EA047046B1 - ANTIBODIES TO ANGPT2 - Google Patents

Description

List of sequences

This application contains a Sequence Listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy in question, created on June 11, 2019, is called 09-0697-US-1_SL.txt and is 78465 bytes in size.

Field of technology to which the invention relates

The present invention generally relates to anti-angiopoietin 2 (ANGPT2) neutralizing antibodies for diagnostic and therapeutic use. More specifically, anti-ANGPT2 antibodies and methods of use are described for the treatment of various diseases or disorders characterized by cells expressing ANGPT2. Pharmaceutical compositions and kits containing antibodies to ANGPT2 are also described.

Prerequisites for creating the invention

Endothelial dysfunction is a characteristic feature of chronic kidney disease (CKD) and associated cardiovascular complications. Basic and clinical studies suggest that improving renal vascular function in CKD will reduce proteinuria and reduce the decline in renal function in addition to SOC. Additionally, it is believed that reducing ANGPT2 will have a beneficial effect on cardiovascular diseases in patients with CKD, including heart failure, MI, stroke and others (Eleuteri 2011, Lukasz 2013, Poss 2015, Lorbeer 2015, Tsai 2016, Gerstein 2015, Chen 2009, Chen 2010a, Chen 2010b, Gurnik 2016).

The human ANGPT-Tie axis consists of two types of tyrosine kinase I receptors (Tie1, Tie2) and two secreted ligands (ANGPT1, ANGPT2). ANGPT1 is a Tie2 receptor agonist that induces phosphorylation of the receptor and activates downstream signaling pathways required to preserve renal vascular function, while ANGPT2 is a functional antagonist of ANGPT1 binding to Tie2. ANGPT1-bound Tie2 translocates to inter-endothelial cell-cell junctions, where multimeric ANGPT1 can form cross-endothelial complexes with Tie2 receptors from adjacent cells to stabilize glomerular capillary structure. Intracellularly, ANGPT1-induced Tie2 phosphorylation leads to the uptake of adapter proteins, which causes AKT activation of pro-survival pathways and suppresses activation of the apoptotic pathway.

A small study examining the role of ANGPT2 in diabetic nephropathy (DN) described a SNP (1233 A/G) associated with a 20% increase in circulating ANGPT2 and subsequent increase in DN severity (Quan, 2012). While ANGPT2 blocking antibodies have not been tested in patients with CKD, ANGPT2 mRNA expression in Pima Indians with CKD has been found to be positively associated with interstitial fibrosis and intimal fibrosis. Other authors have shown that glomerular ANGPT2 mRNA expression is increased in patients with diabetic nephropathy (DN) compared to levels in healthy kidneys (Dessapt-Baradez, 2014). However, both glomerular ANGPT1 mRNA expression (Dessapt-Baradez, 2014) and circulating protein (Chang, 2013; Chang, 2014) were unchanged in DN, thus favoring ANGPT2-bound Tie2 in the context of disease progression.

Key clinical observations linking dysregulation of the ANGPT2-Tie2 pathway with CKD were carried over from studies where patients were stratified by stage (stage 1 to ESRD/HD) and where circulating ANGPT2 was progressively increased, correlating with arterial stiffness (Chang, 2014). and inversely correlated with a decrease in inulin-measured glomerular filtration rate (GFR) (David, 2010). Within the CKD 3-5 cohort, ANGPT2 levels were also associated with the severity of albuminuria and markers of systemic micro-inflammation (Chang, 2013). At least some of the increased ANGPT2 present in CKD may be caused by a decrease in miR-145, which normally represses ANGPT2 transcription and has been shown to be significantly reduced in patients with stages 3–5 CKD (Chen, 2013). A recent poster presented by the American Society of Nephrology (Peters, 2018) for patients with nonproliferative diabetic retinopathy with baseline albuminuria >30 mg/g indicates that stimulation of the Tie2 signaling pathway using vascular endothelial protein tyrosine phosphatase (VE, AKB-9778 (daily , SC injection) was sufficient to reduce the urinary albumin/creatinine ratio (UACR) by approximately 20% in a 3-month Phase 2A study. These results confirm that stimulation of the Tie2 signaling pathway in patients with severe albuminuria is sufficient. reduce the progression of CKD. Clinically important values for categorizing CKD patients are: eGFR 15-60 ml/min/1.73 m ² and UACR 30 mg/g or more.

Consistent with the concept that dysregulation of the ANGPT2-Tie2 axis contributes to CKD, preclinical studies have shown that genetic manipulation of either side of the signaling pathway (decreasing ANGPT1 or increasing ANGPT2) is sufficient to cause disease manifestations. In mice, conditional deletion of ANGPT1 causes proteinuric nephropathy, which is characterized by dysfunction of the glomerular filtration barrier, albuminuria, and the pathological characteristic features observed in humans with progressive diabetic nephropathy (mesengial matrix enlargement and glomerulosclerosis; Jeans

- 1 047046 son, 2013). Additionally, overexpression of podocyte-specific ANGPT2 results in increased albuminuria, apoptosis of the glomerular endothelium, and a decrease in filtration barrier proteins (Davis, 2007). Other researchers have shown that ANGPT2 expression in plasma and kidneys after 5/6 nephrectomy in CD1 mice is combined with increased ANGPT2 staining in the glomeruli (Chang, 2014). The same group showed that the ANGPT2 peptibody, L1-10, after 5/6 nephrectomy blocks vascular expression and upregulates profibrotic and proinflammatory markers, including TGFe1, collagen subtypes, and adhesion molecules, although effects on renal fibrosis were not studied.

ANGPT2 is primarily expressed in tissues undergoing vascular remodeling and is elevated in the circulation in various disease states, including CKD. Endothelial cells (ECs) produce and store ANGPT2 in Weibel-Palad bodies, specialized storage granules from which ANGPT2 can be rapidly released into the bloodstream to bind to blood and lymphatic EC Tie2 receptors (Fiedler, 2004).

In normal human kidneys, ANGPT2 and Tie2 are expressed on ECs, including those facing the glomerular basement membrane, within capillary loops, and ECs within glomeruli. Tie2 is also expressed on ECs adjacent to the subcytic feet (Satchell, 2002). The Tie2 agonist ligand, ANGPT1, is secreted from pericytes (Satchell, 2001), which surround and support underlying endothelial cells, and also importantly from podocytes (Satchell, 2002), specialized kidney cells that make up the glomerular filtration barrier, thus allowing cross-talk interactions between podocytes and adjacent glomerular ECs to stabilize the capillary structure of the glomeruli.

ANGPT2 has limited expression in normal tissues but is widely expressed in actively remodeling human tumor vasculature. Blocking ANGPT2 inhibition of Tie2 signaling is an attractive target for antiangiogenic cancer therapy and vascular-based ocular diseases. Several antibodies that block ANGPT2 binding to Tie2 have been developed for clinical use.

Specifically, based on studies with an ANGPT2-selective antibody (REGN910) administered IV, no dose-limiting safety concerns were found in Phase I clinical trials (Papadopoulos, 2016). A Tie2 stimulator (AKB-9778) was tested in Phase II with no noteworthy adverse events (AEs) (Campochiaro, 2016), and in various Phase III clinical studies, a dual ANGPT1/2 blocker (AMG386) was tested with only mild and reversible AE (Monk, 2014). However, less selective therapeutic approaches with low ratios of ANGPT2:ANGPT1 blockade (eg, AMG386, MEDI3617) have been associated with an increased incidence of clinical edema, which may be associated with dual blockade of lymphatic Tie2 receptors, since both ANGPT1 and ANGPT2 act as Tie2 receptor agonists in the lymphatic vasculature. Tie2 blockade is believed to disrupt the normal lymphatic and venous circulation, leading to extracellular fluid accumulation in general and specifically to lymphedema (Monk, 2013). It is believed that a highly specific ANGPT2 blocking antibody will have a significantly reduced risk of edema; did not observe Scale 3-4 edema in Phase I studies using REGN910 (Papadopoulos, 2015). ANGPT2 blockade may affect vascular and lymphatic response and function.

According to some reports, ANGPT2 is involved in liver regeneration (Hu, 2014); reduces the plasticity of the vascular bed; alters the repair of the liver and other tissues (Gale, 2002), potentially including lung tissue, adipose tissue (An, 2017) and ovarian tissue (Coxon, 2010); and as an autocrine regulator, switches the inflammatory responses of endothelial cells (Fiedler, 2006, Kim, 2016).

The role of ANGPT2 in the adult lymphatic system is not fully known. However, Tie2 is expressed on different types of leukocytes (monocytes, neutrophils and eosinophils) and modulation of the Tie2 signaling pathway may alter immune sensitivity (Grenga, 2015).

ANGPT2 blockade is an attractive mechanism for preventing other respiratory disorders, including increased pulmonary vascular permeability, pulmonary edema, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis (IPF), and advanced exacerbation of IPF, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). High plasma levels of ANGPT2 play a central role in aberrant extravasation associated with increased plasma von Willebrand factor (a standard marker of endothelial damage) in sepsis and ARDS (Calfee, 2012). Plasma levels of ANGPT2 and von Willebrand factor are significantly elevated in patients with sepsis and even higher in patients with ARDS (Van der Heijden, 2008). Circulating ANGPT2 is significantly increased in people with sepsis who also have impaired oxygen saturation. Serum from these patients disrupts endothelial architecture in vitro. This effect of septic serum is reversed by ANGPT-1 (ANGPT2 antagonist) (Parikh, 2006). In a mouse model of hemorrhagic shock-induced ARDS, pre-treatment of mice with anti-ANGPT2 antibody significantly improved lung function, blood saturation

- 2 047046 oxygen and survival rate (Lomas-Neira, 2016).

Increased vascular permeability (vascular hyperpermeability) contributes to various diseases, including ARDS, sepsis, severe sepsis, septic shock, malignancy and inflammation. Reduced increased vascular permeability of the lungs will reduce the accumulation of fluid in the alveolar space (pulmonary edema) and will therefore improve gas exchange between the lung and the vessels, leading to better oxygenation of arterial blood. Improved oxygenation of arterial blood leads to better oxygenation of all organs (eg, brain, heart, liver, kidneys) and reduces the risk of multiple organ failure leading to death.

Increased vascular permeability in sepsis, severe sepsis, and septic shock has also been described in several organs such as the lungs, kidneys, liver, and heart. Fluid accumulation in these organs impairs their proper functioning (eg, causing arrhythmia, impaired glomerular filtration, or metabolic dysfunction) and leads to organ failure followed by death.

Pulmonary edema is a condition in which the lungs fill with fluid. The most common cause of pulmonary edema is congestive heart failure. Other less common conditions that can cause pulmonary edema include sudden increases in blood pressure, pneumonia, kidney failure, lung damage caused by a severe infection, severe blood sepsis, or blood poisoning caused by an infection.

Acute lung injury (ALI) is a pulmonary disorder often caused by smoke inhalation, most recently through the use of E-cigarettes or vaping products.

Acute respiratory distress syndrome (ARDS) is an inflammation of the lungs that is characterized by increased pulmonary vascular permeability and/or pulmonary edema. ARDS is often characterized as mild, moderate, or severe based on the degree of hypoxemia. ARDS can be triggered by several causes, for example, it can be induced by bacterial or viral lung infection, sepsis, inhalation of harmful substances, severe pneumonia, trauma, pancreatitis (inflammation of the pancreas), massive blood transfusions and burns. The most common cause of ARDS is sepsis.

Severe acute respiratory syndrome (SARS) is a viral respiratory disease caused by a coronavirus called SARS-associated coronavirus (SARS-CoV). SARS begins with a high fever (temperature greater than 100.4°F [>38.0°C]). Other symptoms may include sore throat, cough, headache, general discomfort and body pain. Some people may also have mild respiratory symptoms early in the course of the disease. Most patients develop pneumonia. From 2004 until the outbreak of the SARS-CoV-2 pandemic in December 2019, there were no known cases of SARS described worldwide.

Middle East respiratory syndrome (MERS) is a disease caused by a virus (more specifically, a coronavirus) called Middle East respiratory syndrome coronavirus (MERS-CoV). The disease is characterized by severe respiratory distress, including fever, cough and shortness of breath. Approximately three or four out of every 10 patients in whom MERS is described die.

Sepsis, severe sepsis, and septic shock are disorders that develop as a result of a systemic inflammatory response to infection (see Mitchell M. Levy et al., Crit Care Med. 2003 Apr;31(4): 1250-6). Sepsis is a disorder that has both an infectious nature (eg, viral, bacterial, abdominal trauma, intestinal perforation) and a systemic inflammatory response. This leads to increased vascular permeability in some organs such as the kidneys, liver, heart and lungs. Severe sepsis (sepsis with organ dysfunction) refers to sepsis with acute organ failure caused by sepsis. Septic shock refers to stable hypotension unexplained by other causes.

Thus, there is a need for a high affinity neutralizing antibody to ANGPT2 that will limit antagonistic binding of ANGPT2 to cTie2, it is believed that enhanced Tie2 signaling may have various beneficial effects, including, for example, stabilizing glomerular capillary structure, reducing endothelial activation, and restoring filtration integrity. barrier. Overall, these beneficial effects are believed to reduce proteinuria and preserve renal function, resulting in slower disease progression in patients with chronic kidney disease (CKD).

It is also believed that the beneficial effects of the high affinity neutralizing antibody to ANGPT2 help in the treatment of patients suffering from increased pulmonary vascular permeability and associated disorders.

Summary of the invention

The present invention provides monoclonal antibodies that specifically bind to human ANGPT2. In one aspect of the invention, antibodies of the present invention neutralize ANGPT2. Thus, the antibodies according to the invention are suitable, for example, for the treatment and/or prevention of diseases or disorders that can be alleviated by it

- 3 047046 tralization ANGPT2.

In another aspect, the present invention provides an anti-ANGPT2 antibody, particularly a humanized anti-ANGPT2 antibody, having one or more of the properties described later in the present invention.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof comprising:

a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3), and a light chain variable region containing the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 14 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 21 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 25 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3).

In another embodiment, the present invention provides an anti-ANGPT2 antibody or antigen binding fragment thereof comprising:

a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO. 3 and SEQ ID NO. 8, respectively, or a variable heavy chain and a variable light chain containing the amino acid sequences of SEQ ID NO. 4 and SEQ ID NO. 9, respectively, or a variable heavy chain and a variable light chain containing the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 10, respectively, or a variable heavy chain and a variable light chain containing

- 4 047046 amino acid sequences SEQ ID NO. 6 and SEQ ID NO. 11, respectively, or a variable heavy chain and a variable light chain containing the amino acid sequences of SEQ ID NO. 7 and SEQ ID NO. 12, respectively.

In another embodiment, the present invention provides an anti-ANGPT2 antibody or antigen binding fragment thereof comprising:

a heavy chain containing the amino acid sequence of SEQ ID NO. 31 and a light chain containing the amino acid sequence of SEQ ID NO. 32, or a heavy chain containing the amino acid sequence of SEQ ID NO. 33 and a light chain containing the amino acid sequence of SEQ ID NO. 34, or a heavy chain containing the amino acid sequence of SEQ ID NO. 35 and a light chain containing the amino acid sequence of SEQ ID NO. 36, or a heavy chain containing the amino acid sequence of SEQ ID NO. 37 and a light chain containing the amino acid sequence of SEQ ID NO. 38, or a heavy chain containing the amino acid sequence of SEQ ID NO. 39 and a light chain containing the amino acid sequence of SEQ ID NO. 40.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof comprising:

a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 14 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a heavy chain framework region comprising one to four amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO. 41 (H-FR1); amino acid sequence SEQ ID NO. 42 (H-FR2); amino acid sequence SEQ ID NO. 43 (H-FR3); and amino acid sequence SEQ ID NO. 44 (H-FR4).

In another embodiment, the present invention provides an anti-ANGPT2 antibody or antigen binding fragment thereof comprising:

a light chain variable region containing the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a light chain variable region containing the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a light chain variable region containing the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid

- 5 047046 sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a light chain variable region containing the amino acid sequence of SEQ ID NO. 21 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 25 (L-CDR3), or a light chain variable region containing the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3); and a light chain framework region comprising one to four amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO. 45 (L-FR1); amino acid sequence SEQ ID NO. 46 (L-FR2); amino acid sequence SEQ ID NO. 47 (L-FR3); and amino acid sequence SEQ ID NO. 48 (L-FR4).

