administering a LOX1-binding protein (e.g., an anti-LOX antibody or fragment thereof in a pharmaceutical composition described herein) to a subject who has pre-eclampsia or eclampsia. In additional aspects, the subject has high blood pressure and large amounts of protein in the urine. In some aspects, the subject has elevated serum levels of oxLDL and/or sLOX1 and/or elevated LOX1 activity. In additional aspects, the subject has swelling in the feet, legs and/or hands.

[00289] In additional aspects, the disclosure provides methods of stabilizing an atherosclerotic plaque in a subject. In some cases, 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 who needs it. In some aspects, the method reduces signaling from the RhoA/Rac1 signal transduction pathway, nitrogen monoxide, p38MAPK, protein kinases B and C, ERK1/2 and/or NFKB in the plaque. In other aspects, the method reduces apoptosis in the plaque. In additional aspects, the method decreases the activity of caspase 8, caspase 9 and/or BAX, and/or increases the activity of BCL-2 in the plaque. In other aspects, the method decreases the levels of an adhesion molecule or cytokine produced by the plaque. In additional aspects, the method decreases the expression of E-selectin, P-selectin, ICAM-1, VCAM-1, MCP1 and/or CD40/CD40L by the plaque. In additional aspects, the method decreases the size or formation of atherosclerotic plaques, the accumulation of macrophages and/or the expression of MMP (e.g., MMP9) in the atherosclerotic plaque. In additional aspects, the method results in reduced plaque progression or regression.

[00290] Methods of reducing the loss of vascular tone in a subject are also provided. In some cases, 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 who needs it. In some aspects, the method reduces the loss of vascular tone. In additional aspects, the method reduces the loss of vascular tone in a subject by regulating HDL-induced nitric oxide (NO) production (the antibody's ability to stimulate endothelial NO production).

[00291] Methods for improving vascular tone in a subject are additionally provided. In some cases, the method comprises administering a LOX1 binding protein (e.g., an anti-LOX1 antibody or fragment thereof) to a subject in need thereof.

[00292] In some aspects, the disclosure provides methods of treating, preventing and/or improving a pathology associated with hyperglycemia or hypertension. In some cases, the method comprises administering a LOX1-binding protein to a subject who has hyperglycemia or hypertension.

[00293] Methods of treating, preventing and/or improving cancer are additionally provided. In some cases, the disclosure provides a method of treating, preventing and/or ameliorating cancer in a subject comprising administering a LOX1 binding protein to a subject who has 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.

[00294] Methods of inhibiting the proliferation, migration or invasion of tumor cells are also provided. In some cases, the disclosure provides a method for antagonizing LOX1 activity comprising contacting a LOX1-binding protein with a LOX1-expressing tumor cell. 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 of a cancerous lineage.

[00295] 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 pathology associated with pathological angiogenesis. In further aspects, the disclosure provides a method of inhibiting angiogenesis comprising 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.

[00296] Methods of blocking or reducing LOX1 activity are additionally provided. 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, vascular smooth muscle cell and/or platelet. In some aspects, the cell is an endothelial cell, such as a coronary endothelial cell. In further 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 activity of LOX1 is the binding and/or uptake (e.g., internalization) of oxLDL. In additional aspects, the blocked or reduced activity of LOX1 is the induction of the signaling pathway of p38 (MAPK), p44/42 MAPK, protein kinase C (PKC), protein kinase B (PKB), protein tyrosine kinase (PTK), NF-KB and/or AP1 transcription factor. In additional aspects, blocked or reduced activity of LOX1 is the induction of apoptosis. In additional embodiments, induction of apoptosis is mediated by caspase-9, caspase-3 and/or Bcl-2. In additional aspects, the blocked or reduced activity of LOX1 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 LOX1 activity induced by the binding of oxLDL to LOX1.

[00297] Methods of blocking or reducing LOX1 activity in a pathological condition associated with increased levels of LOX1 activity or increased levels of LOX1 expression (e.g., serum protein levels of sLOX1) are further provided. In some cases, the method comprises administering a LOX1-binding protein (e.g., an anti-LOX1 antibody or fragment thereof) to a subject who has increased LOX1 activity or increased levels of LOX1 expression (e.g., protein levels sLOX1 serum levels). In some aspects, the pathologic pathology is systemic lupus erythematosus (SLE), diabetes, hypertension, hyperglycemia, heart failure, vascular injury, organ 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, IL1, 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 K167N variant of LOX1.

[00298] Methods for agonizing or increasing the activity of high-density lipoprotein (HDL) are also provided. In some aspects, the disclosure provides a method for increasing or agonizing 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 respects, increased HDL activity is the promotion of HDL-mediated endothelial NO production. In some aspects, increased HDL activity is inhibition of endothelial cell NFKB signaling activity. In some respects, increased HDL activity is promoting endothelial cell repair. In some ways, increased HDL activity is reduced inflammation.

[00299] 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 evaluation procedure, for example, to determine the effectiveness of a given treatment regimen. For example, detection can be facilitated by coupling the LOX1 binding protein (e.g., an 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, β-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 125I, 131I, 35S or 3H.

VIII. Pharmaceutical Compositions and Methods of Administration

[00300] Methods of preparing and administering a LOX1 binding protein provided herein to a subject in need thereof are known or readily determined by those skilled in the art. The route of administration of LOX1 binding proteins may be, for example, oral, parenteral, inhalation or topical. The term parenteral as used herein includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. While all of these forms of administration are clearly contemplated as being within the scope of the invention, another example of a form for administration would be a solution for injection, in particular for intravenous or intra-arterial injection or drip. Generally, a suitable pharmaceutical composition may comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizing agent (e.g., human albumin), etc. In other methods compatible with the teachings herein, the LOX1-binding proteins provided herein can be delivered directly to the organ and/or site of atherosclerosis or tumor, thereby increasing exposure of the diseased tissue to the therapeutic agent. In one aspect, administration is directly to the airways, for example, by inhalation or intranasal administration.

[00301] As discussed herein, LOX1-binding proteins can be administered in a pharmaceutically effective amount for the in vivo treatment of LOX1-mediated diseases and pathologies, such as atherosclerosis, thrombosis, CAD, ischemia (e.g., ischemia myocardial), 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, pre -eclampsia and cancer. In this regard, it will be recognized that the disclosed LOX1-binding proteins can be formulated so as to facilitate administration and promote stability of the active agent. Pharmaceutical compositions according to the disclosure may comprise a pharmaceutically acceptable non-toxic sterile carrier, such as saline, non-toxic buffers, preservatives and the like. For the purposes of the present application, a pharmaceutically effective amount of a LOX1-binding protein, conjugated or unconjugated, means an amount sufficient to achieve effective binding to LOX1 and achieve a benefit, for example, to improve the symptoms of a disease or pathology or detect a substance or cell. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th edition (1980).

[00302] Certain pharmaceutical compositions provided herein can be administered orally in an acceptable dosage form, including, for example, capsules, tablets, suspensions or aqueous solutions. Certain pharmaceutical compositions may also be administered by aerosol or nasal inhalation. Such compositions can be prepared as saline solutions, using benzyl alcohol or other suitable preservatives, absorption promoters to increase bioavailability, and/or other conventional solubilizing or dispersing agents.

[00303] 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 on the subject treated and the particular route of administration. The composition may be administered as a single dose, or multiple doses over a set period of time in an infusion. Dosage regimens can also be adjusted to provide the desired optimal response (e.g., a therapeutic or prophylactic response).

[00304] Staying within the scope of the present disclosure, a LOX1 binding protein can be administered to a human or other subject in accordance with the aforementioned treatment methods in an amount sufficient to produce a therapeutic effect. The LOX1-binding proteins provided herein can be administered to such a human or other animal in a conventional dosage form prepared by combining the LOX1-binding proteins with a conventional pharmaceutically acceptable carrier or diluent in accordance with known techniques. The form and type of pharmaceutically acceptable vehicle or diluent may be determined by the amount of active ingredient with which it will be combined, the route of administration and other well-known variants. A cocktail comprising one or more different LOX1-binding proteins may also be used.

[00305] By "therapeutically effective dose or amount" or "effective amount" is meant an amount of a LOX1-binding protein (e.g., an anti-LOX1 antibody, such as a full-length LOX1 antibody and a fragment of LOX1-binding antibody, and variants and derivatives thereof) which, when administered, causes a positive therapeutic response in relation to the treatment of a subject with a disease or pathology to be treated.

[00306] Therapeutically effective doses of LOX1-binding protein compositions for treating LOX1-mediated diseases or pathologies, such as atherosclerosis, thrombosis, CAD, ischemia (e.g., myocardial ischemia), infarction (e.g., heart attack heart disease), 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 on many factors different, including routes of administration, target site, physiological state of the subject, whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Typically, 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 skilled in the art to optimize safety and efficacy.

[00307] As used herein, improving the symptoms of a particular disease or condition by administration of a LOX1-binding protein refers to any reduction, permanent or temporary, lasting or transient, that can be attributed to or associated with the administration of a LOX1-binding protein.

[00308] The amount of at least one LOX1 binding protein, e.g., antibody or binding fragment, to be administered is readily determined by a person of ordinary skill in the art without unnecessary experimentation given the present disclosure. Factors that influence the route of administration and the respective amount of at least one LOX1-binding protein include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health status, and physical condition of the individual undergoing therapy. Similarly, the amount of a LOX1-binding protein to be administered will depend on the route of administration and whether the subject receives a single dose or multiple doses of that agent.

[00309] The disclosure also provides 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 medicine, for example, to improve 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.

Combination therapies

[00310] 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 may be administered in combination with the LOX1 binding proteins provided herein include, but are not limited to, platelet inhibitors, anticoagulants, antihypertensives, glycoprotein IIb/IIIa receptor inhibitors, beta-blockers. , calcium channel blockers, HMG-CoA reductase inhibitors (statins), ezetimibe, fibrates (e.g., Gemifibrozil and Fenofibrate), Zetia, bile acid sequestrants, nitrates, and thrombolytic agents.

[00311] In various aspects, a LOX1 binding protein is administered to a subject before, during and/or after a surgical procedure. In some respects, the surgery is an endovascular procedure. In additional aspects, the surgery is vascular surgery, cardiac surgery or angioplasty. In additional aspects, the procedure is transplant or coronary bypass surgery.

IX. Diagnostics

[00312] The present disclosure further provides a useful diagnostic method during the diagnosis of LOX1-mediated diseases and pathologies, 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, pre-eclampsia and cancer, which involves measuring the level of protein expression of LOX1 (including sLOX1) in tissue or body fluid, such as serum, from an individual and compare the measured expression level with a standard LOX1 expression level in normal tissue or body fluid, in which an increased expression level compared The pattern is indicative of a disorder treatable by a LOX1-binding protein provided herein, such as a full-length anti-LOX1 antibody and an antigen-binding antibody fragment as provided herein.

[00313] 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 assess protein levels of LOX1 in a biological sample using classical immunohistological methods known to those skilled in the art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al. al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting LOX1 protein expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blot.

[00314] By "assessing the expression level of LOX1 protein" is intended to mean the qualitative or quantitative measurement or estimation of the level of LOX1 protein in a first biological sample in a direct manner (e.g., determining or estimating the absolute level of protein ) or relative (e.g., comparing with the disease-associated polypeptide level in a second biological sample). The expression level of LOX1 protein in the first biological sample can be measured or estimated and compared to a standard level of LOX1 protein, wherein the standard is taken from a second biological sample obtained from an individual who does not have the disorder or is determined by taking the average levels of a population of individuals who do not have the disorder. As will be recognized in the art, once the "standard" level of LOX1 protein is known, it can be used repeatedly as a standard for comparison.

[00315] By "biological sample" is intended to mean any biological sample obtained from an individual, cell line, tissue culture or other source of cells potentially expressing LOX1. Methods of obtaining biopsies of mammalian tissue and body fluids are known in the art.

X. Kits comprising LOX1-binding proteins

[00316] The present 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 an appropriate packaging and written material, and which can be used to carry out the methods described in this document.

[00317] Written material may include any of the following information: instructions for use, discussion of clinical studies, list of side effects, references to scientific literature, packaging leaflet materials, results of clinical studies and/or summaries of these and the like . Written material may indicate or establish the activities and/or advantages of the composition, and/or describe dosage, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may 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 studies. The kit may additionally 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).

[00318] In certain aspects, a kit comprises at least one purified LOX1-binding protein in one or more containers. In some respects, kits contain all components necessary and/or sufficient to perform a detection assay, including all controls, instructions for performing assays, and any programs necessary for analysis and presentation of results.

XI. Immunoassays

[00319] LOX1-binding proteins (e.g., anti-LOX1 antibodies and LOX1-binding fragments, variants or derivatives thereof) can be assessed for immunospecific binding by any method known in the art. 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 just a few. Such assays are routine and known in the art (see, for example, Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is incorporated herein by reference in its entirety).

[00320] The LOX1-binding proteins (e.g., anti-LOX1 antibodies or LOX1-binding fragments, variants or derivatives thereof) provided herein can be employed histologically, such as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of LOX1 or conserved variants or their peptide fragments. In situ detection can be performed by removing a histological specimen from a subject, and applying labeled LOX1-binding proteins to it by coating a biological sample with the labeled LOX1-binding proteins. Using 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 understand that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

[00321] The binding activity of a given batch 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 test conditions for each determination using routine experimentation.

[00322] Suitable methods and reagents for determining the binding characteristics of an isolated LOX1-binding protein are known in the art and/or are commercially available. Equipment and programs designed for such kinetic analyzes are commercially available (e.g., BIACORE®, BIAevaluation® software, GE Healthcare; KINEXA® Software, Sapidyne Instruments).

[00323] Unless otherwise indicated, the practice of 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 fully explained 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. Patent 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 treatment, 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.).

[00324] The general principles of antibody manipulation are presented in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles of protein manipulation are presented in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The general principles of antibodies and antibody-hapten binding are presented 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 and Co., NY).

[00325] Standard reference works presenting the 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, editors Burden et al., (Elsevere, Amsterdam); Goldsby et al., editors (2000) Kuby Immunology (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 Hall2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach et al. (2003) PCR Primer (Cold Spring Harbor Press).

[00326] The following examples are presented for illustration purposes and not by way of limitation.

EXAMPLES

[00327] Aspects of the present disclosure may be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods for using the antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both in materials and methods, can be made without departing from the scope of the present disclosure.

Example 1. Isolation and identification of anti-LOX1 scFv antibodies

[00328] A large library of human single-chain Fv antibodies (scFv) derived from bone marrow from unexposed ("naïve") adult donors was used and cloned into a phagemid vector based on filamentous M13 phage for selections (Hutchings, C., Generation of Naive Human Antibody Libraries, in Antibody Engineering, Editors R. Kontermann and S. Dubel, 2001, Springer Laboratory Manuals, Berlin p. LOX1-specific scFv antibodies were isolated from the phage display library in a series of repeated rounds of selection on mammalian-expressed recombinant human LOX1 (R&D Systems) essentially as previously described (Vaughan et al., Nat. Biotechnol. 14:309 ( 1996)). Although several antigen-specific scFvs were obtained from different variations of this protocol, the parent clones LOX514 (SEQ ID NO:29 (VH) and SEQ ID NO:33 (VL); also referred to herein as "LOX10514") and LOX696 (SEQ ID NO:54 (VH) and SEQ ID NO:70 (VL); also referred to herein as "LOX10696") were isolated as follows: 10 μg/ml human LOX1 was immobilized on MAXISORP™ plates ( Nunc) and incubated with the phage display library. Unbound phage were removed by washing in a series of washing cycles. The phage particles retained on the antigen were eluted, infected into bacteria and recovered for the next round of selection. Three selection cycles were carried out in this way. A representative number of individual clones from cycle 2 and cycle 3 selection were grown 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.

[00329] The screening hits, i.e. scFv clones that showed an inhibitory effect on the 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)). Single scFvs were reexpressed in bacteria and purified by affinity chromatography (as described in Example 3 of International Application Publication No. WO01/66754), and IC50 values were determined by testing dilution series of purified scFvs in the above assay.

[00330] The most potent scFv clones were converted into the full-length immunoglobulin G1 triple mutant antibody format (IgG1-TM, human IgG1 Fc sequence incorporating the L234F, L235E and P331S mutations) essentially as described in Persic et al., Gene 187:9 (1997) with the following modifications. A fragment of OriP 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 the entire IgG1-TM heavy chain in mammalian cells. Similarly, the VL domain was cloned into a vector for the expression of human light chain constant domains and regulatory elements to express the entire IgG light chain in mammalian cells. To obtain IgGs, vectors expressing heavy and light chain IgG were transfected into mammalian CHO cells. IgGs were expressed and secreted into the medium. Collections were pooled and filtered, and IgG was purified using protein A chromatography. Culture supernatants were loaded onto an appropriately sized ceramic protein A column (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 with the addition of Tris-HCl (pH 9.0). The eluted material was buffer exchanged to PBS using Nap10 columns (Amersham, #17-0854-02) and the IgG concentration was determined spectrophotometrically using an extinction coefficient based on the IgG amino acid sequence (Mach et al., Anal Biochem , 200:74 (1992)). Purified IgGs were analyzed for aggregation and degradation using SEC-HPLC and by SDS-PAGE.

EXAMPLE 2 - Anti-LOX1 antibodies inhibit multiple ligand binding, oxLDL internalization and oxLDL-induced signaling

[00331] Purified IgGs were tested for their ability to specifically inhibit the binding of oxidized low-density lipoprotein ("oxLDL"), advanced glycation end products of bovine serum albumin ("AGE-BSA"), and C-reactive protein ( "CRP") to LOX1 as described in Example 10, Assays 1, 2 and 3. Several isolated clones including LOX10514 and LOX10696, and a commercially available mouse anti-LOX1 antibody (23C11) were tested for their ability to block binding from oxLDL, AGE-BSA and CRP to LOX1. Representative graphs are shown in Figures 1A, 1B and 1C showing the inhibition of oxLDL, AGE-BSA and CRP binding, respectively, by LOX514 and LOX696. To confirm that the antibodies were cross-reactive and had functional activity on LOX1 SNP K167N, the antibodies were tested for their ability to block the binding of oxLDL to LOX1 SNP K167N. LOX1 SNP K167N (GenBank accession number 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 developing myocardial infarction. See, for example, Tatsuguchi et al., Biochem Biophys Res Commun. 28;303(1):247-50 (2003). Representative graphs are shown in Figure 1D showing that both LOX514 and LOX696 block the binding of oxLDL to the LOX1 SNP K167N variant. In addition to binding inhibition, several isolated clones including LOX10514 and LOX10696 were tested for their ability to block oxLDL internalization in cells expressing LOX1 (as described in Example 10, Assay 4) and oxLDL-induced LOX1 signaling (as described in Example 10, Trial 5). Representative graphs are shown in Figures 2A and 2B for inhibition of oxLDL internalization and oxLDL signaling, respectively. The results of these studies are summarized in Table 2. Table 2: Inhibition of oxLDL internalization

[00332] These results demonstrate: (1) the specific inhibition of LOX1 binding to oxLDL, AGE-BSA and CRP by the LOX514 and LOX696 antibodies; (2) that both LOX514 and LOX696 functionally cross-react with the common LOX1 SNP K167N variant; (3) that LOX514 and LOX696 inhibit oxLDL internalization; and (4) that LOX514 and LOX696 inhibit oxLDL-induced LOX1 signaling.