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid regions 117-148 of the carboxy-terminal region of the human ANGPT2 fibrinogen-like domain (FLD) of the sequence SEQ ID NO. 50.

In another embodiment, the invention provides an ANGTP2 antibody or antigen binding fragment thereof that binds to SEQ ID NO: 51.

In one embodiment, the anti-ANGPT2 antibody is a humanized anti-ANGPT2 antibody.

In another embodiment, the anti-ANGPT2 antibody is a chimeric anti-ANGPT2 antibody.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof for medical use.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof for use in the treatment of kidney diseases.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof for use in the treatment of liver diseases.

In one embodiment, the present invention provides a pharmaceutical composition comprising an anti-ANGPT2 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-ANGPT2 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is administered via the parenteral route, intravenous route, intravitreal route, or subcutaneous route. . In one embodiment, the present invention provides isolated polynucleotide or polynucleotides comprising a sequence encoding a light chain or light chain variable region of an antibody or antigen binding fragment thereof and a heavy chain or heavy chain variable region of an antibody or antigen binding fragment thereof.

In one embodiment, the present invention provides an expression vector containing isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or antigen binding fragment thereof and a heavy chain or heavy chain variable region of an antibody or antigen binding fragment thereof.

In one embodiment, the present invention provides a viral vector comprising isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or antigen binding fragment thereof and a heavy chain or heavy chain variable region of an antibody or antigen binding fragment thereof.

In one embodiment, the present invention provides a host cell containing an expression vector or isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or antigen binding fragment thereof and a heavy chain or heavy chain variable region of an antibody or its antigen-binding fragment. In one embodiment, the present invention provides a method for producing an anti-ANGPT2 antibody or antigen-binding fragment thereof, comprising:

obtaining a host cell containing an expression vector or isolated polynucleotide or polynucleotides encoding a light chain or light chain variable region of an antibody or an antigen binding fragment thereof and a heavy chain or heavy chain variable region of an antibody

- 6 047046 or its antigen-binding fragment; and culturing the host cell.

In one embodiment, a method of producing an anti-ANGPT2 antibody or an antigen-binding fragment thereof further comprises reconstituting and purifying the anti-ANGPT2 antibody or an antigen-binding fragment thereof.

In another embodiment, the present invention provides an isolated polynucleotide or polynucleotides comprising:

a heavy chain coding sequence as set forth in SEQ ID NO: 31 or a heavy chain variable region as set forth in SEQ ID NO: 3; and a light chain coding sequence as set forth in SEQ ID NO. 32 or a light chain variable region as set forth in SEQ ID NO: 8, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 33 or a heavy chain variable region as set forth presented in SEQ ID NO: 4; and a light chain coding sequence as set forth in SEQ ID NO. 34 or a light chain variable region as set forth in SEQ ID NO:9, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 35 or a heavy chain variable region as set forth presented in SEQ ID NO: 5; and a light chain coding sequence as set forth in SEQ ID NO. 36 or a light chain variable region as set forth in SEQ ID NO: 10, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 37 or a heavy chain variable region as set forth presented in SEQ ID NO: 6; and a light chain coding sequence as set forth in SEQ ID NO. 38 or a light chain variable region as set forth in SEQ ID NO: 11, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 39 or a heavy chain variable region as set forth presented in SEQ ID NO: 7; and a light chain coding sequence as set forth in SEQ ID NO. 40 or a light chain variable region as set forth in SEQ ID NO: 12.

Non-limiting examples of diseases, disorders or conditions that may be ameliorated by anti-ANGPT2 antibodies of the invention include cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury, stroke hypertension, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, brain-induced disorders trauma, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy-induced hypertension and preeclampsia, sepsis, severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal -segmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic renal failure, end-stage renal disease, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF ), emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), hypervascular permeability (and associated disorders), acute kidney injury (AKI) , renal cell carcinoma, heart failure, lupus nephritis, Raynaud's syndrome, pancreatitis, peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma, retinopathy, diabetic nephropathy, portal hypertension, growth of varicose veins and liver transplantation.

Brief description of the figures

In fig. 1A and 1B show results (in duplicate) of angiopoietin 2 complement-dependent cell-mediated cytotoxicity (CDC) indicating the cytotoxicity of ANGPT2-opt-13 (-□-), REGN910 analog (nesvacumab) (-△-), MEDI3617 analog (^ X and LC06 ('*-.) Figure 2 shows epitope mapping to the FLD domain of antibodies to ANGPT2 (SEQ ID NO: 59): chimeric leader CL-209881, ANGPT2-opt-13, nesvacumab analog, MEDI3617 analog, and LC06. Specific binding sites for each molecule to the extracellular FLD domain of human ANGTP2 (SEQ ID NO: 59) are highlighted in dark gray.

In fig. 3A-3F show results for the ANGPT2 blocking assay for ANGPT2-opt-l) (Figure 3A), ANGPT2-opt-2 (Figure 3B), ANGPT2-opt-13 (Figure 3C), ANGPT2-opt-19 ( Fig. 3D), ANGPT2-opt-31 (Fig. 3E), chimeric leader CL-209881 (Fig. 3F), and the nesvacumab analog (Fig. 3G).

- 7 047046

Detailed Description of the Invention

The general structure of antibodies or immunoglobulins is well known to those skilled in the art; these molecules are heterotetrameric glycoproteins, typically with a molecular weight of approximately 150,000 Daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by a single disulfide bond to form a heterodimer, and a heterotrimeric molecule is formed by a covalent disulfide bond between two identical heterodimer heavy chains. Although the light and heavy chains are linked by a single disulfide bond, the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has evenly distributed intrachain disulfide bridges. Each heavy chain has an amino-terminal variable region (VHR=variable heavy chain), followed by three or four constant domains (CH1, CH2, CH3 and CH4), as well as a hinge region between CH1 and CH2. Each light chain has two regions, an amino-terminal variable region (VL=variable light chain) and a carboxy-terminal constant domain (CL). The VL region is non-covalently linked to the VH region, and the CL domain is usually covalently linked to the CH1 domain via a disulfide bond. It is likely that certain amino acid residues form the interface between the variable regions of the light and heavy chains (Chotia et al., J. Mol. Biol. 186, 1985, pp. 651-663). Certain regions in the variable regions vary significantly among different antibodies, i.e. they are hypervariable. These hypervariable regions contain residues that are directly involved in binding and determine the specificity of each particular antibody with respect to its specific antigenic determinant. Hypervariability in the variable regions, both light chain and heavy chain, is concentrated in three segments, which are designated as complementarity determining regions (hypervariable regions) (CDR) or hypervariable loops (HVL). CDRs are determined by sequence comparison according to the method described by Cabot et al., in: Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991, a HVL is structurally determined based on the three-dimensional structure of the variable region according to the method described by Chotia and Lesk, J. Mol. Biol. 196, 1987, cc. 901-917. If the two methods result in some differences in identifying CDRs, then structure-based identification is preferred. According to Cabot nomenclature, CDR-L1 is located at approximately residues 24-34, CDR-L2 at approximately residues 50-56, and CDR-L3 at approximately residues 89-97 in the light chain variable region; CDR-H1 is located at approximately residues 31-35, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102 in the heavy chain variable region. Thus, CDR1, CDR2, CDR3 of the heavy and light chains determine unique and functional properties specific to a given antibody.

The three CDRs in each of the heavy and light chains are separated by framework regions (FRs) that contain sequences that are less prone to variability. Starting from the amino terminus towards the carboxy terminus, the variable regions of the heavy and light chains of the FRs and CDRs are ordered as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The largely folded conformation of FR allows the CDRs on each strand to be in close proximity to each other, as well as to the CDRs from the other strand. The resulting conformation is inherent to the antigen-binding site (see Cabot et al., NIH Publ. No. 91-3242, Vol. I, 1991, pp. 647-669), although it is not necessary that all CDR residues be directly involved in antigen binding.

The FR residues and Ig constant domains are not directly involved in antigen binding, but are involved in antigen-associated and/or antigen-mediated effector function. Some FR residues are likely to have a profound effect on antigen binding in at least three ways: by noncovalently binding directly to the epitope, by interacting with one or more CDR residues, and by influencing the interface between the heavy and light chains. Constant domains are not directly involved in antigen binding but mediate various Ig effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are classified into one or two distinct classes, kappa (k) and lambda (λ), based on the amino acid sequence of the constant domain. By comparison, mammalian immunoglobulin heavy chains fall into one of five major classes based on the sequence of constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, respectively. The heavy chain constant domains that correspond to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of native immunoglobulin classes are well known.

The terms, antibody, anti-angiopoietin 2 antibody, anti-ANGPT2 antibody, humanized anti-ANGPT2 antibody, and humanized anti-ANGPT2 antibody variant are used in the broadest sense.

- 8 047046 sense, and they refer in particular to monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (for example, bispecific antibodies), antibodies with minor modifications, such as N- or C-terminal truncations, and antibody fragments, such as variable regions and other regions of antibodies that have the desired biological activity, for example, the ability to bind to ANGPT2. The term monoclonal antibody (mAb) refers to an antibody from a population of essentially homogeneous antibodies; those. Individual antibodies in a given population are identical except for naturally occurring mutations, which may be present in very small quantities.

Monoclonal antibodies are highly specific; their target is an individual antigenic determinant, an epitope. Thus, the adjective monoclonal is a distinctive feature of a substantially homogeneous population of antibodies that are targeted by an identical epitope, and the concept is not limited to the requirement that the antibodies be produced by any particular method. It should be obvious that monoclonal antibodies can be produced using any technique or methodology known in the art; including, for example, the hybridoma method (Kohler et al., Nature 256, 1975, p. 495), or recombinant DNA methods known in the art (see, for example, US 4816567), or methods for isolating monoclonal ones obtained using recombinant DNA antibodies using phage antibody libraries as described in Clackson et al., Nature 352, 1991, pp. 624-628 and Marks et al., J. Mol. Biol. 222, 1991, pp. 581-597.

Chimeric antibodies are composed of the heavy and light chain variable regions of antibodies from one species (eg, a non-human mammal, such as a mouse) and the light and heavy chain constant regions of antibodies from another species (eg, human), and can be made by joining sequences DNA encoding antibody variable regions from a first species (eg, mouse), with DNA sequences of antibody constant regions from a second species (eg, human), and transforming the host with an expression vector containing the related sequences, allowing the host to produce the chimeric antibody. Alternatively, a chimeric antibody may also be an antibody in which one or more heavy and/or light chain regions or domains are identical to, homologous to, or a variant of the corresponding sequence in a monoclonal antibody from another immunoglobulin class or isotype or from a consensus or germline sequence . Chimeric antibodies can include fragments of these antibodies, provided that the antibody fragment has the desired biological activity of the parent antibody, for example, the ability to bind to the same epitope (see, for example, US 4816567; and Morrison et al., Proc. Natl. Acad. . Sci. USA 81, 1984, pp. 68516855). The terms antibody fragment, antigen binding fragment, anti-ANGPT2 antibody fragment, humanized anti-ANGPT2 antibody fragment, humanized anti-ANGPT2 antibody variant fragment refer to the portion of the full-length anti-ANGPT2 antibody that retains the variable region or its functional ability, e.g., binding specificity to an ANGPT2 epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragment, diabody, linear antibody, single chain antibody, minibody, diabody formed from antibody fragments , and multispecific antibodies formed from antibody fragments. Full-length antibodies can be treated with enzymes such as papain or pepsin to produce the desired antibody fragments. Papain digestion is used to produce two identical antigen-binding fragments, called Fab'' fragments, each with one antigen-binding site, with the remainder being designated the Fc'' fragment. The Fab fragment also contains a light chain constant domain and a heavy chain C _H1 domain. After treatment with pepsin, an F(ab') ₂ fragment is obtained, which carries two antigen-binding centers and still retains the ability to cross-link with antigen. Fab' fragments differ from Fab fragments by the presence of additional residues, including one or more cysteine residues from the antibody hinge region at the C-terminus of the CHi domain. F(ab') ₂ antibody fragments are pairs of Fab fragments linked via cysteine residues in the hinge region. Other chemical combinations of antibody fragments are known.

The Fv'' fragment contains a complete antigen-recognition and antigen-binding center, consisting of a dimer including the variable region of one heavy and one light chain in close non-covalent association. In this configuration, the three CDRs of each variable region interact with a specific antigen binding site on the surface of the V _H -VL dimer. In general, six CDRs determine the specificity of an antibody for antigen binding.

A single chain Fv or scFv'' antibody fragment is a single chain Fv variant containing the VH and VL regions of the antibody, the regions being present on the same polypeptide chain. Single-chain Fv has the ability to recognize and bind to antigen. The scFv polypeptide may optionally also contain a polypeptide linker located between the VH and VL regions to facilitate the formation of the three-dimensional structure required for antigen binding by the scFv (see, for example, Pluckthun, in: The Pharmacology of monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, 1991, pp. 269-315).

- 9 047046

Other recognition capable antibody fragments include fragments that contain a pair of tandem Fd segments (V _H -C _H1 -V _H -C _H1 ) forming a pair of antigen binding sites. These linear antibodies can be bispecific or monospecific, as described, for example, in Zapata et al., Protein Eng. 8(10), 1995, cc. 1057-1062.

A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody that includes a variant amino acid sequence of an immunoglobulin or fragment thereof that has the ability to bind to a predetermined antigen and that contains one or more FRs that have the amino acid sequence substantially corresponding to the sequence of a human immunoglobulin, and one or more CDRs that have(s) an amino acid sequence that substantially matches the amino acid sequence of a non-human immunoglobulin. Said non-human amino acid sequence, often referred to as an import sequence, is typically derived from the import region of an antibody, in particular the variable region. In general, a humanized antibody includes at least a CDR or HVL of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain.

The present invention describes specific humanized antibodies to ANGPT2 that contain CDRs derived from the murine leader CL-209881 inserted between the FR sequences of the human germline heavy and light chain variable domains.

In one aspect, a humanized anti-ANGPT2 antibody comprises substantially all of at least one, and typically two, variable domains (such as those included in the Fab, Fab', F(ab') ₂ , Fabc, and Fv fragments) wherein all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin, and according to the present invention, the CDRs are murine chimeric CL-209881 leader sequences, and the FRs are FRs that have a consensus or germline sequence of a human immunoglobulin. In another aspect, the humanized anti-ANGPT2 antibody also includes at least a portion of the Fc region of an immunoglobulin, typically such a portion of a human immunoglobulin. Typically, the antibody must contain both a light chain and at least a heavy chain variable domain. The antibody may also include one or more _CH1 , hinge, _CH2 , CH3, and/or CH4 heavy chain regions, as appropriate. The humanized anti-ANGPT2 antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, _IgG2 , IgG3, IgG4, IgA1 and _IgA2 . For example, the constant domain may be a complement-fixing constant domain, where it is desired that the humanized antibody exhibit cytotoxic activity and the isotype is typically IgG1. In cases where such cytotoxic activity is not desired, the constant domain may be another isotype, for example IgG2. An alternative humanized anti-ANGPT2 antibody may contain sequences from more than one class or isotype of immunoglobulins, and the selection of specific constant domains to optimize the desired effector functions is within the skill of ordinary skill in the art.

The FR and CDR or HVL of a humanized anti-ANGPT2 antibody do not need to exactly match the parent sequences. For example, one or more residues in an import CDR or HVL or in a consensus or germline FR sequence may be altered (eg, by mutagenesis) by substitution, insertion, or deletion such that the resulting amino acid residue is not identical to the original residue at the corresponding position of any parental sequence, but nevertheless the antibody retains function, such as the ability to bind to ANGPT2. The change generally should not be extensive and should be a conservative change. Typically, at least 75% of the residues, more often at least 90% and most often more than 95% or more than 98% or more than 99%, of the humanized antibody must match the residues of the parent consensus sequence or germline FR sequence and import CDR sequences. Immunoglobulin residues that affect the interface between the heavy and light chain variable regions (VL-VH interface) are residues that affect the proximity or orientation of the two chains relative to each other. Specific residues that may be involved in interchain interactions are VL residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 and VH residues 35, 37, 39, 45, 47, 91, 93. 95, 100 and 103 (according to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)). US 6,407,213 also discusses that residues VL 43 and 85 and VH residues 43 and 60 may be involved in this interaction. Although these residues are specific to human IgG, they may be considered for other species. Important residues of the antibody that are expected to be involved in interchain interactions are selected for replacement in the consensus sequence.