EXAMPLE 3 - Species cross-reaction and specificity for human LOX1 of anti-LOX1 antibodies in a panel of human related family members

[00333] The cross-reactivity of anti-human LOX1 antibodies with various LOX1 species orthologs was evaluated by a scFv binding ELISA. Extracellular domain constructs for human (Uniprot: P78380), mouse (Uniprot: Q9EQ09), rat (Uniprot: O70156), rabbit (Uniprot: Q9XTA8), and cynomolgus monkey LOX1 were designed with N- or C-terminal Flag and histidine tags and cloned in Target Gateway (Invitrogen) vectors. The constructs were then transfected into mammalian HEK293 EBNA cells for protein expression. The proteins were then subjected to purification by affinity and standard size exclusion chromatography. Briefly, MAXISORP™ plates were coated with human (HisFlag)-LOX1, cynomolgus LOX1-(FlagHis), mouse LOX1-(FlagHis), rat LOX1-(FlagHis) and rabbit LOX1-(FlagHis) at 10.10 , 5, 5, and 5 μg/ml in PBS buffer, respectively. Effective coating with antigen 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% powdered milk, purified anti-LOX1 scFvs in PBS plus 3% powdered milk were incubated with the different antigens by coating the plates for 1 hour. Bound scFv molecules were detected using a europium-labeled anti-Myc tag secondary antibody (Perkin Elmer) at 100 ng/ml. The fluorescence of the plates was read using excitation at 340 nm and emission at 615 nm. Nonspecific binding was determined in bovine serum albumin (New England Biolabs) used to coat the plates at 10 μg/ml.

[00334] As shown in Figure 3A, LOX514 ("LOX10514") and LOX696 ("LOX10696") bind to human and cynomolgus LOX1, but not to mouse, rat or rabbit LOX1 orthologs. The lack of cross-reactivity of the antibody with mouse LOX1 is not unexpected given the low homology along the C-type lectin domain between these species (~62% identity between human and mouse).

[00335] The specificity of human anti-LOX1 antibody molecules relative to other related human C-type lectin and scavenger receptor molecules was assessed by an IgG binding ELISA as described in Example 10, Assay 6. As shown in Figure 3B, LOX514 ("LOX10514") and LOX696 ("LOX10696") bind only to human LOX1 and do not bind to CLEC-7A, CLEC-1A, CLEC-4L, CLEC-1B, SR-A1, and SR-B3 humans. This result demonstrates the specificity of LOX514 and LOX696 in relation to LOX1.

EXAMPLE 4 - Isolation and identification of anti-LOX1 scFv antibodies with optimized potency by targeted randomization and recombination of LOX514 CDRs

[00336] 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 chain (VH) 2 or 3 complementarity determining regions (VH CDR2 or VH CDR3) or 3 chain complementarity determining regions. variable light (VL) (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 higher affinity for human LOX1. The expectation was that these would present enhanced inhibitory activity in relation to the binding of LOX1 to oxLDL and other LOX1 ligands. Selections were carried out essentially as previously described (Thompson et al., J Mol Biol. 256(1):77-88, (1996)). Briefly, scFv-phage particles were incubated with biotinylated recombinant human LOX1 in solution (biotinylated via free amines using EZ LINK™ Sulfo-NHS-LC-Biotin (Thermo/Pierce, product: 21335)). The scFv bound to the antigen were then captured on streptavidin-coated paramagnetic beads (DYNABEADS® M-280) following the manufacturer's recommendations. The selected scFv-phage particles were then rescued as previously described (Osbourn et al., Immunotechnology, 2(3):181-96, (1996)), and the selection process was repeated in the presence of decreasing concentrations of human LOX1. biotinylated (a typical example would be 50 nM to 20 pM for four cycles).

[00337] Periplasmic extracts containing crude scFv were prepared for a representative number of individual scFvs from the CDR-directed selection results and screened in an HTRF® epitope competition assay format against the parent LOX514 antibody as described in Example 10, Assay 7. Screen hits, i.e. scFv variants that showed a significantly enhanced inhibitory effect compared to parent LOX514, were subjected to DNA sequencing, and single variants from parent LOX514 Variable heavy chain CDR2 or CDR3 and variable light chain CDR3 were produced as purified scFv and tested. Some scFvs were then selected and converted into 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 Figure 4).

[00338] To generate further improvements, the results of randomized CDR selection comprising large numbers of scFv variants with the ability to inhibit binding of parent LOX514 to human LOX1 were recombined to form libraries in which clones contained individually randomized paired sequences of CDR2 of VH with CDR3 of VH or VL.

[00339] Periplasmic extracts containing crude scFv were prepared from a representative number of individual scFvs from the VH2/VH3 or VH2/VL3 libraries recombined in the HTRF® epitope competition assay. Screen hits, i.e. scFv variants that showed a significantly enhanced inhibitory effect compared to the parent scFv and results generated before recombination, were subjected to DNA sequencing, and single recombined variants were produced as purified scFv and tested. The most active scFvs were then selected and converted into IgG1 TM for further characterization. Typical examples of optimized LOX514 antibodies obtained from these recombinant libraries were: LX5140092, LX5140093, LX5140094, LX5140108 and LX5140110 (as described in Table 1 or Figure 4).

[00340] Amino acid sequence alignments of optimized heavy chains for selected antibodies from the LOX514 strain are shown in Figure 4A. Amino acid sequence alignments of optimized light chains for selected antibodies from the LOX514 strain are shown in Figure 4B.

EXAMPLE 5 - Isolation and identification of anti-LOX1 scFv variants with optimized potency derived from LOX696

[00341] LOX696 was first optimized using affinity-based ribosome display selection essentially as described by EP494955, US5658754 and Hanes et al (Thompson et al., J. Mol. Biol. 256(1):77-88 (1996 )).

[00342] Large scFv libraries, in ribosome display format, derived from the LOX696 scFv sequence were created by oligonucleotide-directed mutagenesis of the variable heavy chain (VH) complementarity determining regions 2 or 3 (VH CDR2 or CDR3 VH) or variable light chain (VL) complementarity determining regions 3 (VL CDR3) using standard molecular biology techniques as described (Thompson et al., J. Mol. Biol. 256(1):77-88 ( 1996)). At the DNA level, a T7 promoter was added at the 5' end for efficient transcription into mRNA. At the mRNA level, the construct contained a prokaryotic ribosome binding site (Shine-Dalgarno sequence). At the 3' end of the single chain, the termination codon was removed and a portion of bacteriophage M13 gIII (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)).

[00343] The libraries were subjected to affinity-based ribosome display selections in order to select variants with greater affinity for human LOX1. The expectation was that these would present enhanced inhibitory activity in relation to the binding of LOX1 to oxLDL and other LOX1 ligands. scFvs were expressed in vitro using the RIBOMAX™ (T7) large-scale RNA production system (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™ Sulfo-NHS-LC-Biotin (Thermo/Pierce, product: 21335)). Specifically bound tertiary complexes (LOX1:ARM) were captured on streptavidin-coated paramagnetic beads (DYNABEADS® M-280) following the manufacturer's recommendations (Dynal) while unbound ARMs were removed by washing. The mRNA encoding the bound scFvs was then recovered by reverse transcription PCR (RT-PCR). The selection process was repeated on the population obtained by additional cycles of selection with decreasing concentrations of biotinylated human LOX1 (1 μM to a minimum of 2 nM in 4 cycles) in order to enrich with clones with greater affinity for LOX1. The results of selection cycles 2, 3, and 4 were subcloned into pCantab6 (1) for bacterial expression as scFvs, and improved clones were identified as periplasmic extracts in a LOX696 epitope competition (see Example 10, Assay 7) as described. in Example 4.

[00344] Second generation phage display libraries were constructed based on the results or variants of the LOX696 randomized CDR ribosome display. A total of four libraries were generated: (1) By recombining VH CDR2 sequences with VL CDR3 sequences both from cycle 2 results; (2) By performing an additional error-prone PCR on the VH CDR2 X VL CDR3 recombined library (see above) to incorporate additional sequence diversity using the DIVERSIFY™ PCR Random Mutagenesis Kit (BD Biosciences) following the manufacturer's recommendations ; (3) By random mutagenesis of the entire scFv sequence using error-prone PCR as described above using as template a set of 9 scFv DNA hits originating from the initial ribosome display libraries plus the parent LOX696 scFv DNA in a ratio equimolar; (4) By light randomization of the entire VH CDR3 as described in Gallop MA et al., J Med Chem, 37(9):1233-51 (1994) using as a template a set of 6 scFv DNA hits originating from the libraries ribosome display of initial VL CDR3 plus parent LOX696 scFv DNA in an equimolar ratio.