The terms consensus sequence and consensus antibody refer to amino acids

- 10 047046 sequence that contains the most frequently occurring amino acid residue at each position in all immunoglobulins of any class, isotype or in any subunit structure, for example, the variable region of a human immunoglobulin. The consensus sequence may be based on immunoglobulins of a specific species or multiple species. By consensus sequence, structure, or antibody is meant the human consensus sequence specified in particular embodiments of the invention and refers to the amino acid sequence that contains the most frequently occurring residues at each position in all human immunoglobulins of a particular class, isotype, or subunit structure. Thus, the consensus sequence contains an amino acid sequence that has at each position an amino acid that is present in one or more known immunoglobulins, but which may not be an exact copy of the complete amino acid sequence of any individual immunoglobulin. The consensus sequence variable region is not derived from any naturally occurring antibody or immunoglobulin (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and its variants. The heavy and light chain FR consensus sequences and variants thereof are valuable sequences for the production of humanized antibodies to ANGPT2 (see, for example, US 6037454 and 6054297).

Human germline sequences are naturally present in the human antibody population. The combination of these germline genes creates antibody diversity. Antibody light chain germline sequences are derived from conserved human kappa or lambda germline v and j genes. Likewise, heavy chain sequences are derived from the germline v, d, and j genes (LeFranc M-P and LeFranc G, The Immunoglobulin Facts Book, Academic Press, 2001).

An isolated antibody is an antibody that has been identified and separated and/or purified from a component of its natural environment. Contaminants in the natural environment of an antibody are substances that may interfere with the diagnostic or therapeutic use of the antibody and may be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect of the invention, the isolated antibody must be an antibody purified to a degree greater than 95% by weight.

An isolated antibody is an antibody in situ within the recombinant cells in which it is produced if no component of the antibody's natural environment is present. Typically, however, the isolated antibody is produced by at least one purification step during which the recombinant cellular material is removed.

The term antibody performance refers to factors/properties that describe an antibody's recognition of an antigen or the effectiveness of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect properties of the antibody, such as folding, and can affect physical factors such as the initial rate of binding of the antibody to the antigen (ka), the dissociation constant of the antibody from the antigen (kd), the affinity constant of the antibody to the antigen (Kd ), antibody conformation, protein stability, and antibody half-life.

The term neutralizing antibody or blocking antibody refers to an antibody whose binding to ANGPT2 blocks the interaction between ANGPT2 and its receptor (Tie-2) and/or results in inhibition of at least one biological function of ANGPT2. It should be taken into account that the inhibition caused by an antibody that neutralizes or blocks ANGPT2 does not need to be complete if it can be detected using an appropriate assay. Typical assays for detecting inhibition are described in the present invention or known in the art.

As used herein, identical or percent identical to two or more nucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotide or amino acid residue when compared and aligned for maximum consistency. To determine percent identity, sequences are aligned for optimal comparison (eg, gaps can be introduced into the sequence of a first amino acid or nucleotide sequence for optimal comparative analysis of the primary structure with a second amino acid or nucleotide sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence contains the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions in the sequences (ie, % identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). In some embodiments

- 11 047046 of the invention, the two sequences being compared have the same length after introducing, if necessary, gaps in the sequence (eg, excluding additional sequence extending beyond the sequences to be compared). For example, when variable region sequences are compared, leader and/or constant domain sequences are not considered. When comparing the sequences of two sequences, the corresponding CDR refers to the CDR located at the same position in both sequences (eg, CDR-H1 of each sequence). Determining the percent identity or percent similarity between two sequences can be done using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used to compare two sequences is, but is not limited to, the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87, 1990, cc. 2264-2268, modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 1993, cc. 5873-5877. This algorithm is included in the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215, 1990, cc. 403-410. Nucleotide BLAST analysis can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST analysis of proteins can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To perform comparative analyzes of the primary structure including gaps, the Gapped BLAST program described in Altschul et al., Nucleic Acids, 25, 1997, cc. 3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search to determine distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, you can use the default settings of the corresponding programs (for example, XBLAST and NBLAST). Another preferred example, but not limited to, of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Myers and Miller, CABIOS, 1989. This algorithm is included in the ALIGN program (version 2.0), which is part of the software package for comparative analysis of the primary structure of GCG sequences. When ALIGN is used to compare amino acid sequences, the PAM120 weighted residue table, gap length penalty of 12, and gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, described in Torellis and Robotti, Comput. . Appl. Biosci. 10, 1994, cc. 3-5; and FASTA, described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 1988, cc. 2444-2448. In the FASTA program, the ktup parameter denotes a control parameter that specifies the sensitivity and speed of the search. If ktup = 2, then similar regions in the two sequences to be compared are identified by looking at pairs of aligned residues; if ktup = 1, then individual aligned amino acids are evaluated. Protein sequences can have a ktup value of 2 or 1, and DNA sequences can have values ranging from 1 to 6. Alternatively, comparative primary structure analysis of protein sequences can be performed using the CLUSTAL W algorithm described in Higgins et al., Methods Enzymol. 266, 1996, cc. 383-402.

A nucleic acid sequence is operably linked if it is located in a functional relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader sequence is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned in such a way as to facilitate translation. In general, operably linked means that the DNA sequences that are linked are contiguous and, in the case of a secretory leader sequence, contiguous and in reading frame. However, enhancers are not necessarily contiguous. Linking can be accomplished by ligation at suitable restriction sites. If such sites do not exist, then synthetic oligonucleotide adapters or linkers can be used.

As used in the present invention, the expressions cell, cell line and cell culture are used interchangeably and all such designations include their progeny. Thus, transformants and transformed cells include the primary cells in question and the cultures derived from them, regardless of the number of transfers.

The term "mammal to be treated" refers to any animal that belongs to the class of mammals, including humans, domestic and farm animals, as well as zoo, sporting or pet animals such as dogs, horses, cats, cows, etc. .d. Preferably the mammal is a human.

A disorder, as used in the present invention, is any condition that will benefit from treatment using a humanized anti-ANGPT2 antibody,

- 12 047046 described in the present invention. It includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder.

As used herein, the term ANGPT2-related disorder or ANGPT2-related disease refers to a condition in which modification or activation of ANGPT2-expressing cells is indicated. ANGPT2-associated disorders include diseases and disorders such as age-related macular degeneration, geographic atrophy, diabetic retinopathy, diabetic macular edema, retinal pigmentary degeneration, hereditary retinal dystrophy, hereditary macular degeneration, myopic degeneration, retinal vein occlusions, retinal artery occlusions , endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane due to any retinal disease, optical neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, and traumatic retinopathy, as well as prodromal and mild to moderate Alzheimer's disease, delayed disease progression in patients with Alzheimer's disease, Huntington's disease, Parkinson's disease, major depressive disorder, schizophrenia, cognitive impairment associated with schizophrenia, prevention of first episode of psychosis in individuals with attenuated psychotic syndrome, prevention of recurrence in patients with schizophrenia, treatment-resistant depression, and metabolic diseases , such as hyperphagia, obesity or metabolic syndrome.

ANGPT2-related disorders also include cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury, stroke hypertension, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, trauma-induced brain disorders, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy-induced hypertension and preeclampsia, sepsis, severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal segmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic renal failure, end-stage renal disease, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF), emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), hypervascular permeability (and associated disorders), acute kidney injury, renal cell carcinoma, heart failure, lupus nephritis, Raynaud's syndrome, pancreatitis , peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma, retinopathy, diabetic nephropathy, portal hypertension, varicose veins and liver transplantation. The term intravitreal injection has a common meaning in the art and refers to the introduction of an antibody to ANGPT2 or an antigen-binding fragment thereof into the vitreous body of a patient.

The term specifically binds or the like means that an anti-ANGPT2 antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether two molecules are specifically bound are described herein or known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and others. In one embodiment, specific binding is characterized by a KD of approximately 1 x 10 ^-8 M or less in accordance with the Affinity Binding Method described in the Examples section of the present invention. In another embodiment, specific binding is characterized by a KD of approximately 1x 10 ^-9 M or less in accordance with the Affinity Binding Method described in the Examples section of the present invention. However, an isolated antibody that specifically binds human Ang-2 may cross-react with other antigens, such as ANGPT2-2 molecules from other species. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemicals.

The term subcutaneous administration refers to the introduction of an anti-ANGPT2 antibody or antigen-binding fragment thereof under the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by a relatively slow, sustained injection from a drug-containing reservoir. Pinching or pulling the skin upward and away from underlying tissue can create a pocket.

Subcutaneous infusion refers to the introduction of a drug into the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by relatively slow, sustained administration from a drug-containing reservoir over a period of time of, but not limited to, 30 minutes. or less or 90 minutes or less. Optionally, the infusion can be carried out by subcutaneous implantation on

- 13 047046 pump for administering a medicinal product implanted under the skin of a sick animal or human, wherein the pump provides administration of the medicinal product in a predetermined amount over a predetermined period of time of 30 minutes, 90 minutes or a period of time corresponding to the duration of the treatment regimen.

The term subcutaneous bolus refers to the administration of a drug under the skin of a diseased animal or human, wherein the duration of administration of the drug as a bolus is less than about 15 minutes; in another object of the invention for less than 5 minutes, and in another object of the invention for less than 60 s. In a further aspect of the invention, administration is made into a pocket between the skin and the underlying tissue, where the pocket can be created by pinching or pulling the skin upward and away from the underlying tissue.

The term therapeutically effective amount is used to refer to the amount of an anti-ANGPT2 antibody or antigen-binding fragment thereof that reduces or alleviates one or more symptoms of the disorder being treated. The active substance is used in such a way that in the specified amount it has a beneficial effect on the patient. In one aspect, the therapeutically effective amount has a neuroprotective or neuroregenerative effect. In another aspect, a therapeutically effective amount refers to the target concentration in a patient's serum at which it appears effective, for example, in slowing the progression of a disease. Efficacy can be assessed in conventional ways depending on the condition being treated. For example, effectiveness can be measured by determining the response rate, such as vision recovery, or by assessing the delay time to disease progression.

The terms treatment and therapy and the like, as used herein, refer to therapeutic as well as prophylactic or suppressive measures for a disease or disorder that result in any clinically important or beneficial effect, including, but not limited to, alleviation or relief of one or more symptoms, regression, slowing or cessation of the progression of a disease or disorder. Thus, for example, treatment includes administration of an anti-ANGPT2 antibody or an antigen-binding fragment thereof before or after the onset of a symptom of a disease or disorder, resulting in the prevention or elimination of one or more symptoms of the disease or disorder. In another example, the concept refers to the administration of an anti-ANGPT2 antibody or an antigen-binding fragment thereof after the clinical manifestation of a disease to combat symptoms of the disease. Moreover, as used herein, the term treatment or therapy refers to the administration of an anti-ANGPT2 antibody or an antigen-binding fragment thereof after the onset and development of clinical symptoms, where the administration has an effect on clinical parameters of the disease or disorder, such as the extent of tissue damage or the number and extent of metastases , regardless of whether the treatment leads to relief of the disease or not. In addition, if the use of the compositions of the invention, either alone or in combination with another therapeutic agent, relieves or alleviates at least one of the symptoms of the disorder being treated compared to the specified symptom without the use of the anti-ANGPT2 antibody composition or antigen-binding fragment thereof, the result should be considered as effective treatment of the required disorder, whether or not all symptoms of the disorder are relieved. The term package insert refers to instructions typically included in commercial packages of therapeutic products that contain information regarding the indications, methods of use, administration, contraindications and/or precautions for the use of said therapeutic products.

Antibodies.

The antibodies described and disclosed herein are anti-ANGPT2 antibodies, particularly humanized anti-ANGPT2 antibodies, as well as compositions and articles containing anti-ANGPT2 antibodies according to the present invention. Antigen-binding fragments of antibodies to ANGPT2 have also been described. Antibodies to ANGPT2 and antigen binding fragments thereof can be used to treat various diseases or disorders that are characterized by reduced activity of the ANGPT2 pathway. An anti-ANGPT2 antibody and an antigen-binding fragment thereof each comprise at least a portion that specifically recognizes an ANGPT2 epitope.

During initial characterization, the anti-ANGPT2 chimeric leader CL-209881 was selected based on its much superior antibody performance. A library of variants was created by placing the mouse CDR leader sequence into the FR of the human heavy and light chain consensus variable domains, and by constructing the FR with various changes.

This resulted in 33 sequences, which were further optimized and subjected to fixation, yielding 6 final candidates with improved properties as described in the present invention. The sequences of the antibodies according to the invention are presented below:

VH sequences.

CL-209881_VH (chimeric leader), variable heavy chain, SEQ ID NO: 1

- 14 047046

QVOLKOSGAELVKPGSSVKISCRASGYIFIDYFINWVKQRPGOGLEWIGKIGPGS

GSSSSNEKFKGKATLTADKS S S T A YMQL S SLTSEDSAVYFCAREAFDYDGDYYG

MAYWGQGTSVTVSS

ANGPT2-opt-1 (humanized) variable heavy chain, SEQID NO: 3

OVOLVOSGAEVKKPGSSVKVSCKASGYIFIDYFINWVROAPGOGLEWMGKIGP

GSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYEGDY

YGMAYWGOGTLVTVSS

ANGPT2-opt-2 (humanized) variable heavy chain, SEQ ID NO: 4

QVOLVOSGAEVKKPGSSVKVSCKASGYIFIEYFINWVROAPGQGLEWMGKIGP

GSGSSSSNEKFKGRVTITADKSTS T A YMEL S S LRSEDTAVYYCAREAFDYEGDY

YGMAYWGOGTLVTVSS

ANGPT2-opt-13 (humanized) variable heavy chain, SEQ ID NO: 5

OVOLVOSGAEVKKPGSSVKVSCKASGYIFIDYFINWVROAPGOGLEWMGKIGP

GSGSSSSNEKFKGRVTITADKSTSTAYMEL SSLRSEDTAVYYCAREAFDYDGDY

YGMAYWGOGTLVTVSS

ANGPT2-opt-19 (humanized) variable heavy chain, SEQ ID NO: 6

OVOLVOSGAEVKKPGSSVKVSCKASGYIFIDYFINWVROAPGOGLEWMGKIGP

GSGSSSSNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREAFDYDGDY

YGMAYWGOGTLVTVSS

ANGPT2-opt-31 (humanized) variable heavy chain, SEQ ID NO: 7

OVOLVOSGAEVKKPGSSVKVSCKASGYIFIDYFINWVROAPGOGLEWMGKIGP

GSGSSSSNEKFKGRVTITADKSTS TAYMELSSLRSEDTAVYYCAREAFDYDGDY

YGM AYWGOGTL VT VS S

VL SEQUENCES.

CL-209881_VL (chimeric leader), variable light chain, SEQ ID NO: 2

DIVMTOSPSSLSVSAGEKVTMSCKSSOSLLNSGNOKNFLAWYOQKPGOPPKLLI

YGASTRESGVPDRFTGSGSGTDFTLTITSVOAEDLAVYYCQNDHSYPITFGSGTK

LEIK

ANGPT2-opt-l (humanized) variable light chain, SEQ ID NO: 8

EIVMTOSPATLSVSPGERATLSCKSSOSLLASGNOKNFLAWYOOKPGQAPRLLI

YGASTRESGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCQNDHSYPITFGOGTKL

EIK

ANGPT2-opt-2 (humanized) variable light chain, SEQ ID NO: 9

EIVMTOSPATLSVSPGERATLSCKSSOSLLASGNOKNFLAWYOOKPGQAPRLLI

YGASTRESGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCQNDHSYPITFGOGTKL

EIK

ANGPT2-opt-13 (humanized) variable light chain, SEQ ID NO: 10

EIVMTOSPATLSVSPGERATLSCKSSOSLLSSGNOKSFLAWYOQKPGOAPRLLIY

GASTRETGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCQNDHSYPITFGOGTKLE

IK

ANGPT2-opt-19 (humanized) variable light chain, SEQ ID NO: 11

EIVMTOSPATLSVSPGERATLSCRASOSVLSSGNOKSFLAWYOQKPGOAPRLLIY

GASTRETGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCOQDHSYPITFGOGTKLE

IK

ANGPT2-opt-31 (humanized) variable light chain, SEQ ID NO: 12

EIVMTOSPATLSVSPGERATLSCKSSOSLLSSGNOKSFLAWYOQKPGOAPRLLIY

GASTRESGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCQNDHSYPITFGOGTKLEI

TO

The underlined portions of the sequences described above correspond to the CDR regions of ba

- 15 047046 light and heavy chain riable regions. The humanized anti-ANGPT2 antibodies of the present invention are antibodies that have light and heavy chain sequences as shown in the table below.

Table 1

Full-length LC and HC sequences of humanized antibodies to ANGPT2

Antibody Subsequence SEQ ID NO: ANGPT2-opt-l (humanized) heavy chain Q VQL VQ S G AE VKKP GS S VK V S SK AS GYIFIDYFIN W VRQ APGOGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCAREAFDYEGDYYGMAYWGOG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 31 ANGPT2-opt-2 (humanized) heavy chain OVOLVQSGAEVKKPGSSVKVSCKASGYIFIEYFINWVRQ APGOGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCAREAFDYEGDYYGMAYWGOG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 33 ANGPT2-opt-13 (humanized) heavy chain OVQLVOSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQ APGOGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCAREAFDYDGDYYGMAYWGOG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 35 ANGPT2-opt-19 (humanized) heavy chain OVOLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQ APGOGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCAREAFDYDGDYYGMAYWGOG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 37 ANGPT2-opt-31 (humanized) heavy chain OVOLVQSGAEVKKPGSSVKVSCKASGYIFIDYFINWVRQ APGOGLEWMGKIGPGSGSSSSNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCAREAFDYDGDYYGMAYWGOG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 39 ANGPT2-opt-l (humanized) light chain EIVMTQSPATLSVSPGERATLSCKSSOSLLASGNQKNFLA WYOQKPGOAPRLLIYGASTRESGIPARFSGSGSGTEFTLTI SSLQSEDFAVYYCONDHSYPITFGOGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY A CEVTHQGLSSPVTKSFNRGEC 32

- 16 047046

Antibody Subsequence SEQ ID NO: ANGPT2-opt-2 (humanized) light chain C EVTHQGLSSPVTKSFNRGEC 34 ANGPT2-opt-13 (humanized) light chain EIVMTOSPATLSVSPGERATLSCKSSOSLLSSGNOKSFLA WYQQKP GOAPRLLIYGASTRETGIPARF S GS GS GTEFTLTI SSLQSEDFAVYYCONDHSYPITFGOGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYA CEVTHQGLSSPVTKSFNRGEC 36 ANGPT2-opt-19 (humanized) light chain EIVMTOSPATLSVSPGERATLSCRASOSVLSSGNOKSFLA WYQQKP GOAPRLLIYGASTRETGIPARF S GS GS GTEFTLTI SSLQSEDFAVYYCQQDHSYPITFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYA CEVTHQGLSSPVTKSFNRGEC 38 ANGPT2-opt-31 (humanized) light chain EIVMTOSPATLSVSPGERATLSCKSSOSLLSSGNQKSFLA WYQQKPGOAPRLLIYGASTRESGIPARFSGSGSGTEFTLTI SSLQSEDFAVYYCONDHSYPITFGOGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 40

table 2

CDR of humanized antibodies to ANGPT2 according to the numbering systems of CHEMICAL COMPUTING ______________________GROUP (CCG), Cabot and Chotia______________________

Antibody Subsequence SEQ ID NO: H-CDR1 from CL-209881 ANGPT2-opt-l ANGPT2-opt-13 ANGPT2-opt-19 ANGPT2-opt-31 GYIFIDYFIN 13 H-CDR1 from ANGPT2-opt-2 GYIFIEYFIN 14 H-CDR2 from CL-209881 ANGPT2-opt-l ANGPT2-opt-2 ANGPT2-opt-13 ANGPT2-opt-19 ANGPT2-opt-31 KIGPGSGSSSSNEKFKG 15 H-CDR3 from CL-209881 ANGPT2-opt-13 ANGPT2-opt-19 ANGPT2-opt-31 EAFDYDGDYYGMAY 16 H-CDR3 from ANGPT2-opt-l ANGPT2-opt-2 EAFDYEGDYYGMAY 17 L-CDR1 from CL-209881 KSSOSLLNSGNQKNFLA 18 L-CDR1 from ANGPT2-opt-l ANGPT2-opt-2 KSSOSLLASGNQKNFLA 19

- 17 047046

L-CDR1 from ANGPT2-opt-13 ANGPT2-opt-31 KSSOLLLSSGNQKSFLA 20 L-CDR1 from ANGPT2-opt-19 RASOSVLSSGNOKSFLA 21 L-CDR2 from CL-209881 ANGPT2-opt-l ANGPT2-opt-2 ANGPT2-opt-31 GASTRES 22 L-CDR2 from ANGPT2-opt-13 ANGPT2-opt-19 GASTRET 23 L-CDR3 from CL-209881 ANGPT2-opt-l ANGPT2-opt-2 ANGPT2-opt-13 ANGPT2-opt-31 ONDHSYPIT 24 L-CDR3 from BI00767086 QQDHSYPIT 25 H-CDR1 for SEQ ID NO. 15 (Cabot) DYFIN 26 H-CDR1 for SEQ ID NO. 16 (Cabot) EYFIN 27 H-CDR1 for SEQ ID NO. 15 (Chotia) GYIFIDY 28 H-CDR1 for SEQ ID NO. 16 (Chotia) GYIFIEY 29 H-CDR2 for SEQ ID NO. 17 (Chotia) GPGSGS thirty

The CDRs presented above according to Chemical Computing Group (CCG) numbering are underlined (Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al., Proteins 2014; 82:1599-1610). Sequences defined according to Cabot's nomenclature are in bold, and those defined according to the Chotia numbering system are in italics.

Humanization and variants of amino acid sequences.

Further variants of ANGPT2 antibodies and antibody fragments can be designed based on the set of CDRs presented in SEQ ID NO: 13-25. It is understood that in the amino acid sequence variants of anti-ANGPT2 antibodies and antibody fragments, the CDRs remain unchanged, but the surrounding regions, such as the FR regions, can be engineered. Anti-ANGPT2 antibody amino acid sequence variants can be generated by introducing appropriate nucleotide changes into the anti-ANGPT2 antibody DNA or by peptide synthesis.

These variants include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the anti-ANGPT2 antibodies exemplified herein. Any combination of deletions, insertions and substitutions is used to obtain the final construct, provided that the final construct has the required characteristics. Amino acid changes may also alter post-translational processing of the humanized anti-ANGPT2 antibody or variant thereof, such as changing the number or position of glycosylation sites.

In some embodiments, the present invention includes ANGPT2 antibodies or antibody fragments thereof having a variable heavy chain and a variable light chain, wherein the amino acid sequence of the variable heavy chain and the amino acid sequence of the variable light chain are at least 80%, at least 90% identical. %, at least 95%, at least 98%, or at least 99% identical to the amino acid sequences presented in SEQ ID NO. 3 or 8; or 4 or 9; or 5 or 10; or 6 or 11; or 7 or 12, respectively.

In some embodiments, the present invention includes anti-ANGPT2 antibodies or antibody fragments thereof having a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain and the amino acid sequence of the light chain are at least 80%, at least 90%, at least at least 95%, at least 98%, or at least 99% identical to the amino acid sequences set forth in SEQ ID NO: 31 or 32; or 33 or 34; or 35 or 36; or 37 or 38; or 39 or 40, respectively.

Another type of producing an amino acid variant of an antibody is to change the original glycosylation pattern of the antibody. The term change in the context of the present invention means the deletion of one or more carbohydrate moieties present in the antibody and/or the addition of one or more glycosylation sites not previously present in the antibody.

In some aspects, the present invention includes nucleic acid molecules that encode variant amino acid sequences of the anti-ANGPT2 antibodies described in the present invention. Nucleic acid molecules encoding amino acid variants in sequence

- 18 047046 antibodies to ANGPT2 are prepared using various methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or production by oligonucleotide mutagenesis (or site-directed mutagenesis), PCR mutagenesis, and cassette mutagenesis of a previously obtained variant or non-variant version. antibodies to ANGPT2.

In certain embodiments, the anti-ANGPT2 antibody is an antibody fragment. There are techniques that have been developed to obtain antibody fragments. Fragments can be obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24, 1992, pp. 107-117 and Brennan et al., Science 229, 1985, p. 81). Alternatively, fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be isolated directly from E. coli and chemically cross-linked to produce F(ab') ₂ fragments (see, for example, Carter et al., Bio/Technology 10, 1992, pp. 163- 167). According to another approach, F(ab') ₂ fragments can be isolated directly from a recombinant host cell culture. Other techniques for preparing antibody fragments will be apparent to one skilled in the art.

Antibodies to ANGPT2 and their antigen binding fragments may include modifications.

In certain embodiments, it may be desirable to use an anti-ANGPT2 antibody fragment rather than an intact antibody. It may be desirable to modify an antibody fragment to increase its serum half-life. This can be accomplished, for example, by incorporating a salvage receptor binding epitope into an antibody fragment. In one method, a suitable region of the antibody fragment can be changed (eg, mutated), or the epitope can be incorporated into a peptide tag, which is then fused to the antibody fragment at either end or in the middle, for example, by DNA synthesis or peptide synthesis. See, for example, WO 96/32478.

In other embodiments, the present invention includes covalent modifications of anti-ANGPT2 antibodies. Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino-terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl or threonyl residues. Another type of covalent modification involves chemical or enzymatic cross-linking of glycosides to the antibody. These modifications can be accomplished by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if possible. To introduce other types of covalent modifications into the antibody molecule, interaction of labeled amino acid residues of the antibody with an organic derivatizing agent that can react with selected side chains or amino-terminal or carboxy-terminal residues can be used.

Removal of any carbohydrate moieties present on the antibody can be accomplished chemically or enzymatically. Chemical deglycosylation is described in Hakimuddin et al., Arch. Biochem. Biophys. 259, 1987, p. 52 and in Edge et al., Anal. Biochem., 118, 1981, p. 131. Enzymatic cleavage of carbohydrate moieties on antibodies can be accomplished using endo- and exoglycosidases according to the method described in Thotakura et al., Meth. Enzymol., 138, 1987, p. 350.

Another type of suitable covalent modification involves coupling the antibody to one of a variety of non-protein polymers, such as, for example, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, using the methods described in one or more of US 4,640,835, US 4,496,689, US 4,301,144, US 4,670,417. US 4791192 and US 4179337.

Epitope binding.

In another aspect, the invention provides an antibody that recognizes a specific epitope of an ANGPT2 antigen and an ANGPT2 epitope. Specifically, the antibody of the invention binds to an epitope in the terminal fibrinogen-like domain (FLD) of the human ANGTP2 domain with the sequence SEQ ID NO: 50.

In one aspect, the invention relates to an ANGTP2 antibody or antigen binding fragment thereof that binds to at least one amino acid residue within the amino acid region 117-148 of the human ANGTP2 FLD domain (which is located at the C-terminus of the full-length human ANGPT2 protein), as set forth in SEQ ID NO: 50.

In another aspect, the invention relates to an ANGTP2 antibody or antigen binding fragment thereof that binds to SEQ ID NO: 51.

The sequences of SEQ ID NO: 50 and 51 are presented in the table below.

- 19 047046

Table 3

FLD home of human ANGPT2 and ANGPT2 epitope of antibodies according to the invention

Name Subsequence SEQ ID NO: FLD domain ANGTP2 person KEEQISFRDC AEVFKSGHTT NGIYTLTFPN STEEIKAYCD MEAGGGGWTIIQRREDGSVD FQRTWKEYKV GFGNPSGEYW LGNEFVSQLT NQQRYVLKIH LKDWEGNEAY SLYEHFYLSS EELNYRIHLK GLTGTAGKIS SISQPGNDFS TKDGDNDKCI CKCSQMLTGG WWFDACGPSN LNGMY YPQRQ NTNKFNGIKW YYWKGSGYSL KATMMIRPA DF SEQ ID NO: 50 ANGTP2 epitope YLSSEELNYR IHLKGLTGTA GKISSISQPG ND SEQ ID NO: 51

As used herein, the terms ANGPT2 antigen epitope and ANGPT2 epitope refer to a molecule (eg, peptide) or fragment of a molecule capable of binding to an anti-ANGPT2 antibody or antigen-binding fragment thereof. These terms further include, for example, an ANGPT2 antigenic determinant that is recognized by any of the antibodies or antibody fragments of the present invention that has a combination of light and heavy chain CDRs selected from the light chain CDRs set forth in SEQ ID NO. 18-25, and the heavy chain CDRs shown in SEQ ID NO. 13-17. In a further embodiment, the ANGPT2 antigenic determinant that is recognized by any of the antibodies or antibody fragments of the present invention has a combination of light and heavy chain CDRs selected from the light chain CDRs set forth in SEQ ID NO. 19-25, and the heavy chain CDRs shown in SEQ ID NO. 13-17.

Epitopes of the ANGPT2 antigen may be included in proteins, protein fragments, peptides or the like. Epitopes are most often proteins, short oligopeptides, oligopeptide mimics (ie, organic compounds that mimic the antibody-binding properties of the ANGPT2 antigen), or combinations thereof.

Antibodies or antibody fragments of the present invention have been found to bind to a unique epitope in the FLD domain of human ANGPT2. Preferably, the anti-ANGPT2 antibody or antigen-binding fragment thereof binds to at least one amino acid residue within the amino acid region of the human ANGPT2 FLD domain of the sequence SEQ ID NO: 50.

In one embodiment, the present invention provides an anti-ANGPT2 antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid regions of the human ANGPT2 FLD domain of the sequence SEQ ID NO: 51.

Thus, in the context of epitope binding, the expression binds within the amino acid region X-Y... means that the anti-ANGPT2 antibody or antigen binding fragment thereof binds to at least one amino acid residue within the amino acid region specified in the sequence.

If, for example, an anti-ANGPT2 antibody or an antigen-binding fragment thereof binds to at least one amino acid residue within the amino acid region 117-148, this means that the anti-ANGPT2 antibody or an antigen-binding fragment thereof binds to at least one amino acid residue or within the amino acid region region 117-148 of the human ANPT2 FLD domain of SEQ ID NO: 50. In another aspect, an anti-ANGPT2 antibody or antigen binding fragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid residues within amino acid regions 117-148 of the human ANGPT2 FLD domain of SEQ ID NO: 50.

In fig. 2 shows epitope mapping results for the chimeric leader CL_209881, ANGPT2-opt-13 (an exemplary anti-ANGTP2 antibody of the invention), the nesvacumab analog (where the lysine residue at position 219 is replaced by arginine, K219R), the MEDI3617 analog, and LC06. The specific binding sites for each molecule to the extracellular FLD domain of human ANGTP2 are highlighted in dark gray. The chimeric leader CL_209881 and ANGPT2-opt-13 (which is derived from CL209881) bind to an epitope that is different from the epitopes to which the reference antibodies bind.

The anti-ANGPT2 antibodies of the invention block the physical interaction between ANGPT2 and Tie2-expressing cells, and reverse the complete inhibition of Tie2 phosphorylation mediated by the full-length ANGPT2 oligomer.

Antibodies to ANGPT2 according to the invention are highly selective. There is no binding to the FLD domain of human ANGPT1 that can be detected at the highest concentration tested (500 nM). In addition, the anti-ANGPT2 antibodies of the invention do not exhibit nonspecific binding to charged or hydrophobic surfaces when tested down to 1 μM.