[00345] 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 cycles). Periplasmic extracts containing crude scFv were prepared from a representative number of individual scFvs from the 2nd generation libraries and tested in HTRF® epitope competition (as described in Example 10, Assay 7). The scFv variants that provided the greatest competition for binding were again sequenced, and purified scFvs were prepared as described in Example 1 for evaluation. The most potent scFvs from those from the second generation libraries were converted to IgG1 TM format for further characterization as described in later examples.

[00346] 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, 102_ngl1 and LX6960116_ngl1 (as described in Table 1 or Figure 5).

[00347] Optimized VH amino acid sequence alignments for selected antibodies from the LOX696 strain are shown in Figure 5A. The optimized VL amino acid sequence alignments for selected antibodies from the LOX696 strain are shown in Figure 5B.

EXAMPLE 6 - Germlining and manipulation for stability of anti-LOX1 antibodies in the form of IgG1-TM

[00348] The VH and VL amino acid sequences of LOX514 were aligned with known human germline sequences in the VBASE database (Tomlinson, I., VBASE. 1997, MRC Center of Protein Engineering, Cambridge, United Kingdom) 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 VÀ1-e (DPL-8) and JL3. There is only one difference in the two alignments, located in framework 4 of the heavy chain. This unique residue (R105 - Kabat numbering) was reverted to the germline (Q) residue during the IgG conversion process by standard molecular biology, meaning that all optimized IgGs from the LOX514 strain were in fact produced in the modified format for germline. Additionally, an N96D heavy chain mutagenesis (Kabat numbering) was performed to remove a potential N-deamidation motif in the CDR3 of both LX5140092 and LX5140093. The variants were named LX5140092_N>D and LX5140093_N>D, respectively (see Table 1 or Figure 4). An alignment of the heavy and light chain amino acid sequences for LX5140092_N>D and LX5140093_N>D is shown in Figures 4A and 4B, respectively.

[00349] Similarly, a germline analysis of the LOX696 antibody was also performed. The closest germline was identified as VH3-09 (DP-31) and JH3 for the heavy chain, and VÀ2a2 (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 VH, were detected: two in framework 1, one in framework 3, and two in framework 4. Differences in the optimized LOX696 IgG1-TM sequences were reverted to the closest germline residues with the exception of V89 by standard molecular biology techniques: Q1E, Q6E, R105Q, and T108M. Additionally, a G82bS heavy chain mutagenesis (Kabat numbering) was performed to remove a potential N-deamidation motif in backbone 3 of germline modified LX6960073_gl (see Table 1 or Figure 5). The variant was named LX6960073_G82bS_gl (see Table 1 or Figure 5). An alignment of the heavy and light chain amino acid sequences for germline unmodified LX6960073_ngl1, germline modified LX6960073_gl and LX6960073_G82bS_gl is shown in Figures 5A and 5B, respectively.

EXAMPLE 7- Specificity and cross-species cross-reaction of optimized anti-LOX1 antibodies

[00350] The specificity of anti-human LOX1 antibodies optimized as IgG1-TM toward other related human C-type lectin and scavenger receptor 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 used for coating as a positive binding control. The following optimized antibodies were tested: LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D, LX6960073_gl and LX6960073_G82bS_gl. The human IgG1-TM isotype control antibody NIP228 was included in the antibody panel for testing to determine the nonspecific background level.

[00351] As shown in Figure 6, all optimized anti-LOX1 antibodies bind to human LOX1 but do not bind to 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 in relation to human LOX1.

[00352] Cross-reactivity of anti-human LOX1 antibodies with various LOX1 species orthologs was assessed by an IgG binding ELISA (as described in Example 10, Assay 7). Briefly, MAXISORP™ plates were coated with human (HisFlag)-LOX1, cynomolgus LOX1-(FlagHis), mouse LOX1-(FlagHis), rat LOX1-(FlagHis) and rabbit LOX1-(FlagHis) at 5 μg/ ml in PBS buffer. After blocking with PBS containing 3% powdered milk, purified anti-LOX1 IgG1-TM at 10 μg/ml in blocking buffer were incubated with the different antigens used in the coatings. Bound IgG molecules were detected using a europium-labeled anti-human IgG secondary antibody (Perkin Elmer) at 100 ng/ml. The fluorescence of the plates was read using excitation at 340 nm and emission at 615 nm. Nonspecific binding was determined on the CD86 FlagHis antigen also used to coat the plates at 5 μg/ml. The following optimized LOX1 antibodies were tested, as well as human IgG1-TM NIP228 as a negative control: LX5140108, LX5140110, LX5140092_N>D, LX5140093_N>D, LX6960073_gl and LX6960073_ G82bS_gl. As shown in Figure 7a, all tested antibodies (LX5140108, LX5140110, LX5140092_N> D, LX5140093_N> D, LX6960073_gl and LX6960073_ G82BS_GL) to. Some of these 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 lower fluorescence counts.

[00353] Further characterization of the cross-reactivity between human and cynomolgus LOX1 was performed for the optimized anti-LOX1 IgG1-TM antibodies LX5140108, LX5140110 and LX6960073_G82bS_gl by a competition ELISA.

[00354] Briefly, biotinylated human LOX1 was used at 0.125 μg/ml in PBS to coat plates with streptavidin (Abgene). After blocking with PBS containing 3% milk powder, purified anti-LOX1 IgG1-TM at 12.5 ng/ml in blocking buffer were coincubated with competing 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 2 μM for LX6960073_G82bS_gl. Bound anti-LOX1 IgG molecules were detected using a europium-labeled anti-human IgG secondary antibody (Perkin Elmer) at 100 ng/ml. The fluorescence of the plates was read using excitation at 340 nm and emission at 615 nm.

[00355] As shown in Figure 7B and Table 3 below, all three optimized anti-LOX1 IgG1-TM (LX5140108, LX5140110 and LX6960073_G82bS_gl) compete for cynomolgus LOX1 and exhibit an IC50 within 5 times the IC50 for a competition for human LOX1, demonstrating that the cynomolgus cross-reactivity of the anti-LOX1 antibodies LX5140108, LX5140110 and LX6960073_G82bS_gl is strong. Table 3: Optimized antibody binding to cynomolgus LOX1 and human LOX1

EXAMPLE 8 - Affinity of hLOX1-optimized anti-LOX1 antibodies as determined by BIACORE™ and KinExATM

[00356] The affinity of the anti-LOX1 antibody for recombinant human and cynomolgus orthologs of the extracellular domain (ECD) of LOX1 was determined at 37 ° C by monitoring the interaction in real time by Biacore (for human and cynomolgus) and in the balance using KinExA (for human LOX1 affinity measurements).

[00357] In the Biacore assay, recombinant G protein + the anti-LOX1 antibody captured by G protein were immobilized by amine binding to the surface of a CM5 chip, and the association and dissociation profiles of LOX1 orthologs passed through the surface were collected. The tests were carried out with the IFC temperature set at 37°C and the sample compartment left at room temperature. All experiments were performed with the HBSEP running buffer passing through the IFCs at a constant flow rate of 30 ul/min. At the end of each application of LOX1, any bound antibodies and antibody complexes were removed from the G protein by two 40-second pulses of 10 mM glycine pH 1.5. The amount of anti-LOX1 antibody captured on the surface of the G protein was adjusted 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 interactor was sufficient to ensure that the entire range of binding from antibody saturation to no detectable binding was observed. Data were analyzed using the BiaEval evaluation program after double reference subtraction from an antibody body profile only.

[00358] The solution equilibrium assay was performed at a single concentration of anti-LOX1 antibody, in which an ECD titration of recombinant human LOX1 was performed from 100 times above to 100 times below the antibody concentration. Free antibody concentrations were determined by capturing unoccupied antibody which was subsequently detected by a fluorescently labeled human Fc-specific probe. Table 4 shows details of the observed rate constants and total affinities. Table 4: Rate and affinity constants of LX5140110

EXAMPLE 9 - Optimized anti-LOX1 antibodies demonstrate inhibition of multiple ligand binding, oxLDL internalization and oxLDL-induced signaling

[00359] Purified IgGs were tested for their ability to specifically inhibit the binding of oxLDL, AGE-BSA and CRP to LOX1 (see Example 10; assays 1, 2 and 3). Several isolated clones including LX5140110 and LX6960073_G82bS_gl were tested for their ability to block the binding of oxLDL, AGE-BSA and CRP to LOX1. Representative graphs are shown in Figures 8A, 8B and 8C showing the inhibition of oxLDL, AGE-BSA and CRP binding, respectively, by LX5140110 and LX6960073_G82bS_gl. To confirm that the antibodies were cross-reactive and had functional activity on the LOX1 SNP K167N, the antibodies were tested for their ability to block the binding of oxLDL to LOX1 SNP K167N. Representative graphs are shown in Figure 8D showing that both LX5140110 and LX6960073_G82bS_gl block the binding of oxLDL to the LOX1 SNP K167N variant. In addition to binding inhibition, several isolated clones including LX5140110 and LX6960073_G82bS_gl were tested for their ability to block oxLDL internalization in LOX1-expressing cells (see Example 10, Assay 4) and oxLDL-induced LOX1 signaling (see Example 10, Essay 5). Representative graphs are shown in Figures 8A and 8F showing the inhibition of oxLDL internalization and oxLDL-induced LOX1 signaling, respectively, by LX5140110 and LX6960073_G82bS_gl. The results of these studies are summarized in Table 5. Table 5: Inhibition of oxLDL internalization and signaling by optimized anti-LOX1 antibodies

[00360] These results demonstrate the specific inhibition of various ligands of LOX1 binding to oxLDL, AGE-BSA and CRP by the antibodies LX5140110 and LX6960073_G82bS_gl, and that both LX5140110 and LX6960073_G82bS_gl functionally cross-react with the common variant LOX1 SNP K167N and inhibit oxLDL internalization and oxLDL-induced LOX1 signaling.