Binding affinity data indicate that the anti-ANGTP2 antibodies of the invention have high affinity and are highly selective for blocking ANGPT2. For example, antibodies

- 20 047046 to ANGPT2 according to the invention have high binding affinity to the FLD domains of human, cynomolgus and rabbit ANGPT2.

Since the anti-ANGPT2 antibodies of the invention do not exhibit binding affinity for recombinant mouse ANGPT2 proteins, efficacy studies were performed in mice with a mouse cross-reacting instrumental molecule (nesvacumab analogue) that recognizes the FLD domain of mouse ANGPT2 ( _KD : ~200 pM). In these studies, using a preclinical and mechanistically relevant disease model (db/db UNX mice), treatment with the analogue nesvacumab for 8–10 weeks, and at dosage levels that suppressed freely circulating ANGPT2, resulted in a significant increase in glomerular Tie2 phosphorylation and a decrease in disease phenotype, including significant improvements in kidney structure (reduction of glomerulosclerosis and interstitial fibrosis).

While the anti-ANGPT2 antibodies of the invention cannot be tested in mice for reducing the progression of nephropathy, the weak binding affinity of the anti-ANGPT2 antibodies of the invention to the FLD domain of rat ANGPT2 provides the opportunity to test the molecule in a strong mechanistic in vivo model of permeability in rats known as Miles research. Miles' study takes advantage of the VEGF-mediated release of ANGPT2 from storage granules in endothelial cells, leading to rapid vascular destabilization in vivo, an effect that can be quantified; the model was validated with the analogue nesvacumab. In the Miles study in rats, an antipermeability effect of ANGPT2-opt-13 was observed (49% reduction in permeability relative to IgG control), confirming in vivo activity in blocking ANGPT2-mediated vascular destabilization.

The cytotoxicity of anti-ANGPT2 antibodies according to the invention was studied using the ANGPT2 complement-dependent cell-mediated cytotoxicity (CDC) assay. The CDC study includes two ANGPT1/ANGPT2 cross-reacting antibodies (MEDI3617 analog and LC06), a comparator antibody that does not cross-react with ANGPT1 (nesvacumab analog), and an exemplary anti-ANGPT2 antibody of the invention (ANGPT2-opt-13).

The results of the CDC study (Figures 1A and 1B) demonstrate that the ANGPT1/ANGPT2 cross-reacting comparator antibodies (MEDI3617 analogue and LC06) and the ANGPT2 specific comparator antibody (nesvacumab analogue) all exhibit higher cytotoxicity compared to ANGPT2-opt13.

Therapeutic applications.

In one embodiment, anti-ANGPT2 antibodies of the invention, or antigen-binding fragments thereof, are useful for treating or preventing diseases or disorders that may be ameliorated by neutralizing ANGPT2 (ANGPT2-related diseases or disorders).

In another embodiment, the anti-ANGPT2 antibodies of the invention, or antigen binding fragments thereof, are useful as a drug.

In one embodiment, the ANGPT2 disease or disorder is selected from the group consisting of a disorder associated with ANGPT2 also includes cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury, stroke hypertension, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, brain disorders induced trauma, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy-induced hypertension and preeclampsia, sepsis, severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal -segmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic renal failure, end-stage renal disease, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF ), emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), increased vascular permeability (and associated disorders), acute kidney injury, renal cellular carcinoma, heart failure, lupus nephritis, Raynaud's syndrome, pancreatitis, peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma, retinopathy, diabetic nephropathy, portal hypertension, growth varicose veins and liver transplantation. In one embodiment, the present invention provides a method for treating NASH, cirrhosis, portal hypertension, variceal growth, variceal bleeding, and hepatic encephalopathy.

In another embodiment, the present invention relates to a method for treating chronic kidney disease. The term chronic kidney disease generally refers to a patient having decreased kidney function, as measured by glomerular filtration rate

- 21 047046 (GFR) or. An estimated GFR (eGFR) of 90 or greater (Stage 1) is considered normal renal function; eGFR or 89-60 (Stage 2) is considered moderate loss of kidney function; eGFR 59 to 45 (Stage 3a) is considered moderate to moderate loss of kidney function; eGFR 44 to 30 (Stage 3b) is considered moderate to severe loss of kidney function; and GFR 29 to 15 (Stage 4) is considered severe loss of kidney function.

In another embodiment, CKD patients have an albumin/creatinine ratio (UACR) 1 >30 mg/g and an eGFR of 20-75 ml/min/1.73 m ² or 15-60 ml/min/1.73 m ²

In one embodiment, the invention relates to the treatment of Stage 2 chronic kidney disease; in another embodiment, the invention relates to the treatment of Stage 3a chronic kidney disease; in another embodiment, the invention relates to the treatment of Stage 3b chronic kidney disease; and in another embodiment, the invention relates to the treatment of Stage 4 chronic kidney disease. In another embodiment, the invention relates to the treatment of chronic kidney disease in a patient having >eGFR 60; in another embodiment, the invention relates to the treatment of chronic kidney disease in a patient having >eGFR 45; in another embodiment, the invention relates to the treatment of chronic kidney disease in a patient having >eGFR 30; and in another embodiment, the invention relates to the treatment of chronic kidney disease in a patient having >eGFR 20.

In another embodiment, the invention relates to the treatment of chronic kidney disease in a patient having an eGFR of 20-75 ml/min/1.73 m ² .

In another embodiment, the invention relates to a method of treating chronic kidney disease in a patient having a UACR >30 mg/g.

In another embodiment, the invention relates to a method of treating chronic kidney disease in rapidly progressing (dynamic progression) patients. As used in the present invention, the term rapidly progressing CKD patients have an eGFR of 20-75 ml/min/1.73 m ² and a decrease of >3 ml/min/1.73 m ² /year. In another embodiment, rapidly progressing CKD patients have an eGFR of 20-75 ml/min/1.73 m ² and a decrease of >4 ml/min/1.73 m /year.

In another embodiment, the invention relates to a method of reducing progression to end-stage renal disease (ESRD), dialysis and/or cardiovascular complications in a CKD patient having an eGFR of 20-75 ml/min/1.73 m ² . In another embodiment, the invention relates to a method of reducing the risk of a cardiovascular complication in a CKD patient, for example, a patient having an eGFR of 20-75 ml/min/1.73 m ² .

In another embodiment, the present invention relates to a method for treating diabetic nephropathy.

In another embodiment, the present invention relates to methods for treating increased vascular permeability and/or associated disorders. These include (i) leaky respiratory disorders not primarily caused by infection (Group 1), and (ii) leaky respiratory disorders caused by certain bacterial, viral, or fungal pathogens. parasitic infections (Group 2).

Non-limiting respiratory disorders associated with Group 1 hypervascular permeability include pulmonary edema, idiopathic interstitial pneumonia, IPF and severe exacerbation of IPF, ARDS not associated with infection, and ALI; And

Non-limiting respiratory disorders associated with Group 2 hypervascular permeability include infection-related ARDS, SARS, MERS, sepsis, severe sepsis, and septic shock.

Non-infection related ARDS is understood as ARDS that is not triggered or caused by an infection, such as ARDS caused by inhalation of harmful substances (eg, toxic smoke), trauma, pancreatitis, gastric reflux, massive blood transfusions, or burns.

Infection-related ARDS is understood as ARDS that is triggered or caused by an infection, such as ARDS caused by sepsis or severe pneumonia.

In one embodiment, the present invention relates to the treatment of increased vascular permeability.

In another embodiment, the present invention relates to the treatment of increased vascular permeability due to or caused by bacterial infection, including, but not limited to, Legionella pneumophila, Haemophilus influenzae, Sterptococcus pneumonia, Klebsiella, Mycoplasma pneumonia, and Staphylococcus aureus.

In another embodiment, the present invention relates to the treatment of increased vascular permeability due to or caused by fungal infection, including, but not limited to, fungal pneumonia and parasitic pneumonia.

In another embodiment, the present invention relates to the treatment of increased vascular permeability resulting from or caused by viral infections, including but not

- 22 047046 but limited to H1N1 influenza virus, respiratory syncytial virus, parainfluenza virus, adenovirus and human coronavirus (CoV) infections such as SARS-CoV, SARS-CoV-2 and MERS-CoV.

In another embodiment, the present invention relates to the treatment of increased vascular permeability associated with ALI, ARDS, or SARS.

Non-therapeutic uses.

The antibodies presented herein can be used as affinity-based purification agents.

In this process, antibodies are immobilized on a solid phase, such as a Protein A resin, using methods well known in the art. The immobilized antibody is contacted with a sample containing the ANGPT2 protein (or fragment thereof) to be purified, and the support is then washed with a suitable solvent that should remove substantially all of the material in the sample except the ANGPT2 protein associated with the immobilized antibody. Finally, the support is washed with another suitable solvent, which should separate the ANGPT2 protein from the antibody.

Antibodies to ANGPT2 are also useful in diagnostic studies to detect and/or quantify ANGPT2 protein, for example, detecting ANGPT2 expression in specific cells, tissues or serum.

In some embodiments, it is advantageous, for example for diagnostic purposes, to label an antibody with a detectable moiety. Numerous detectable tags are known and available, such as radioisotopes, fluorescent tags, enzyme substrate tags, and the like. The label can be indirectly conjugated to the antibody using various known techniques. For example, an antibody can be conjugated to biotin, and any of the above three broad categories of labels can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin and therefore the label can be conjugated to the antibody indirectly. Alternatively, to achieve label-antibody indirect conjugation, the antibody can be conjugated to a small hapten (such as digoxin) and one of the various types of labels above conjugated to an anti-hapten antibody (for example, an anti-digoxin antibody). In this way, indirect conjugation of the label with the antibody can be achieved.

Examples of radioisotope tracers are ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I.

The antibody can be labeled with a radioisotope using techniques described, for example, in Current Protocols in Immunology, vol. 1 and 2, 1991, ed. Coligen et al., Wiley-Interscience, New York, N.Y., 1991. Radioactivity can be measured, for example, using a scintillation counter. Examples of fluorescent tags are known tags based on rare earth metal chelates (europium chelates), or tags based on fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas red. Fluorescent tags can be conjugated to the antibody using known techniques, for example, those described in Current Protocols in Immunology. Fluorescence can be quantified using a fluorimeter.

Various well-characterized enzyme-substrate tags are known in the art (see, for example, the review given in US 4,275,149). The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be assessed using a variety of techniques. For example, the change may be a change in the color of the substrate, which can be assessed spectrophotometrically. Alternatively, the enzyme can change the fluorescence or chemiluminescence of the substrate. Fluorescence quantification techniques are described above. As a result of a chemical reaction, an electronically excited state arises in the chemiluminescent substrate, as a result of which it can emit light, which can be assessed, for example, using a chemiluminometer, or it acts as an energy donor for a fluorescent acceptor.

Examples of enzymatic tags are luciferases such as firefly luciferase and bacterial luciferase (US 4737456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase and glucose-6phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. Methods for conjugating enzymes with antibodies are described, for example, in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in: Methods in Enzym., ed. J. Langone and N. Van Vunakis, Academic press, N.Y., 73, 1981, cc. 147-166.

Examples of enzyme-substrate combinations are, for example: horseradish peroxidase (HRPO) and hydrogen peroxidase as substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) and para-nitrophenylphosphonate as a chromogenic substrate; and β

- 23 047046

D-galactosidase (β-D-Gal) and a chromogenic substrate, such as para-nitrophenyl-β-D-galactosidase, or a fluorogenic substrate, such as 4-methylumbelliferyl-β-galactosidase. Numerous other enzyme-substrate combinations are known to those skilled in the art. A general overview is presented, for example, in US 4275149 and US 4318980.

In another embodiment, the anti-ANGPT2 antibody is used unlabeled and detected with a labeled antibody that binds the anti-ANGPT2 antibody.

The antibodies provided herein can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, ed. in CRC Press, Inc., 1987, pp. 147-158). Diagnostic kits. The humanized anti-ANGPT2 antibody may be used in a diagnostic kit, which is a package that contains a combination of reagents in predetermined quantities along with instructions for performing the diagnostic assay. If the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme, such as a precursor substrate that forms a detectable chromophore or fluorophore. In addition, other additives such as stabilizers and buffers (eg, blocking buffer or lysis buffer) and the like may be included. The relative amounts of the various reagents can vary widely, providing concentrations of reagents in solution that greatly optimize the sensitivity of the assay. The reagents may be dry powders, typically lyophilized, including excipients which, when dissolved, should form a reagent solution having the appropriate concentration.

Diagnostic kits.

Antibody to ANGPT2 can be used in a diagnostic kit, which is a package that contains a combination of reagents, taken in predetermined quantities, in combination with instructions for performing the diagnostic assay. If the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme, such as a precursor substrate that forms a detectable chromophore or fluorophore. In addition, other additives such as stabilizers and buffers (eg, blocking buffer or lysis buffer) and the like may be included. The relative amounts of the various reagents can vary widely, providing concentrations of reagents in solution that greatly optimize the sensitivity of the assay. The reagents may be dry powders, typically lyophilized, including excipients which, when dissolved, should form a reagent solution having the appropriate concentration.

Compositions and their introduction.

A composition containing an anti-ANGPT2 antibody or an antigen-binding fragment thereof can be administered to a subject who suffers from or is at risk of developing the ANGPT2-related diseases or disorders described in the present invention. The invention further provides the use of an anti-ANGPT2 antibody or an antigen-binding fragment thereof for the preparation of a medicament for the prevention or treatment of an ANGPT2 disease. The term subject, as used herein, refers to any mammalian patient to whom an anti-ANGPT2 antibody or antigen-binding fragment thereof can be administered, including, for example, humans and certain non-human mammals such as primates and dogs. Subjects specifically intended for treatment using the methods disclosed in the present invention include humans. The anti-ANGPT2 antibody or antigen-binding fragment thereof can be administered either alone or in combination with other compositions.

Preferred antibodies for use in such pharmaceutical compositions are those that include an antibody of the invention.

Various delivery systems are known and can be used to administer an anti-ANGPT2 antibody or an antigen-binding fragment thereof. Routes of administration include, but are not limited to, intravitreal, eye drops, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The anti-ANGPT2 antibody or antigen-binding fragment thereof may be administered, for example, by infusion, bolus, or injection, and may be administered in conjunction with other biologically active agents. Administration may be systemic or local. In preferred embodiments, administration is by intravitreal injection. Preparations for such injections can be prepared, for example, in pre-filled syringes.

The anti-ANGPT2 antibody or antigen-binding fragment thereof can be administered in the form of pharmaceutical compositions containing a therapeutically effective amount of the anti-ANGPT2 antibody or antigen-binding fragment thereof and one or more pharmaceutically compatible ingredients. According to typical embodiments of the invention, the pharmaceutical composition is prepared according to conventional procedures to obtain a pharmaceutical composition suitable for

- 24 047046 intravenous or subcutaneous administration to humans. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may also contain a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the ingredients are supplied either separately or mixed to form a unit dose of the drug, for example, as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active ingredient. In cases where the pharmaceutical agent is intended for administration by infusion, it can be filled into infusion bottles containing sterile water or pharmaceutical grade saline solution. In cases where the pharmaceutical agent is administered by injection, an ampoule of sterile water for injection or saline may be used so that the ingredients can be mixed before administration. In addition, the pharmaceutical composition can be included in a pharmaceutical kit that contains (a) a container containing an anti-ANGPT2 antibody or antigen binding fragment thereof in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (eg, sterile water) for injection. A pharmaceutically acceptable diluent can be used to reconstitute or dilute the lyophilized anti-ANGPT2 antibody or antigen binding fragment thereof. Optionally, the specified container(s) may be accompanied by a notice in a form approved by an official agency authorizing the manufacture, use, or sale of pharmaceuticals or biological products, where the notice reflects the agency's approval of the manufacture, use, or sale for the purpose of administration to humans.

The amount of anti-ANGPT2 antibody or antigen-binding fragment thereof that is effective for treating or preventing diseases or disorders associated with ANGPT2 can be determined by conventional clinical research methods. In addition, in vitro assays may not necessarily be used, the results of which may help determine optimal dosage limits. The exact dose to be used in the formulation should also depend on the route of administration and the stage of the disorder, and should be based on the recommendations of the treating physician and the circumstances specific to each patient. Effective doses can be determined by extrapolation from dose response curves obtained from in vitro experiments or animal model test systems.

For example, the toxicity and therapeutic efficacy of an anti-ANGPT2 antibody or antigen-binding fragment thereof can be determined in cell cultures or experimental animals by standard pharmaceutical methods used to determine the ED50 (the therapeutically effective dose for 50% of the population). Preferred is an anti-ANGPT2 antibody or antigen-binding fragment thereof that exhibits a high therapeutic index.

Data obtained from cell culture assays and animal studies can be used to determine dose limits for human use. Typically, the dose of anti-ANGPT2 antibody or antigen-binding fragment thereof is within the range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dose can be varied within the specified limits depending on the dosage form used and the route of administration used. For any anti-ANGPT2 antibody or antigen-binding fragment thereof used in the method, a therapeutically effective dose may initially be determined based on the results of cell culture assays. The dose can be determined based on data obtained in animal models, reaching plasma concentration limits that include the IC ₅₀ (i.e., the concentration of the test compound at which half the maximum inhibition of symptoms is achieved) based on data obtained in culture cells. Such information can be used to more accurately determine suitable doses for humans. Plasma concentrations can be determined, for example, by high performance liquid chromatography, ELISA, etc. For intravitreal injection of anti-ANGPT2 antibody, longer treatment intervals are generally preferred. Due to their improved efficacy, the anti-ANGPT2 antibodies of the present invention can be administered at longer intervals. In one embodiment, the ANGPT2 antibody is administered every 6 weeks, or every 7 weeks, or every 8 weeks, or every 9 weeks, or every 10 weeks, or every 11 weeks, or every 12 weeks. In a further embodiment, the ANGPT2 antibody of the invention is administered once every 3 months.

The antibodies of the present invention can be formulated in dosages that include, but are not limited to, 20 mg/ml to 180 mg/ml; or 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml. Preferably, the antibodies of the present invention can be formulated in a liquid formulation of from about 50 mg/ml to about 150 mg/ml.

In some embodiments, pharmaceutical compositions comprising an anti-ANGPT2 antibody or antigen-binding fragment thereof may further include a therapeutic vehicle

- 25 047046 product, conjugated or unconjugated with a binding agent. Such combination therapy may have additive or synergistic effects on disease features (eg, symptom severity, number of symptoms, or relapse rates).

When using combination administration treatment regimens, in a particular embodiment of the invention, the anti-ANGPT2 antibody or antigen-binding fragment thereof is administered simultaneously with the therapeutic agent. In another specific embodiment of the invention, the therapeutic agent is administered before or after administration of the anti-ANGPT2 antibody or antigen-binding fragment thereof, for from 1 hour to several months, for example, for at least 1 hour, 5 hours, 12 hours, days, weeks, a month or three months before or after administration of an anti-ANGPT2 antibody or antigen-binding fragment thereof.

The compounds of the invention may be used alone or in combination with one or more additional therapeutic agents. Non-limiting examples of additional therapeutic agents may include:

antidiabetic drugs such as alpha-glucosidase inhibitors (eg, miglitol and acarbose), amylin analogues (eg, pramlintide), dipeptidyl peptidase 4 inhibitors (eg, alogliptin, sitagliptin, saxagliptin, and linagliptin), incretin mimetics (eg, liraglutide, exenatide, liraglutide , exenatide, dulaglutide, albiglutide and lixisenatide), insulin, meglitinides (eg repaglinide and nateglinide), biguanides (eg metformin); SGLT-2 inhibitors (eg, canagliflozin, empagliflozin, and dapagliflozin), sulfonylureas (eg, chlorpropamide, glimepiride, glyburide, glipizide, glyburide, tolazamide, and tolbutamide), and thiazolidinediones (eg, rosiglitazone and pioglitazone);

angiotensin II receptor antagonists (angiotensin receptor blockers (ARBs)), such as candesartan, eprosartan, candesartan, irbesartan, losartan, olmesartan, telmisartan, valsartan, azilsartan and medoxomil;

angiotensin-converting enzyme inhibitors (eg, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril and perindopril);

anticoagulants (eg dabigatran, actilise, warfarin, heparin and acetylsalicylic acid);

bronchodilators, including short-acting and long-acting beta-agonists (eg, albuterol, levalbuterol, salmeterol, formoterol, arformoterol, vilanterol, indacaterol, and olodaterol);

short-acting and long-acting anticholinergics (ipratropium, tiotropium, umeclidinium, glycopyrrolate and aclidinium);

steroids such as fluticasone and budesonide;

antimalarials such as hydroxychloroquine or chloroquine;

viral nucleoside analogues such as remdesivir; and HIV protease inhibitors such as lopinavir-ritonavir;

Polynucleotides, vectors, host cells and recombinant methods.

The present invention relates to isolated polynucleotides, which include the anti-ANGPT2 antibody coding sequence, vectors and host cells containing the polynucleotides, and recombinant techniques for producing the antibody. The isolated polynucleotides may encode any desired form of anti-ANGPT2 antibody, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab') ₂ , and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from fragments antibodies. The polynucleotide(s) that contain a sequence encoding an anti-ANGPT2 antibody or a fragment or chain thereof may be conjugated to one or more regulatory or control sequences, as known in the art, and can be contained in suitable expression vectors or host cells, as known in the art. Each of the polynucleotide molecules encoding the heavy or light chain variable domains can be independently paired with a polynucleotide sequence encoding a constant domain, such as a human constant domain, enabling the production of intact antibodies. Alternatively, the polynucleotides, or portions thereof, can be conjugated together to provide a template for the production of a single chain antibody.

For recombinant production, a polynucleotide encoding an antibody is inserted into a replicable vector for cloning (DNA amplification) or expression. Various suitable vectors for expression of recombinant antibodies are available. Vector components typically include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Antibodies to ANGPT2 can also be prepared as fusion polypeptides in which the antibody is fused to a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence is typically selected from a sequence that is located

- 26 047046 is known and processed (that is, cleaved by signal peptidase) by the host cell. For prokaryotic host cells that do not recognize or process the anti-ANGPT2 antibody signal sequence, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein, thermostable enterotoxin II leader sequences, and the like. For secretion by yeast, the native signal sequence can be replaced, for example, by a leader sequence derived from yeast alpha-factor invertase (including the leader sequences of Saccharomyces and Kluyveromyces alpha-factors), acid phosphatase, C. albicans glucoamylase, or the signal sequence described in WO90/ 13646. In mammalian cells, mammalian signal sequences as well as viral secretory leader sequences, for example the herpes simplex gD signal sequence, can be used. The DNA for such a precursor region is ligated in frame to DNA encoding a humanized anti-ANGPT2 antibody.

Expression and cloning vectors contain a nucleotide sequence that enables the vector to replicate in one or more selected host cells. In general, in cloning vectors, this sequence is the sequence that enables the vector to replicate independently of host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for various bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the origin of replication is 2-υ. Plasmids are suitable for yeast, and various viral origins of replication (SV40, polyoma, adenovirus, VSV and BPV) are suitable as cloning vectors in mammalian cells. In general, the origin of replication component is not necessary for mammalian expression vectors (the SV40 origin of replication can typically be used only because it contains an early promoter).

Expression and cloning vectors may contain a gene that encodes a selectable marker to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, or alternatively, complement axotrophic deficiencies, or in other alternatives, supplement specific nutrients that are not present in complex media, for example, the gene encoding the D-alanine racemate for Bacilli.

One example of a selection scheme uses a drug to stop the growth of a host cell. Those cells that are successfully transformed with the heterologous gene produce the drug resistance protein and therefore survive the selection scheme. Examples of such dominant selection are the use of the drugs neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those that provide the ability to identify cells competent to accept a nucleic acid encoding a humanized anti-ANGPT2 antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and -II (such as the primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and others. Cells transformed by a DHFR-selectable gene are first identified by culturing all transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. A suitable host cell used, if wild-type DHFR is used, is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (eg, DG44). Alternatively, host cells (preferably wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an anti-ANGPT2 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APN) , can be selected by growing cells in a medium containing a selectable agent for the selectable marker, such as an aminoglycoside antibiotic, for example kanamycin, neomycin, or G418. see, for example, US patent 4,965,199.

When recombinant production is carried out in a yeast cell as a host cell, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1 gene provides a selection marker for a mutant yeast strain that lacks the ability to grow on tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). Subsequently, the presence of the trp1 lesion in the host yeast cell genome provides an efficient environment for detecting transformation by growth in the absence of tryptophan. Likewise, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented with known plasmids carrying the LEU2 gene.

Additionally, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, an expression system for the industrial production of recombinant bovine chymosin for K. lactis has been described (Van den Berg,

- 27 047046

1990, Bio/Technology 8:135). Stable multicopy expression vectors for the secretion of mature recombinant human serum albumin using commercial Kluyveromyces strains have also been described (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vectors typically contain a promoter that is recognized by the host and is operably linked to a nucleic acid molecule encoding an anti-ANGPT2 antibody or polypeptide chain thereof. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgamo (S.D.) sequence operably linked to DNA encoding a humanized anti-ANGPT2 antibody. Many eukaryotic promoter sequences are known. In fact, all eukaryotic genes have an AT-rich region located approximately 25-30 bp upstream of the transcription start site. Another sequence, 70-80 bases upstream from the start of transcription of many genes, is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes there is an AATAAAA sequence, which can be a signal for the addition of a poly-A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other hydrolytic enzymes such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Inducible promoters have the added benefit of transcription under the control of growth conditions. These include the yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use for expression in yeast are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

Transcription of anti-ANGPT2 antibody from vectors in mammalian host cells is controlled, for example, by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus , retrovirus, hepatitis B virus and simian virus 40 (SV40), from heterologous mammalian promoters, for example, actin promoter or immunoglobulin promoter, or from heat shock promoters, provided that such promoters are compatible with host cell systems. The SV40 early and late promoters are readily obtained as an SV40 restriction fragment, which also contains the SV40 origin of replication. The immediate early promoter of human cytomegalovirus is readily obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is described in US Pat. No. 4,419,446. A modification of this system is described in US Patent No. 4,601,978. See also Reyes et al., 1982, Nature 297:598-601, which describes the expression of human p-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to enhance transcription of DNA encoding an anti-ANGPT2 antibody by higher eukaryotes. Many enhancer sequences are now known from mammalian genes (eg, globin, elastase, albumin, afetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 late origin enhancer (100-270), cytomegalovirus early promoter enhancer, late polyoma enhancer, and adenoviral enhancers. Also see Yaniv, 1982, Nature 297:17-18 for a description of enhancer elements for activation of eukaryotic promoters. The enhancer can be spliced into the vector at a position 5' or 3' to the anti-ANGPT2 antibody coding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells from other multicellular organisms) may also contain sequences necessary for transcription termination and for mRNA stabilization. Such sequences are publicly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-ANGPT2 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation site. See WO94/11026 and the expression vector described therein. In some embodiments, antibodies to ANGPT2 can be expressed using the CHEF system. (See, for example, US Pat. No. 5,888,809; the contents of which are incorporated herein by reference.)

Suitable host cells for cloning or expressing DNA in the vectors of the present invention are prokaryotic, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis (for example, B. licheniformis 41 P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas, such as P. aeruginosa , and Streptomyces. One of the preferred hosts for cloning E. coli is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. . These examples are illustrative and not restrictive.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for cloning or expression of anti-ANGPT2 antibody-encoding vectors. Saccharomyces cerevisiae, or the common baker's yeast, is the most commonly used lower eukaryotic microorganism host. However, various other genus species and strains are publicly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906 ), K. thermotolerans, and K. marxianus; yarnrnowia (EP 402.226); Pichia pastors (EP 183,070); Candida; Trnichodernma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for expressing glycosylated anti-ANGPT2 antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells, including, for example, various baculovirus strains and variants and corresponding insect-permitted host cells from organisms such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various viral strains are publicly available for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potatoes, soybeans, petunia, tomato and tobacco can also be used as hosts.

Antibodies to ANGPT2 or antigen binding fragments thereof according to the invention can also be incorporated into viral vectors, that is, a polynucleotide encoding an antibody to ANGPT2 or an antigen binding fragment thereof is introduced into a viral vector and then expressed in the patient after infection with the virus.

In another aspect, anti-ANGPT2 antibodies are expressed in vertebrate cells. Propagating vertebrate cells in culture (tissue culture) has become a common procedure and techniques are widely available. Examples of suitable mammalian host cell lines include the monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), newborn hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g. DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), Buffalo rat kidney cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 44-68) , MRC 5 cells, FS4 cells, and a human hepatoma line (Hep G2).

Host cells are transformed using the expression or cloning vectors described above to produce anti-ANGPT2 antibodies and cultured in a suitable growth medium modified, if appropriate, to induce promoters, select transformants, or amplify genes encoding the desired sequences. The host cells used to produce the anti-ANGPT2 antibody described herein can be cultured once

- 29 047046 personal nutrient media. Commercially available media such as Ham F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium (MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's modified Eagle's medium ((DMEM), Sigma-Aldrich Co.) are suitable for culturing host cells. Additionally, any of the media described in one or more of: Ham et al., 1979, Meth. 58: 44, Barnes et al., 1980, Anal. Biochem. 102: 255, US Patent No. 4,767,704, US Patent No. 4,657,866, US Patent No. 4,927,762, US Patent No. 4,560,655, US Patent No. 5,122,469, WO 90/103430, and WO 87/00195, can be used as a culture medium for host cells. Any of these media can be supplemented, if necessary, with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range) , and glucose or an equivalent energy source. Other additives may also be included in suitable concentrations as will be apparent to one skilled in the art. Culture conditions, such as temperature, pH, and others, are conditions that have previously been used for the host cell selected for expression and will be understood by one skilled in the art.

Using recombinant techniques, the antibody can be produced intracellularly, into the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, the cells can be disrupted to release the protein as a first step. Particulate waste, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 10:163-167 describes a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell mass is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cellular debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis and antibiotics may be included to prevent the growth of incidental contaminants. Various methods can be used to isolate an antibody from a host cell.

The antibody composition prepared from cells can be purified, for example, by hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, where a typical purification technique is affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (see, eg, Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all murine isotypes and for human gamma3 (see, eg, Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene offer the possibility of faster flow rates and shorter processing times, which can be achieved with agarose. If the antibody includes a C _H3 domain, then Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, NJ) is suitable for purification. Other technologies for protein purification are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin chromatography SEPHAROSE™ chromatography on anion or cation exchange resin (such as a polyaspartic acid), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody being reduced. After any preparatory purification step(s), the mixture containing the antibody of interest and contaminants can be subjected to hydrophobic interaction chromatography at low pH using an elution buffer at a pH in the range of 2.5-4.5, typically carried out at low salt concentrations (eg, from about 0-0.25 M salt).

The invention also relates to nucleic acids that hybridize under relaxed, mild or stringent conditions, as defined herein, with all or part (for example, a part that encodes a variable region) of a nucleotide sequence that is an isolated polynucleotide(s). ) sequence(s) encoding an anti-ANGPT2 antibody or antibody fragment. The hybridizing region of the hybridizing nucleic acid typically consists of at least 15 (eg, 20, 25, 30, or 50) nucleotides. The hybridizing region of the hybridizing nucleic acid is at least 80% identical, for example, at least 90%, at least 95%, or at least 98% identical to the sequence of the target

- 30 047046 the sequence or entire nucleic acid encoding a polypeptide to ANGPT2 (for example, a heavy chain or light chain variable region), or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, for example a PCR primer, or a diagnostic probe.