EXAMPLE 10 - Tests and Methods

[00361] The following materials and methods were used in the experiments described in Examples 1 to 9.

Materials

[00362] Doxycycline hyclate (Sigma #D9891-1G); CarboxyH2DCFDA (MolecularProbes #C400); Hoechst dye (Molecular probes #33342); AGE-BSA (Biovision #2221-10); PBS (Gibco #14190); Recombinant C-reactive protein (R&D #1707-CRCF); 30% BSA (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 CellBIND 384 Black Breadboard (Corning Costar #3683); Delfia Enhancement solution (Perkin Elmer #4001-0010); europium-labeled streptavidin (Perkin Elmer #1244-360); Zeba 0.5 ml desalination columns (Pierce #89883); CHO-TREx (Invitrogen #R718-07); oxLDL (Intracel #RP049); DiI-oXLDL (Intracel #RP-173); Anti-LOX1 antibody (Biovision #3659); Hoechst dye (Molecular probes #33342); PBS (Gibco #14190); Culture medium: Hams:F12-GlutaMax-I (Gibco # 31765-027); supplemented with 10% fetal bovine serum (Invitrogen #16000-044); Blasticidin (Invitrogen #46-1120); Zeocin (Invitrogen #R25001); Lipofectamine (Invitrogen #11668-019); Doxycycline hyclate (Sigma #D9891-1G).

Reagent modifications Tagging oxLDL with Cypher 5E

[00363] The pH of the ox-LDL (1 mg/ml) was adjusted to pH 8.5 using 1/10th of the volume of 0.5 M sodium borate buffer and the oxLDL was labeled according to the instructions in the kit. manufacturer at a protein:dye molar ratio of 40:1 (1 hour at room temperature in the dark). Unreacted dye was removed using Zeba centrifuge columns according to the manufacturer's instructions (Cypher 5E Mono NHS Ester from GE healthcare) and its buffer exchanged to PBS. The labeled protein was kept at 4°C in the dark. For calculation purposes, it is assumed that more than 95% of the protein has been recovered and that there are no dilution factors to consider.

Labeling of oxLDL and AGE-BSA with DyLight 649

[00364] The pH of ox-LDL or AGE-BSA (1 mg/ml) was adjusted to pH 8.5 using 1/10th of the volume of 0.5 M sodium borate buffer, and were labeled according to the instructions from the manufacturer (DyLight 649 NHS ester from Thermo Scientific for 1 hour at room temperature in the dark). Unreacted dye was removed using Zeba spin columns according to the manufacturer's instructions and its buffer exchanged to PBS. For calculation purposes, it is assumed that more than 95% of the protein is recovered and that there are no dilution factors to consider.

Labeling of human anti-LOX1 antibodies with Dylight 649

[00365] The pH of the anti-human LOX1 antibody (1 mg/ml) was adjusted to pH 8.5 using 1/10th of the volume of 0.5 M sodium borate buffer, and was labeled according to the manufacturer's instructions (DyLight 649 NHS Ester from Thermo Scientific for 1 hour at room temperature in the dark). Unreacted dye was removed using Zeba spin columns according to the manufacturer's instructions and its buffer exchanged to PBS. For calculation purposes, it is assumed that more than 95% of the protein is recovered and that there are no dilution factors to consider.

Labeling of CRP with biotin

[00366] CRP C-reactive protein (R&D systems; carrier free) was reconstituted in distilled water to a concentration of 4 mg/ml and biotinylated according to kit instructions provided with the Lightning-Link™ Biotin Conjugation Kit (Type A).

CHO-TREX cell line 3A9 expressing human LOX1

[00367] 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 synthesized with the addition of an Afl II restriction site and a Kozak sequence at the 5' end, and an EcoRI restriction site at the 3' end. pAM2037 was then transfected into Chinese hamster ovary cells expressing the tetracycline repressor (CHO-TREx cells) using lipofectamine 2000. CHO-TREx is a CHO-K cell line that contains pcDNA6/TR which has the gene for the repressor. of tetracycline (Tet), which will block the expression of genes cloned in pcDNA4/TO. When these cells are treated with tetracycline, repression is released and the LOX1 gene will be expressed.

[00368] The mixed transfected cell population was separated using a FACS apparatus into 96-well plates to obtain a single cell in each well. pcDNA6/TR was selected with blasticidin at 10 μg/ml, and zeocin at 300 μg/ml was used to select the pcDNA4TO-LOX1 plasmid, and 200 μg/ml for maintenance. Individual cells were propagated and stable clones were selected for their ability to bind and internalize DiI-labeled xLDL, using a High Content Analysis approach to quantify the fluorescence taken up by the cells. A clone was selected for DiI-oxLDL uptake and stability over several culture passages. Surface LOX1 protein expression was verified by immunostaining using a specific anti-LOX1 antibody from Biovision. The functional activity of the LOX1 protein was demonstrated by uptake of oxLDL and generation of an oxLDL-mediated ROS response in CHO-TREx-LOX1 cells.

ASSAYS 1 and 2 - LOX1: oxLDL and LOX1:AGE-BSA binding assays Bond saturation curve

[00369] Both oxLDL and AGE-BSA labeled with DyLight 649 (1 mg/ml) were diluted 1:50 in HBSS and titrated in a 384-well plate using a 1+1 serial dilution in 16 spots in triplicate. For determination of total binding, 10 μl of HBSS was added to all wells, followed by the addition of 10 μl of CHO TREX cells transfected with human LOX1 (4000 cells per well). To determine nonspecific binding, 10 μl of HBSS was replaced by 10 μl of unlabeled oxLDL (1 mg/ml). The assay plate was incubated for 1 hour at room temperature. After incubation, plates were read on an Applied Biosystems 8200 Cell Detection System (FMAT plate reader) using a PMT1 setting of 422 with a minimum count set to 10; Color<0.4 and FL1<5600. Specific binding was determined by subtracting the average total binding signal from the average nonspecific binding signal and plotting the resulting binding isotherm using the Graphpad Prism program using a site-specific binding algorithm. The calculated concentration at which the KD was determined was used for subsequent high-throughput screening and antibody competition experiments.

Assessment of anti-hLOX1 monoclonal antibodies

[00370] The competition between anti-LOX1 antibodies and ligand (oxLDL; AGE-BSA) labeled with DyLight 649 for binding to the LOX1 receptor expressed in CHO TREX cells was performed as follows.

[00371] Competing antibodies were used without any prior dilution and were titrated in 384-well plates using a 1+1 serial dilution in assay buffer (HBSS) at 24 points in duplicate (10 μl per well). Dylight 649-labeled ligands (at their respective KD concentrations) were added to all antibody-containing wells (10 μl per well) followed by the addition of CHO TREX cells transfected with human LOX1 (4000 cells per well in 10 μl) or CHO TREX transfected with K167N SNP.

[00372] The assay plates were incubated for 1 hour at room temperature. After incubation, plates were read on an Applied Biosystems 8200 Cell Detection System (FMAT plate reader) using a PMT1 setting of 422 with a minimum count set to 10; Color<0.4 and FL1<5600. Data were analyzed using a 4-parameter logistic equation with GRAPHPAD™ Prism version 5 (GraphPad Inc, California) to determine apparent IC50 values. Y= Bottom + (Top-Bottom)/ (1+10A ((Log IC50-X)*HillSlope factor)) where X is the logarithm of concentration and Y is the % of specific binding . The IC50 is the sample concentration that produces 50% inhibition of specific binding. Table 6. Stock concentrations used in oxLDL and AGE-BSA binding assays

ASSAY 3 - LOX1:CRP binding assay Bond saturation curve

[00373] LOX1 (R&D systems) was immobilized on the surface of a 384-well Nunc Maxisorp plate at a concentration of 3 μg/ml in PBS, overnight at 4 °C (25 μl per well). The next day, the assay plate was washed 12x in PBS (without calcium and magnesium). A working solution of biotinylated CRP was prepared at 20 μg/ml (769 nM) in PBS/0.1% BSA/0.01% tween 20, and titrated in a 384-well plate using a 1+ serial dilution. 1 in 16 spots in triplicate (12.5 μl per well). An additional 12.5 μl 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 16x in PBS/0.01% Tween 20, followed by the addition of 50 μl of europium-labeled streptavidin (1:1000 dilution). The assay plate was incubated for an additional 1 hour at room temperature followed by 16 washes in PBS/0.01% Tween 20. The plate was developed by adding 100 μl/well of DELFIA Enhancement solution, and read on a Wallac Victor V plate reader using a factory-installed DELFIA europium protocol. The resulting binding isotherm was plotted using Graphpad's Prism program using a 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.