In one embodiment, the present invention provides an isolated polynucleotide or polynucleotides comprising:

a heavy chain coding sequence as set forth in SEQ ID NO: 31 or a heavy chain variable region as set forth in SEQ ID NO: 3; and a light chain coding sequence as set forth in SEQ ID NO. 32 or a light chain variable region as set forth in SEQ ID NO: 8, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 33 or a heavy chain variable region as set forth presented in SEQ ID NO: 4; and a light chain coding sequence as set forth in SEQ ID NO. 34 or a light chain variable region as set forth in SEQ ID NO: 9, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 35 or a heavy chain variable region as set forth presented in SEQ ID NO: 5; and a light chain coding sequence as set forth in SEQ ID NO. 36 or a light chain variable region as set forth in SEQ ID NO: 10, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 37 or a heavy chain variable region as set forth presented in SEQ ID NO: 6; and a light chain coding sequence as set forth in SEQ ID NO. 38 or a light chain variable region as set forth in SEQ ID NO: 11, or an isolated polynucleotide or polynucleotides containing a heavy chain coding sequence as set forth in SEQ ID NO: 39 or a heavy chain variable region as set forth presented in SEQ ID NO: 7; and a light chain coding sequence as set forth in SEQ ID NO. 40 or a light chain variable region as set forth in SEQ ID NO: 12.

It is intended that in said anti-ANGPT2 antibodies and antibody fragments, the nucleic acid sequence encoding the CDRs remains unchanged (unchanged with respect to the amino acid they encode, possible DNA sequence equivalents due to the degeneracy of the genetic code), but the surrounding regions, e.g., FR regions, can be designed.

Products.

In another aspect, an article containing substances useful for treating the disorders described above is covered. The product includes a container and a label. Acceptable containers include, for example, bottles, vials, syringes and laboratory tubes. Containers can be made from various materials such as glass or plastic. The container contains a composition that is effective for treating a particular condition and may have a sterile access opening. For example, the container may be a bag or vial for an intravenous solution having a closure device permeable to a hypodermic injection needle. The active substance in the composition is an antibody to ANGPT2 or its antigen-binding fragment. The label on or associated with the container states that the composition is used to treat the selected condition. The article may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may additionally include other substances whose presence is desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

The invention is described below using the examples provided, which are not intended to limit the scope of the invention.

Examples

The antibodies ANGPT2-opt-1, ANGPT2-opt-2, ANGPT2-opt-13, ANGPT2-opt-19, and ANGPT2-opt-31 were characterized along with the reference antibodies nesvacumab analogue, MEDI3617 analogue, and LC06. These reference antibodies are generated using standard procedures based on published sequences, as described below.

- 31 047046

Table 4

Comparative antibodies to ANGPT2

Comparison antibody Published sequence Nesvacumab analogue (Regeneron) Heavy chain: SEQ ID NO. 53 Light chain: SEQ ID NO. 54 See USAN drug name file number ZZ-34 (2012) for nesvacumab. For the nesvacumab analog described herein, the lysine residue at position 219 in the nesvacumab heavy chain was replaced with arginine (K219R). MEDI3617 analog (Medimmune) Heavy Chain: SEQ ID NO. 55 Light chain: SEQ ID NO. 56 The structure of the MEDI3617 analogue used in this application is based on the description in US patent No. 8,507,656. LC06 (Roche) Heavy chain: SEQ ID NO. 57 Light chain: SEQ ID NO. 58 The structure of LC06 used in this application is based on the description in US patent No. 9,340,609.

Data for these antibodies are described below.

Example 1. Creation of antibodies (immunization).

Wild-type CD1 mice were immunized with recombinant human and mouse ANGPT2 DNA and human and mouse ANGPT2 protein. Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, Titermax, or Gerbu were used as adjuvants at various time points to enhance the humoral immune response. Serology was then assessed using ELISA. Selected serologically positive mice received a terminal boost before B cell isolation.

The selected mice all exhibited positive serum antibody titers except. After completion of the immunization regimen, splenocytes were collected to recover antigen-specific B cells.

Example 2. Preparation of humanized antibodies.

The chimeric leader CL-209881_VL was selected for further optimization. The chimeric leader had a variable light chain corresponding to SEQ ID NO: 2 and a variable heavy chain corresponding to SEQ ID NO: 1. Thirty-three sequences (leader) were selected and optimized. Subsequent studies were carried out to select six antibodies for further production on a larger scale.

Example 3. Susceptibility sequences in CDRs.

CDR sequences were checked for the presence of any potential exposures such as N-glycosylation sites, strong deamidation motifs (NG, NS, NH, NA, ND, NT, NN), aspartate isomerization motifs (DG), defragmentation motifs (DG, DS), cysteine. These amino acids or motifs may undergo a chemical reaction and impart undesirable heterogeneity to the product, also with the potential to negatively impact target binding and function. For these reasons, it is preferable to remove such amino acids or motifs (if necessary) from the CDR.

Example 4: Immunogenicity.

Sequence immunogenicity was assessed virtually using an algorithm licensed from EpiVax, Inc., Providence, Rhode Island. The EpiMatrix Treg-adj score takes into account T cell epitopes and regulatory T cell epitopes. The lower the immunogenicity score, the less likely the sequence is to be immunogenic. In general, a negative score is considered to indicate a low risk of immunogenicity, while a positive score is considered to indicate potential immunogenicity.

Example 5: Epitope information.

Materials:

Water (Sigma Aldrich, part number 37877-4L).

Acetonitrile (Sigma Aldrich, part number 34998-4L).

Formic acid (Fluka, catalog number 94318).

Urea (Sigma Aldrich, part number 51456-500G).

TSER-HC1-1O g (Thermo Scientific-Pierce, catalog number 20491).

Dibasic sodium phosphate (Sigma Aldrich, part number S7907-100G).

Monobasic sodium phosphate (Sigma Aldrich, part number S8282-500G).

Pre-column ACQUITY UPLC VEN C18 VanGuard, 130A, 1.7 µm, 2.1 mm X 5 MM (Waters Technologies Corp, 186003975).

Poroszyme® immobilized pepsin cartridge, 2.1 mm x 30 mm (Life Technologies Corp, 2313100).

Column Acquity UPLC VEN C18 1.7 µm, 1 mmx50 mm (Waters, 186002344).

Solvent A: 0.1% Formic acid/99% water/1% acetonitrile.

Solvent B: 0.1% Formic acid/5% water/95% acetonitrile.

Aqueous buffer: H ₂ O 10 mM sodium phosphate pH 7.4.

Deuterium buffer: D2O 10 mM sodium phosphate pH 7.4.

- 32 047046

Quenching buffer: Water 8 M Urea, 0.4 M TCEP-HCl.

In control samples for epitope mapping, antigen was run with and without antibody. To determine the list of antigenic peptides, a first run was first performed in this puncture using aqueous buffer instead of deuterium buffer. 4 μl of sample was mixed with 40 μl of deuterium buffer. This mixture was incubated at 20°C for several time points (1, 2 and 4 minutes). After this, 40 μl of the mixture was transferred to 40 μl of 4°C quenching buffer (4 M Urea, 0.4 M Tser-HCl) and mixed. 60 μl of quenching protein was injected, where it was digested on a pepsin column for 2 minutes by washing with 200 μl/ml solvent A: 0.1% Formic acid/99% water/1% acetonitrile. Subsequent peptides were desalted on a Vanguard precolumn for 3 minutes. Pepsin-digested peptides were sent to a reverse-phase VEN C18 column inside a temperature-controlled compartment column/valve. A gradient solvent system was used consisting of: solvent A: 0.1% Formic acid/99% water/1% acetonitrile and solvent B: 0.1% Formic acid/5% water/95% acetonitrile. The percentage of solvent B was increased from 10% to 15% after 5.1 minutes, to 50% after 11 minutes, to 90% after 11.5 minutes, holding until 12.5 minutes, to 0% B after 13 minutes, holding until 14 minutes. Chromatographic separation occurred at 4°C with a flow rate of 180 μL/min. After chromatographic separation, the sample was fed to a Thermo Scientific Orbitrap Fusion mass spectrometer operating in positive electrospray ionization mode. The method used included CID and ETD activation types in identifying reference peptides using a separation of 120k, a minimum signal of 10k, a separation width of 1.0, and a normalized collision energy of 35.0V. The RF level of the S-lenses was set to 60%. For control peptides, the data acquisition type was profiles for full MS scans and centroid for CID MS/MS data. For deuterated samples, no MS/MS was collected. Data were collected over the mass range of 280–1800 Da. For analysis of raw LC-MS/MS fragmentation data, control samples (with CID and ETD MS/MS) were analyzed using Proteome Discover 1.4 (Thermo Scientific) and PMi Byonic (Protein Metrics) against a given sequence to generate a peptide list and time holding. The raw data files were pre-processed and converted to ASCII format using proprietary SHARC software. The identified peptides were then noted and the data summarized using our own developed SHAFT software. Epitopes were determined based on differences in the average mass shift induced by binding after deuterium labeling. At the peptide level, protection greater than 0.4 Da was considered significant.

Epitope mapping results are presented for an exemplary antibody of the invention (ANGPT2-opt-13) (FIG. 2), a nesvacumab analog, a MED3617 analog, and LC06. Nesvacumab does not appear to cross-react with ANGPT1, while the MED3617 and LC06 analogues do not appear to cross-react with ANGPT1. The specific binding sites of antibodies to the FLD domain of human ANGPT2 with the sequence SEQ ID NO: 50 (Fig. 2) are highlighted in dark gray. The data presented indicate that the reference antibodies (nesvacumab analogue, MED3617, and LC06) bind to epitopes that are distinct and distinct from the ANGPT2-opt-13 binding epitope. Example 6 CDC Assay The Fc region of the IgG heavy chain confers effector functions to the antibody through interaction with Clq and, therefore, may have the ability to induce complement-dependent cytotoxicity (CDC). This effect can be examined in vitro by exposing target cells to complement in the presence of an antibody that specifically binds to the target cell. The CDC assay can be used to evaluate the activity of monoclonal antibodies to mediate CDC. (See Mapping of the Clq binding site on Rituxan, a chimeric antibody with a human IgG1 Fc, Idusogie, Esohe E., et al., Journal of Immunology (2000), 164 (8), 4178-4184; and Utilization of Complement -Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus, Konishi, Eiji et al., Clin Vaccine Immunol (2008) Jan 15(1): 88-94.)

CHO cells expressing membrane-bound human ANGPT2 (CHO-GPI-ANGPT2) were cultured in RPMI-1640 supplemented with HI FBS, 1x glutamax, and 1000 μg/ml Geneticin (used as target cells). CDC activity was determined by measuring the release of LDH from target cells using the Cytotoxicity Detection Kit Plus from Roche®. Samples were prepared in triplicate in 96-well round-bottom plates. Samples consisted of 50 μl antibody, 50 μl target cells (50,000/well), and 100 μl human complement (Cedarlane®), at a final dilution of 1:12 in cytotoxicity assay media (Cedarlane®). The background control was 200 µl of cytotoxicity assay media (Cedarlane®) only. The maximum release control (Tmax) was 50 μL of target cells and 150 μL of cytotoxicity assay media. The target cell control (Tspon) consisted of 50 μl of target cells and 150 μl of cytotoxicity assay media. The plate was incubated at 37°C in a humidified CO ₂ incubator for 3 hours. Thirty minutes before the end of incubation (after incubation for 2.5 hours), 10 μl of lysis solution (provided in the Cytotoxicity Detection Kit-Roche®) was added to the wells under maximum release control (Tmax). After completion of incubation, 100 μl of supernatants were transferred accordingly

- 33 047046 flat-bottomed 96-well plate for LDH detection. 100 μl of the reaction mixture (provided in the Cytotoxicity Detection Kit) was added to each well and the plate was incubated at room temperature for 15 minutes in the dark. After completion of this second incubation, the reaction was stopped by adding 50 μl of stop solution (provided in the Cytotoxicity Detection Kit). Absorbance was measured at 490 nm with 650 nm as reference on a Biotek® plate reader (Biotek, Synergy). CDC% was calculated using the equation:

%CDC=((Ab induced release)-[(T)_spon])/((T_max)-(T_spon))*100.

The CDC study described above was used to determine the cytotoxicities caused by anti-ANG antibodies: ANGPT2-opt-13-IgG, nesvacumab analogue, MEDI3617 analogue, and LC06. The results of studies in duplicate are presented in Fig. 1A and 1B. The results indicate that the exemplary anti-ANGPT2 antibody of the invention (ANGPT2-opt-13-IgG) is less toxic than each of the nesvacumab analog, MEDI3617 analog, and LC06.

Example 7. ANGPT2 Blocking analysis.

Human ANGPT2 dimer (CC-FLD) was preincubated with test antibodies. The ANGPT2/Antibody mixture was then incubated with HEK293 human Tie2 cells at 4°C. After washing, cells were stained with anti-His-AF647 (from GenScript) to detect the His tag on the ANGPT2 protein. Binding of secondarily labeled ANGPT2 to human Tie2 cells was detected by flow cytometry. The averaged values from two replicates for each concentration point were used to generate curve fitting plots. Antibodies that protect ANGPT2 from binding to Tie2 cells were considered blocking antibodies. The results are presented in Fig. 3A-3G and EC50 values are presented in table. 5 based on the average of two replicates.

Table 5

ANGPT2 blocking assay

Antibody to ANGPT2 EC50, nM Ang2-opt-l 13.22 Ang2-opt-2 14.81 Ang2-opt-13 12.98 Ang2-opt-19 15.08 Ang2-opt-31 13.03 Analogue Nevescumab 3.77

The results of the ANGPT2 blocking assay indicate that the antibodies of the invention block ANGPT2 interaction with the Tie2 receptor on the cell surface.

Example 8 Functional analysis of Tie2 phosphorylation.

Functional analysis of Tie2 phosphorylation was performed as described below.

Unless specifically stated otherwise, the following reagents or materials were used:

HEK293/huTie2 cells;

0.25% trypsin/EDTA (Gibco part no. 25200-056);

96-well black clear bottom plate coated with poly^-lysine (BioCoat catalog no. 35460); PathScan Sandwich ELISA lysis buffer (Cell Signaling, cat. no. 7018);

HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Cat. No. 78443, Lot No. QK226996, 100x stock);

Activated sodium orthovanadate, 200 mM stock solution (Five photon, catalog no. ActVO-4, lot no. 26716-4);

Clear polystyrene 96-well high-binding microplates (R&D Systems part no. DY990, lot no. 316940);

ELISA blocking buffer: Phosphate buffered saline (PBS) wt/2% BSA, diluted from 10% stock solution (R&D Systems, catalog no. DY995);

ELISA Assay Diluent: PBS w/1% BSA diluted from 10% stock solution (R&D Systems, Cat. No. DY995);

ELISA Wash Buffer Concentrate: 25X Stock (R&D Systems, Cat. No. WA126);

Packaged ELISA substrate reagent (R&D Systems, cat. DY999);

ELISA stop solution (R&D Systems, cat. DY994); and rh Angiopoietin-2 (R&D systems cat. 623-AN/CF; lot no. SUL61 at 169 µg/ml stock solution).

Cell growth medium:

Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/L Glucose with L-Glutamine (500 ml) (Gibco Part No. 11995-065);

Fetal bovine serum (FBS) (50 ml) (Hyclone cat. SH30071.03, batch no. AC10219630);

100 mM nonessential amino acid (NEAA) solution (5 ml) (Gibco: Cat. No. 11140-050);

100 mM sodium pyruvate (5 ml) (Gibco part no. 11360-070);

PenStrep (5 ml) (Gibco part no. 15140-122); And

- 34 047046

1M No. 2-hydroxyethylpiperazine-I-2-ethane sulfonic acid (Hepes) (6.25 ml) (Gibco catalog no. 15630-080); Geneticin (10 ml) (Gibco part no. 10131-035).

Depleted environment:

DMEM with 4.5 g/l Glucose with L-Glutamine;

FBS (25 ml) 5%; NEAA (5 ml);

Sodium pyruvate (5 ml); PenStrep (2.5 ml);

Hepes (6.25 ml); And

Geneticin (10 ml) Dulbecco's Phosphate Buffer Saline (dPBS), Calcium and Magnesium Free (Gibco Part # 14190).

Growing cells.