Evaluation of human anti-LOX1 monoclonal antibodies

[00374] Competition between antibody and biotin-labeled CRP for binding to recombinant human LOX1 was carried out as follows: LOX1 (R&D systems) was immobilized on the surface of a 384-well Nunc Maxisorp assay plate at a concentration of 3 μg/ ml in PBS overnight at 4°C (25 μl per well). The next day, the assay plate was washed 12x in PBS (without calcium and magnesium). Competing antibodies were used without any prior dilution and were titrated in a 384-well dilution plate using a 1+1 serial dilution in assay buffer (PBS/0.1% BSA/0.01% tween 20) in 24 dots in duplicate (30 μl per well). Biotin-labeled CRP (30 μl per well at the KD concentration) was then added to all antibody-containing wells. Samples (50 μl) were then transferred using a MiniTrak 384 liquid handling robot from the dilution plate to the assay plate containing immobilized recombinant human LOX1. The plates were incubated for 2 hours at room temperature. The plate was washed 16x in PBS/0.01% Tween 20, followed by the addition of 50 μl of europium-labeled streptavidin (1:1000 dilution). The assay plate was incubated for an additional 1 hour at room temperature followed by 16 washes in PBS/0.01% Tween 20. The plate was developed by adding 100 μl/well of DELFIA Enhancement solution, and read on a Wallac Victor V plate reader using a factory-installed DELFIA europium protocol. Data were analyzed using a 4-parameter logistic equation with GRAPHPAD™ Prism version 5 (GraphPad Inc, California) to determine apparent IC50 values. Y= Bottom + (Top-Bottom)/ (1+10A ((Log IC50-X)*HillSlope factor)) where X is the logarithm of concentration and Y is the % of specific binding . The IC50 is the sample concentration that produces 50% inhibition of specific binding. Table 7. Stock concentrations used in CRP binding assays

ASSAY 4 - LOX1:oxLDL internalization assay Bond saturation curve

[00375] Cypher 5E-labeled OxLDL (40:1 molar ratio at 1 mg/ml) was diluted 1:50 in HBSS and titrated in a 384-well plate using a 1+1 serial dilution at 16 points in triplicate. For determination of total binding, 10 μl of HBSS was added to all wells, followed by the addition of 10 μl of CHO TREX cells transfected with human LOX1 (4000 cells per well). To determine nonspecific binding, 10 μl of HBSS was replaced by 10 μl of unlabeled oxLDL (1 mg/ml).

[00376] The assay plate was incubated for 1 hour at 37°C. A parallel experiment was carried out in which all reagents were kept at 4°C in order to demonstrate that internalization was a metabolically active process. After incubation, plates were read on an Applied Biosystems 8200 Cell Detection System (FMAT plate reader) using a PMT1 setting of 422 with a minimum count set to 10; Color<0.4 and FL1<5600. Specific binding was determined by subtracting the average total binding signal from the average nonspecific binding signal and plotting the resulting binding isotherm using the Graphpad Prism program using a site-specific binding algorithm. The calculated concentration at which the KD was determined was used for subsequent high-throughput screening and antibody competition experiments.

Assessment of anti-hLOX1 monoclonal antibodies

[00377] Competition between anti-LOX1 antibodies and Cypher5E-labeled ox-LDL for internalization in CHO TREX cells expressing the LOX1 receptor was performed as follows.

[00378] Competing antibodies were used without any prior dilution and were titrated in a 384-well plate using a 1+1 serial dilution in assay buffer (HBSS) at 24 points in duplicate (10 μl per well). Dylight 649-labeled ligands (at their respective KD concentrations) were added to all antibody-containing wells (10 μl per well) followed by the addition of CHO TREX cells transfected with human LOX1 (4000 cells per well in 10 μl) or CHO TREX transfected with K167N SNP.

[00379] The assay plates were incubated for 1 hour at room temperature. After incubation, plates were read on an Applied Biosystems 8200 Cell Detection System (FMAT plate reader) using a PMT1 setting of 422 with a minimum count set to 10. Color<0.4 and FL1<5600. Data were analyzed using a 4-parameter logistic equation with GRAPHPAD™ Prism version 5 (GraphPad Inc, California) to determine apparent IC50 values. Y= Bottom + (Top-Bottom)/ (1+10A ((Log IC50- X)*HillSlope factor)) where X is the logarithm of concentration and Y is the % of specific binding . The IC50 is the sample concentration that produces 50% inhibition of specific binding. Table 8. Stock concentrations used in oxLDL internalization assays

ASSAY 5 - Induction of ROS in recombinant CHO-LOX1 cells Cell preparation

[00380] CHO TREX cells transfected with human LOX1 were induced 24 hours before conducting the experiments with 1 μg/ml (final concentration) of doxycycline. On the day of the experiment, the cells were scraped and centrifuged for 5 minutes at 1200 rpm. The supernatant was discarded and the cell pellet was resuspended in 50 ml of PBS and centrifuged at 1200 rpm for 5 minutes. This procedure was repeated twice. The supernatant was discarded and the cell pellet was resuspended in 5 ml of HBSS. Cells were counted on a hemocytometer using the Trypan blue exclusion method. The number of cells was adjusted to provide 4x105 cells in total. Cells were kept on ice until use.

Bond saturation curve

[00381] Cypher 5E-labeled OxLDL (40:1 molar ratio at 1 mg/ml) was diluted 1:50 in HBSS and titrated in a 384-well plate using a 1+1 serial dilution at 16 points in triplicate. For determination of total binding, 10 μl of HBSS was added to all wells, followed by the addition of 10 μl of CHO TREX cells transfected with human LOX1 (4000 cells per well). To determine nonspecific binding, 10 μl of HBSS was replaced by 10 μl of unlabeled oxLDL (1 mg/ml).

[00382] The assay plate was incubated for 1 hour at 37°C. A parallel experiment was carried out in which all reagents were kept at 4°C in order to demonstrate that internalization was a metabolically active process.

[00383] After incubation, the plates were read on an Applied Biosystems 8200 cell detection system (FMAT plate reader) using a PMT1 setting of 422 with a minimum count set to 10; Color<0.4 and FL1<5600.

[00384] Specific binding was determined by subtracting the average total binding signal from the average nonspecific binding signal and plotting the resulting binding isotherm using the Graphpad Prism program using a site-specific binding algorithm. The calculated concentration at which the KD was determined was used for subsequent high-throughput screening and antibody competition experiments.

[00385] CHO-TREx cells transfected with LOX1 were seeded in assay plates at 7000 cells/well (100 μl/well in medium + 10% FBS (tetracycline-free)), and incubated overnight at 37°C , CO2 at 5%. The following day, doxycycline was added at a final concentration of 50 ng/ml per well to induce LOX expression and the plates were incubated overnight at 37°C in a 95% O2/CO2 atmosphere. 5%. For each screening experiment, three separate plates were prepared, and anti-LOX antibodies were analyzed in triplicate with one replicate per well. The next day, anti-LOX1 antibodies were prepared by serially diluting the antibodies in warm cell culture medium as concentrated 2x solutions in a separate dilution plate. All medium was removed from the assay plates, and 25 μl aliquots of a dilution series of anti-LOX1 antibodies (2x concentrated solution) were added to each well of the assay plate and incubated for 20 min at 37°C. °C in a 95% O2/5% CO2 atmosphere. This was followed by the addition of 25 μl per well of oxLDL (at a fixed final concentration of 25 μg/ml) and the plates were incubated for 60 min at 37°C in 95% O2a/5% CO2.

[00386] The wells were then carefully washed with 1x 100 μl of warm BSS/Ca/Mg and then 50 μl/well of carboxy-H2DCFDA (1.5 μM in warm HBSS/Ca/Mg) was added, and the plates test samples were incubated for 30 minutes. During the last 5 min of incubation with carboxy-H2DCFDA, cells were counterstained with Hoechst dye (final concentration 8.3 μg/ml) in warm HBSS/Ca/Mg by adding 10 μl of 50 μg/ml Hoechst in warm HBSS/Ca/Mg to carboxy- H2DCFDA already present in the wells.

[00387] Assay plates were then washed 2x with warm HBSS + Ca/Mg and read immediately on an Arrayscan high-content plate reader (ThermoFischerScientific, Cellomics) using the settings for XF53-Hoechst (Ch1) and XF53-FITC ( Ch2). For image analysis, the compartmental bioapplication algorithm ("Compartmental BioApplication Algorithm") was used and the fluorescence generated by the ROS probe was quantified by the circle and dot mean intensity parameter ("CircSpotAvgIntensity parameter"). Competitive data and resulting IC50s were plotted and calculated in ExcelFit v.5.1 using a zero-site sigmoid dose-response model using a 4-parameter logistic model (Model 903).

ASSAY 6 - LOX1 specificity ELISAs

[00388] IgG specificity ELISAs were performed essentially as follows on the LOX1 and LOX1-related molecules mentioned in Table 9 below. MAXISORB™ plates (NUNC) were coated with antigen at 5 μg/ml in PBS and incubated overnight at 4°C, except 10 μg/ml was used for human SR-B3. Plates were washed 3x with PBS and blocked with 200 μl/well blocking buffer (PBS + 3% dry milk) for 1 hour. After the plates were washed 3x with PBS, IgGs were diluted to 0.2 μg/ml in blocking buffer, added to 50 μl/well, and incubated for 1 hour. The plates were then washed 3x with PBS-Tween, the detection reagent (anti-lambda light chain (Sigma A5175, diluted to 0.23 μg/ml) or kappa light chain (Sigma A7164, diluted to 0.8 μg/ml). ) of peroxidase-conjugated human IgG) was added (50 μl/well in blocking buffer), and the plates were incubated for 1 hour. Plates were washed 3x with PBS-Tween, 50 μl/well of TMB was added, and plates were allowed to develop for 5 to 15 minutes. The reaction was stopped with 50 μl/well of 0.1 M H2SO4, and the plates were read on an ENVISION™ plate reader, or similar equipment, at 450 nm. Table 9. Reagents used in LOX1 specificity ELISAs

ASSAY 7 - Competition for epitope

[00389] LOX514 and LOX696 were labeled with DyLight 649 as described above in the protein modifications section and used as competitor molecules for high-throughput screening and profiling in parallel with ligand binding assays. Labeled antibodies were used at various concentrations above their respective KD concentrations in order to make it more difficult for them to compete against higher affinity unlabeled antibodies.

Determination of the KD of LOX514 and LOX696 labeled with DyLight 649

[00390] Anti-LOX1 antibodies labeled with DyLight 649 were diluted 1:50 in HBSS and titrated in a 384-well plate using a 1+1 serial dilution in 16 spots in triplicate. For determination of total binding, 10 μl of HBSS was added to all wells, followed by the addition of 10 μl of CHO TREX cells transfected with human LOX1 (4000 cells per well). To determine nonspecific binding, 10 μl of HBSS was replaced with 10 μl of unlabeled anti-human LOX1 antibody (1 mg/ml).

[00391] The assay plate was incubated for 1 hour at room temperature.

[00392] After incubation, the plates were read on an Applied Biosystems 8200 cell detection system (FMAT plate reader) using a PMT1 setting of 422 with a minimum count set to 10; Color<0.4 and FL1<5600.

[00393] Specific binding was determined by subtracting the average total binding signal from the average nonspecific binding signal and plotting the resulting binding isotherm using the Graphpad Prism program using a site-specific binding algorithm. Antibodies were used at concentrations that are multiples of and above the calculated KD concentration. Table 10. Stock concentrations for LOX514 and LOX696 epitope competition assays

EXAMPLE 11 - Effects of anti-LOX1 antibodies on endothelial lipid uptake, cell signaling and nitric oxide homeostasis in tissue culture and human blood vessels

[00394] The LOX1 receptor acts as one of the immediate causes of the atherogenic process by specific binding to and internalization of OxLDL with subsequent activation of several intracellular signal transduction cascades. See, for example, Twigg et al., Cardiol Res Pract. 2012:632408 (2012). Activation of the LOX1 receptor on endothelial cells contributes to endothelial dysfunction including increased ROS production and impaired nitric oxide (NO) signaling induced, in part, by increased arginase 2 expression. (See, for example, Ryoo et al. , Atherosclerosis 214(2):279-287 (2011), Ryoo et al., Circ Res. 102(8):923-932 (2008); 60 (2006). Furthermore, activation of the LOX1 receptor results in the initiation and perpetuation of an inflammatory process in the vascular wall with increased production of cytokines and expression of adhesion molecules. , an effective way to attenuate the development of the atherogenic process. Here, we demonstrate that LX5140110 blocks the uptake of OxLDL by human aortic endothelial cells (HAECs) in a dose-dependent manner. which was previously shown to be LOX1 dependent. Furthermore, LX5140110 prevents an Ox-LDL-dependent decrease in NO production and an increase in superoxide (ROS) production. Furthermore, using an NFkB luciferase reporter construct, we demonstrate that LX5140110 significantly attenuates the activation of NFkB - a master inflammatory regulator in atherogenesis. See, for example, 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 for barrier function in endothelial cells. Thus, we demonstrate that LX5140110 blocks several LOX1-dependent processes that result in endothelial activation and initiate and promote atherogenesis. The results of these experiments are discussed in turn.

A. LX5140110 blocks OxLDL uptake in HAECs (Figure 9A) Methods: Conjugation of OxLDL with Alexa Fluor-568

[00395] 500 microliters of human oxidized LDL (1 mg/ml, Intracel, Frederick, Maryland) was labeled with Alexafluor-568 using the Alexafluor-568 protein labeling kit ("Alexafluor-568 Protein Labeling Kit", Molecular probes , Eugene, Oregon) 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. OxLDL conjugated with Alexafluor-568 was then purified with column purification resin.

Uptake of OxLDL conjugated with Alexa Fluor-568 by HAEC

[00396] HAEC were plated on coverslips coated with fibronectin (10 μg/ml) for eight hours in serum-containing medium before serum deprivation for 18 hours.

[00397] The cells were then incubated with 0, 0.5, 1, 5 or 10 nM of LX5140110 ("514") or 10 nM of the NIP control antibody for one hour in serum-free medium. The antibody was removed completely by washing with fresh serum-free medium before approximately 50 ng/ml of Alexa Fluor 568-conjugated OxLDL was added to the medium 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-conjugated phalloidin (Life Technologies, Grand Island, NY) and DAPI (Life Technologies) and were observed on a Nikon TE-200 epifluorescence microscope. Images were captured with a Rolera EMCCD camera (QImaging, Vancouver, Canada) with the Volocity program (PerkinElmer, Lexington, MA). The images were additionally analyzed with the Volocity program by counting the red fluorescent particles of OxLDL conjugated with Alexafluor-568 (exclusion of particles smaller than 0.1 μm2) inside HAEC cells (images not shown). Approximately 12 images for each set were acquired and analyzed for the average number of red fluorescent particles of Alexafluor-568-conjugated OxLDL in each cell. Dose-response data are shown in Figure 9A (number of vesicles per cell with standard derivatization). As shown in Figure 9A, 5 nM or 10 nM LX5140110 significantly inhibited OxLDL uptake by HAECs (p=0.0003 or p=0.0002, respectively).

B. LX5140110 attenuates NFkB signaling in HAECs (Figure 9B)

[00398] Methods: Confluent 6-well plates of HAECs that were coexpressing NFKB-LUCIFERASE and GFP were serum starved for 24 hours before being subjected to the following conditions (numbers indicate bars in Figure 9B from left to right): 1. Control; 2. OxLDL alone (50 μg/ml); 3. OxLDL + LX5140110 ("514") (10 nM); 4. OxLDL+ NIP (control antibody) (10 nM).

[00399] Antibodies were added 1 hour before OxLDL (50 μg/ml). Incubation with OxLDL continued 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 5x Promega lysis buffer and supernatants were plated on white plates containing 50 μl 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, sodium deoxycholate at 1%, 1 mM Na3VO4, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 μg/ml leupeptin, and 1:1000 diluted protease inhibitor cocktail (Sigma). Loading buffer was added to a final concentration of 2x, boiled for 5 min, centrifuged for 3 min and loaded onto a gel. GFP fluorescence was used as a normalization control. N=5 for each group; * indicates P<0.05 (compared to untreated control); # indicates P<0.05 (compared to OxLDL + 0 nM from ac 514 (LX5140110)). As shown in Figure 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, for example, Zhao W, et al., "Lipopolysaccharide induced LOX-1 expression via TLR4/MyD88/ROS activated p38MAPK-NF-KB pathway." Vascul Pharmacol. (2014).

C. LX5140110 inhibits arginase activation in HAECs (Figure 9C)

[00400] Methods: Confluent 6-well plates of HAECs were serum starved for 24 hours before being subjected to the following conditions (numbers indicate bars in Figure 9C from left to right): 1. Control; 2. OxLDL (50 μg/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 HAECs were incubated with OxLDL (50 μg/ml) for an additional 3 hours. Cells were lysed and arginase activity was determined using the urea assay using α-isonitrosopropiophenone. Briefly, supernatants from extracted cell lysates were prepared after incubation in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, and protease inhibitors) for 30 min at 4°C and centrifuged for 20 min at 14,000 x g at 4°C. The supernatants were then incubated with 150 mM L-arginine for 1 hour at 37°C. After 1 hour, the reaction was stopped using 400 μl of a mixture of acidic solutions (H2SO:H3PO4:H2O 1:3:7) and then 25 μl of 9% α-isonitrosopropiophenone (in 100% EtOH) was added. and heated for 30 min at 95 °C, and reading was taken after 10 min at 540 nm. N=3 for each group; * indicates P<0.05 compared to untreated controls; # indicates that P<0.05 compared to 0 nM of ac 514 (LX5140110). As shown in Figure 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 recently shown to be coupled to LOX-1 in vascular endothelium. See, for example, Ryoo et al., Atherosclerosis 214:279-87 (2011).

D. LX5140110 blocks OxLDL-dependent reduction of nitric oxide production (Figure 9D)

[00401] Methods: NO production was determined using DAF fluorescence. Briefly, to measure NO, HAEC cells were plated in white 96-well plates (ThermoLabsystems) at a density of about 5x104 cells per well and serum starved (1% serum) overnight. Cells were investigated in 7 experimental groups (numbers indicate bars in Figure 9D from left to right): 1. Control; 2. OxLDL (50 μg/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 HAECs were incubated with OxLDL (50 μg/ml) for 24 hours. The medium was then removed and cells were placed at 37°C in EBM2 medium containing DAF-FM DA (5 μM) for 30 min at 37°C. The medium was then exchanged for fresh medium and the cells were incubated for an additional 20 min before measuring total fluorescence with a FlexStation 3 microplate reader (Molecular Devices) with excitation at 495 nm and emission at 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 P<0.05 (compared to untreated control). As shown in Figure 9D, addition of 5 nM or 10 nM LX5140110, but not 10 nM NIP (control antibody), significantly inhibited in a dose-dependent manner the OxLDL-dependent reduction of nitric oxide production in HAECs.

E. LX5140110 blocks OxLDL-dependent increases in ROS production (Figures 9E and 9F)

[00402] Methods: Superoxide production was determined using the Luminol analogue L-012. To measure superoxide (ROS), HAEC cells were plated in TC-treated white 96-well plates (ThermoLabsystems) at a density of about 5x104 cells per well and serum starved (1% serum) overnight. The 7 study populations were as follows (numbers indicate bars in Figure 9E from left to right): 1. Control; 2. OxLDL (50 μg/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 HAECs were incubated with OxLDL (50 μg/ml) for 24 hours. The medium was then removed and cells were further incubated at 37°C in phenol-free DMEM (Sigma) containing 400 μM of the luminol analogue L-012 (Wako) for a minimum of 20 minutes before adding 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 provided virtually undetectable levels of luminescence under control or OxLDL-stimulated conditions (see Figure 9F). Relative light units (RLU) quantified from L-012 luminescence therefore indicate changes in superoxide production. 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 Figures 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. Therefore, LX5140110 prevents OxLDL-mediated uncoupling of eNOS and the subsequent increase in ROS production by preventing the activation of LOX-1 and the subsequent activation of arginase 2. See, for example, Ryoo et al., Atherosclerosis 214:279- 87 (2011).

F. LX5140110 blocks OxDL-mediated phosphorylation of focal adhesion kinase (FAK), Y397 (Figures 9G and 9H).

[00403] Methods: HAECs were cultured in ECM medium (ScienCell, Carlsbad, CA) with 5% serum for one day before serum deprivation for 18 hours. LX5140110 or the control antibody NIP was then added to the cells at the concentration indicated in Figure 9G and incubated for one hour. Cells were washed with fresh medium to remove antibodies before being incubated with 50 μg/ml 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, 2mM EDTA, 1mM PMSF, 2mM sodium vanadate, leupeptin 20 mg/ml and aprotinin 20 mg/ml in PBS). Cell lysates were clarified by centrifugation at 15,000 x g at 4°C for 10 minutes. Protein concentration was then quantified by the bicinchoninic assay (Pierce, Rockford, IL). 10 μg of protein lysates were mixed with 2X Laemmli sample buffer, boiled, and then subjected to SDS-PAGE using 4 to 15% gradient polyacrylamide gels, followed by transfer to nitrocellulose membranes for Western blotting.

[00404] The primary antibodies used in the experiments described in Figures 9G and 9H include anti-pY397-FAK (rabbit polyclonal, Life Technologies, Grand Island, NY) and anti-GAPDH (mouse monoclonal, Novus Biologicals, Littleton, CO) . Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from ICN Biochemicals, Inc. (Costa, Mesa, CA).

[00405] A Student's t test was used to analyze differences in FAK phosphorylation at Tyr397 in control cells, treated with OxLDL and pre-incubated with antibody. p-values are provided in Figure 9H and significance (*) was judged to be present at p-values less than 0.05 for all data. Figures include standard error bars. A representative blot showing changes in FAK phosphorylation at Tyr397 and GAPDH expression in HAECs is shown in Figure 9G, while quantification of the percentage increase in FAK phosphorylation at Tyr397 (pY397-FAK) normalized to GAPDH expression is shown in Figure 9H. These results demonstrate that addition of 5 nM or 10 nM LX5140110, but not 10 nM NIP (control antibody), significantly inhibited OxLDL-mediated and OxLDL-dependent phosphorylation of FAK at Tyr397 in HAECs. * indicates that P<0.05 (compared to the untreated control); # indicates P<0.05 compared to LX5140110 (514) 0 nM. These data suggest that LX5140110 inhibits OxLDL-mediated induction of LOX-1-dependent phosphorylation of FAK, a process that regulates endothelial cytoskeletal functions including cell-cell and cell-matrix adhesion with subsequent effects on endothelial barrier function.

G. LOX1 is the main receptor responsible for OxLDL signaling in HAECs (Figures 9I, 9J and 9K). Methods: Construction of adenovirus shRNA for LOX1:

[00406] Viruses encoding non-targeting Ad-sh and Ad-sh LOX1 were generated using a pAdBLOCK-iT kit (Life Sciences). Briefly, non-targeting and other oligonucleotides targeting the 5' UTR region of human LOX1 were designed with the Life Sciences proprietary program and cloned into pU6-ENTR. The 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); Low, 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); Low, 5'-AAA AGC TTC ACT CTC TCA TTC TTA GTT CGC TAA GAA TGA GAG AGT GAA GC -3' (SEQ ID NO:82).

[00407] Non-targeting pU6-sh and pU6-LOX1shRNA plasmids were tested for function in transient transfection experiments with HAEC cells. Constructs showing the greatest inhibition were recombined with pAD/BLOCK-iTDEST (Invitrogen) to generate pAd-nontargeting and Ad-shLOX1. Viruses were amplified, purified and concentrated using a Millipore kit.

[00408] To confirm that LOX1 is required for OxLDL signaling in HAECs, LOX1 RNA expression was inhibited using a viral vector expressing small interfering hairpin RNAs (shRNA) targeting the 5' UTR region of the LOX1 gene human (LOX1shRNA). Briefly, 60 percent confluent HAECs were transduced with 25 MOI (multiplicity of infection, a measure of virulence) of adenovirus expressing shRNA for LOX1 ("LOX1-shRNA" or "Ad-LOX-1shRNA"). 24 hours later, the medium was changed to low serum medium (1%) containing virus encoding NFKB-LUC. The day after viral transduction, cells were treated with 50 μg/ml OxLDL and incubated for an additional 8 hours, followed by measurement of luciferase activity by chemiluminescence. Cell lysates were then immunoblotted with anti-Lox-1 or anti-GAPDH antibodies. As shown in Figures 9J and 9K, addition of shRNA to LOX1 significantly reduced LOX1 protein expression (see, for example, lane 3 of Figure 9J compared to lanes 1 and 2), confirming that the shRNA to LOX1 effectively inhibited the expression of LOX1 in HAECs. Furthermore, as shown in Figure 9I, AdshLOX1 significantly inhibited OxLDL-mediated NFkB signaling in HAECs compared to cells incubated with OxLDL and a non-targeting shRNA control construct (Ad-NTsh). * indicates P<0.05 (compared to untreated control); # indicates P<0.05 compared to OxLDL + Ad-NTsh.

[00409] A summary of the signaling pathways involved in LOX1 receptor signaling and blocked by the anti-LOX1 antibodies disclosed herein (including, for example, LX5140110; "Ac 514") is shown in Figure 10.

[00410] The foregoing description of the specific aspects thus fully discloses the general nature of the disclosure that others may, by application of knowledge within the art, readily modify and/or adapt for various applications such as specific aspects, without undue experimentation, without depart from the general concept of this disclosure. Therefore, these adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teachings and guidelines presented here. It is to be understood that the phraseology or terminology herein is for the purpose of description and not 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.

[00411] 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 publication, patent, patent application and/or other document individually designated as being incorporated by reference for all purposes.

Claims (11)

1. Antibody isolated from lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) characterized in that it comprises a heavy chain variable region (VH) comprising SEQ ID NO: 4 and a light chain variable region (VL) comprising SEQ ID NO: 33. 2. Isolated LOX1 antibody according to claim 1, characterized in that the LOX1 antibody reduces, inhibits or antagonizes LOX1 activity. 3. Isolated LOX1 antibody according to claim 1, characterized in that the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody or a LOX1-binding antibody fragment. 4. Isolated LOX1 antibody according to claim 3, characterized in that the LOX1-binding antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment , an Fv fragment, a diabody or a single-chain antibody molecule. 5. Isolated LOX1 antibody according to claim 1, characterized in that the LOX1 antibody 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 constant domain of human IgE; (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 constant domain of human IgM. 6. Isolated LOX1 antibody according to claim 1, characterized in that the LOX1 antibody further comprises a light chain immunoglobulin constant domain selected from the group consisting of: (a) a human Ig cap constant domain ; and (b) a human Ig lambda constant domain. 7. Isolated LOX1 antibody according to claim 1, characterized in that the LOX1 antibody further comprises a human IgG1 heavy chain constant domain and a human lambda light chain constant domain. 8. Isolated LOX1 antibody according to claim 7, characterized by the fact that the IgG1 constant Fc region domain contains a mutation in positions 234, 235 and 331, wherein the numbering of the positions is according to the index from EU as in Kabat. 9. Isolated LOX1 antibody according to claim 8, characterized by the fact that the IgG1 heavy chain constant domain contains the mutations L234F, L235E and P331S, wherein the numbering of the positions is according to the EU index as in Kabat. 10. Pharmaceutical composition characterized by the fact that it comprises a LOX1 antibody, as defined in claim 1, and a pharmaceutically acceptable carrier. 11. Use of an effective amount of a pharmaceutical composition comprising a LOX1 antibody as defined in claim 1, characterized by the fact that it is in the preparation of a medicament for the treatment, prevention and/or improvement of a cardiovascular disease or condition associated with LOX1 in an individual in need thereof, wherein the disease or condition is selected from the group consisting of: atherosclerosis, thrombosis, coronary artery disease (CAD), ischemia, infarction, acute coronary syndrome (ACS), stroke, reperfusion injury, restenosis, peripheral vascular disease, hypertension, heart failure and angiogenesis.