HEK293/huTie2 cells were washed with PBS, detached with 0.25% Trypsin, and counted using a countess cell counting device (Invitrogen). 5 x 104 cells were seeded per well in a 96-well clear-bottom tissue culture plate with Ploy D-Lysine in 100 µl Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, NEAA, Sodium Pyruvate, PenStrep, Geneticin and HEPES. Cells were incubated overnight at 37°C, 5% CO ₂ incubator. After approximately 18 hours, the cell growth media was replaced with 100 μl of depleted medium (Dulbecco's modified Eagle's medium (DMEM) with 5% FBS, NEAA, Sodium Pyruvate, PenStrep, Geneticin and HEPES, returned the plate to the incubator, and incubated for nights.

ELISA plate coating.

The captured antibody (anti Tie2 (AB33) from Cell signaling) was diluted to 1 μg/ml working solution in coating buffer (eBioscience). The working solution was immediately added to a high-binding (R&D) 96-well polystyrene microplate to obtain 100 μL per well. The wells were sealed and incubated overnight at 4°C. The plate was washed 3 times with 300 μl per well using 1XELISA wash buffer and then blocked with 200 μl blocking buffer for 2 hours at room temperature while shaking.

Cell processing.

Ang2 stock solution (rh Angiopoietin-2 from R&D systems catalog no. 623-AN/CF; lot no. SUL61) was diluted to 6 μg/ml in depleted medium. (Depletion medium was also used as an antibody diluent.) Separately, anti-ANGPT2 antibody solutions were prepared by diluting to 66 nM followed by a 1:3 serial dilution to 0.27 nM. Antibodies to ANGPT2 with rhAng2 were incubated at room temperature for 30 minutes. Cell culture medium (50 μl) was removed from each well in the cell plate. Cells were treated with 50 μl aliquots of anti-ANGPT2 antibody pre-incubation solution for 20 minutes at 37°C, 5% CO ₂ incubator. The supernatant was discarded and the cells were washed once with cold PBS (containing 1 mM activated sodium orthovanadate). The wash buffer was discarded and PathScan lysis buffer (cell signaling) with 1 mM activated sodium orthovanadate, protease and phosphatase inhibitors was added to each cell. The plate was shaken at high speed for 1-3 hours at 4°C.

ELISA.

The blocking buffer was removed from the ELISA plate, cell lysate was added to each well, and the plate was incubated overnight at 4°C with shaking. The plate was washed four times with ELISA wash buffer, and each well was treated with detection antibody: Biotin Conjugate-4G10 platinum anti-Phospotyrosine (from Millipore) at a 1:300 dilution in ELISA assay diluent. The plate was incubated for 2 hours on a shaker at room temperature and then washed four times using ELISA wash buffer. Streptavidin-HRP conjugate (from Millipore) diluted 1:300 in ELISA assay diluent was added to each well and incubated on a shaker for 1 hour at room temperature. The plate was washed four times with ELISA wash buffer. After the washing step, the substrate solution from the ELISA Substrate Reagent Pack (R&D Systems) was added to each well at room temperature for 5-10 minutes. Stop solution was added followed by gentle tapping for 5 minutes to ensure complete mixing. The plate was then analyzed on a plate reader with absorbance at 450 nm corrected from 650 nm.

Table 6

Functional analysis of Tie2 phosphorylation

Antibody to ANGPT2 EC50, nM EC50, nM Ang2-opt-l 3.0 3.2 Ang2-opt-2 1.8 1.9 Ang2-opt-13 4.2 4.0 Ang2-opt-19 5.0 4.6 Ang2-opt-31 4.4 3.8 Ang2-opt-34 5.8 7.4 Analogue Nevescumab 4.4 2.2 Analogue MEDI3617 - 2.7 LC06 1.3 -

- 35 047046

The results of the Tie2 functional assay suggest that anti-ANGPT2 antibodies inhibit LCVPT2-induced Tie2 phosphorylation and may block Tie2-mediated phosphorylation and downstream signaling.

Example 9: Binding affinity.

Binding affinities for various ANGPT2 analytes were determined by surface plasmon resonance (SPR) using ProteOn XPR36 (Bio-Rad). Unless specifically stated otherwise, all reagents were obtained from Bio-Rad. The running buffer for all assays and dilutions (except where specifically noted) was phosphate buffered saline (PBS)/ethylenediaminetetraacetic acid (EDTA) with 0.01% Tween20 (PBS-T-EDTA). PBS-T-EDTA was prepared by adding 100 μL of 100% Tween20 to 2 L of PBS-T-EDTA with an initial concentration of 0.005% Tween20 to obtain a final Tween20 concentration of 0.01%. The general linearized model (GLM) sensor chip was normalized, pretreated, and activated with equilibrated 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDCyN-hydroxysulfosuccinimide (s-NHS)) in the horizontal direction for 300 seconds at flow rate 30 μl/min. This was followed by horizontal immobilization with recombinant protein A/G (Thermo Scientific) (60 μg/ml in 10 mM acetate pH 4.5) for 300 seconds at a flow rate of 30 μl/min, which resulted in to approximately 4370-4875 response units (RU) Protein A/G on the surface The sensor chip was then deactivated using 1 M ethanolamine HCl in a horizontal direction for 300 seconds at a flow rate of 30 μl/min. The sensor chip was stabilized for 18 s 0. .85% phosphoric acid at a flow rate of 100 µl/min 3 times horizontally and 3 times vertically.

Each ANGPT2 antibody was captured onto the surface of Protein A/G vertically for 300 s at a flow rate of 30 μL/min, resulting in a capture level of approximately 2500 RU. Initial conditions were stabilized by injecting PBS-T-EDTA for 60 s at a flow rate of 100 μL/min horizontally with a dissociation time of 120 s. The analyte (e.g., HuANGPT2) was injected horizontally over the captured antibody for 300 s at a flow rate of 30 μL/min and dissociated for 1800 s. The analyte concentrations were 0 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. The surface was regenerated by injecting 0.85% phosphoric acid for 18 s at a flow rate of 100 μL/min once horizontally and once vertically. PBS-T-EDTA was injected over 60 s at a flow rate of 100 μL/min once vertically and once horizontally. Crosstalk (interactions with the sensor surface) and blank data (PBS-T-EDTA with 0.01% Tween20 or 0 nM analyte (in this case HuANGPT2)) were subtracted from the raw data. The sensorgrams were then fitted globally to 1:1 Langmuir binding, yielding association (k _a ), dissociation (k d ), and affinity (K _D ) values. The above procedure was used to measure binding affinity for the following analytes: human ANGPT1, human ANGPT2, cyno ANGPT2, rabbit ANGPT2, and rat ANGPT2. For the anti-ANGPT2 antibodies of the invention and the analogue nesvacumab, no binding to human ANGPT1 was observed (K _D > 100 nM). The results of binding to human ANGPT2 and cyno ANGPT2 are presented in table. 7.

Table 7

Affinity binding properties of anti-ANGPT2 antibodies in human and cyno macaque models

Antibody k _a (1/Ms) k _d (1/s) K _D (nM) k _a (1/Ms) k _d (1/s) kD (nM) human ANGPT2 cyno ANGPT2 CL 209881 1.51E+05 2.34E-05 0.155 1.64E+05 3.93E-05 0.240 ANGPT2-opt-l 1.44E+05 4.25E-05 0.294 1.52E+05 5.48E-05 0.360 ANGPT2-opt-2 1.25E+05 8D1E-05 0.649 1.35E+05 8.45E-05 0.626 ANGPT2-opt-13 1.32E+05 1.89E-05 0.144 1.42E+05 2.08E-05 0.146 ANGPT2-opt-19 8D8E+04 5.83E-05 0.712 8.77E+04 7.40E-05 0.844 ANGPT2-opt-31 1.25E+05 1D4E-05 0.091 1.35E+05 1.96E-05 0.145 analogue Nesvacumab 1.48E+05 1.93E-05 0.130 1.51E+05 1.93E-05 0.128 analogue MEDI3617 6D4E+05 1.66E-02 27 nd nd nd LC06 1.05E+05 1.25E-02 12 nd nd nd

ND = not available/not measured.

For studies performed in the rabbit ANGPT2 model, ANGPT2-opt-13 and the nesvacumab analog exhibit K _D values of 0.133 nM and 0.137 nM, respectively.

For studies performed in a rat model, the anti-ANGPT2 antibodies of the invention exhibit only weak binding (K _D >118 nM), while the nesvacumab analog exhibits a K _D value of 0.159 nM.

The results obtained indicate that the anti-ANGPT2 antibodies of the invention have high affinity for human ANGPT2 and no measurable affinity for human ANGPT1.

This disclosure covers, in particular, the following:

Embodiment 1: Anti-ANGPT2 antibody or antigen-binding fragment containing:

variable region of the heavy chain containing the amino acid

- 36 047046 sequence SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3), and a light chain variable region containing the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 14 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 21 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 25 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3).

Embodiment 2: Anti-ANGPT2 antibody of embodiment 1, wherein said antibody comprises a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO. 3 and SEQ ID NO. 8, respectively; SEQ ID NO. 4 and SEQ ID NO. 9, respectively; SEQ ID NO. 5 and SEQ ID NO. 10, respectively; SEQ ID NO. 6 and SEQ ID NO. 11, respectively; or SEQ ID NO. 7 and SEQ ID NO. 12, respectively.

Embodiment 3: Anti-ANGPT2 antibody of embodiment 2, wherein said antibody comprises a variable heavy chain and a variable light chain having at least 90%, at least 95%, at least 98%, or at least 99% identity to amino acid sequences SEQ ID NO. 3 and SEQ ID NO. 8, respectively; SEQ ID NO. 4 and SEQ ID NO. 9, respectively; SEQ ID NO. 5 and SEQ ID NO. 10, respectively; SEQ ID NO. 6 and SEQ ID NO. 11, respectively; or SEQ ID NO. 7 and SEQ ID NO. 12, respectively.

Embodiment 4: Anti-ANGPT2 antibody of embodiment 1, wherein the antibody comprises a heavy chain and a light chain comprising SEQ ID NO. 31 and SEQ ID NO. 32, respectively; SEQ ID NO. 33 and SEQ ID NO. 34, respectively; SEQ ID NO. 35 and SEQ ID NO. 36; respectively; SEQ ID NO. 37 and SEQ ID NO. 38; respectively; or SEQ ID NO. 39 and SEQ ID NO. 40.

Embodiment 5: Anti-ANGPT2 antibody of embodiment 4, wherein said antibody comprises a heavy chain and a light chain having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity SEQ ID NO. 31 and SEQ ID NO. 32, respectively; SEQ ID NO. 33 and SEQ ID NO. 34, respectively; SEQ

- 37 047046

ID NO. 35 and SEQ ID NO. 36; respectively; SEQ ID NO. 37 and SEQ ID NO. 38; respectively; or SEQ ID NO. 39 and SEQ ID NO. 40.

Embodiment 6: Anti-ANGTP2 antibody or antigen-binding fragment thereof according to one of Embodiments 1-5, wherein the anti-ANGTP2 antibody or antigen-binding fragment thereof that binds to at least one amino acid residue of SEQ ID NO: 51, or to SEQ ID NO: 51.

Embodiment 7: An anti-ANGTP2 antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid regions of the human ANGPT2 FLD domain of the sequence SEQ ID NO: 51, or to SEQ ID NO: 51.

Embodiment 8: An anti-ANGTP2 antibody or an antigen-binding fragment thereof that competes for binding of SEQ ID NO: 51 with an anti-ANGTP2 antibody according to one of embodiments 1-7.

Embodiment 9: An isolated polynucleotide having a sequence that encodes an antibody as defined in one of embodiments 1-8.

Embodiment 10: A vector containing a polynucleotide according to Embodiment 9.

Embodiment 11: A host cell transformed or transfected with a polynucleotide according to embodiment 9 or a vector according to embodiment 10.

Embodiment 12: A method for producing an antibody according to one of embodiments 1-8, comprising (a) culturing a host cell according to embodiment 11 under conditions allowing expression of the antibody according to one of embodiments 1-8 and (b) recovery of the produced antibody from the culture.

Embodiment 13: A pharmaceutical composition comprising an anti-ANGPT2 antibody or antigen binding fragment according to one of embodiments 1-8, and a pharmaceutically acceptable carrier.

Embodiment 14: The pharmaceutical composition according to embodiment 13, wherein the composition further comprises a second selected therapeutic agent.

Embodiment 15: The pharmaceutical composition according to embodiment 13 or 14, wherein the composition is administered via the parenteral route, intravenous route or subcutaneous route.

Embodiment 16: An anti-ANGPT2 antibody or antigen binding fragment according to one of embodiments 1-8 for use as a drug.

Embodiment 17: A method of treating diseases or disorders that may be ameliorated by treatment with an anti-ANGPT2 antibody, wherein the method comprises administering to a patient in need thereof a pharmaceutically effective amount of an anti-ANGPT2 antibody or antigen binding fragment in accordance with one of Embodiments 1-8 .

Embodiment 18: An anti-ANGPT2 antibody or antigen-binding fragment according to one of embodiments 1-8 for use in the treatment of diseases or disorders that may be ameliorated by treatment with an anti-ANGPT2 antibody.

Embodiment 19: Use of an anti-ANGPT2 antibody or an antigen-binding fragment according to one of claims 1 to 8 for the preparation of a medicament for the treatment of diseases or disorders that can be ameliorated by treatment with an anti-ANGPT2 antibody.

Embodiment 20: The method of Embodiment 17, the anti-ANGPT2 antibody or antigen-binding fragment of Embodiment 18, or the use of an anti-ANGPT2 antibody or antigen-binding fragment of Embodiment 19, wherein the disease or disorder is selected from the group consisting of cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury injury, stroke hypertension, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, trauma-induced brain disorders, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy- and preeclampsia-induced hypertension, sepsis, severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal segmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic kidney failure, end-stage renal disease failure, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF), emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), increased vascular permeability (and associated disorders), acute kidney injury, renal cell carcinoma, heart failure, lupus nephritis, Raynaud's syndrome, pancreatitis, peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma,

-

Claims (23)

retinopathy, diabetic nephropathy, portal hypertension, varicose veins and liver transplantation. Embodiment 21: The method of Embodiment 20, an anti-ANGPT2 antibody or an antigen-binding fragment of Embodiment 20, or the use of an anti-ANGPT2 antibody or an antigen-binding fragment of Embodiment 20, wherein the disease or disorder is selected from the group consisting of chronic kidney disease, non-alcoholic steatohepatitis (NASH), and sepsis. Embodiment 22: The method of embodiment 17 or 20, which further includes administering the selected second therapeutic agent. Embodiment 23: The method of embodiment 17 or 20, wherein said antibody is administered via the parenteral route, intravenous route or subcutaneous route. Embodiment 24: A method of blocking human ANGPT2 function in a human patient, which comprises administering to said human patient a composition comprising an anti-ANGPT2 antibody or antigen-binding fragment according to one of Embodiments 1-8 in an amount sufficient to block the ANGPT2-mediated response in the specified human patient. Embodiment 25: An anti-ANGPT2 antibody or antigen-binding fragment according to one of embodiments 1-8 for use in blocking the function of human ANGPT2. Embodiment 26: Use of an anti-ANGPT2 antibody or antigen-binding fragment according to one of Embodiments 1-8 to prepare a medicament for blocking the function of human ANGPT2. Embodiment 27: An isolated polynucleotide encoding an amino acid sequence of a heavy chain variable region or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region comprises any of SEQ ID NO: 3-7, SEQ ID NO: 13-17; SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39; and wherein the amino acid sequence of the light chain variable region comprises any of SEQ ID NO: 812, SEQ ID NO: 19-26, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40. CLAIM 1. An antibody to ANGPT2 or an antigen-binding fragment thereof, containing: a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3), and a light chain variable region containing the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 14 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 17 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 19 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region containing the amino acid - 39 047046 sequence SEQ ID NO. 21 (L-CDR1); amino acid sequence SEQ ID NO. 23 (L-CDR2); and amino acid sequence SEQ ID NO. 25 (L-CDR3), or a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 13 (H-CDR1); amino acid sequence SEQ ID NO. 15 (H-CDR2); and amino acid sequence SEQ ID NO. 16 (H-CDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 20 (L-CDR1); amino acid sequence SEQ ID NO. 22 (L-CDR2); and amino acid sequence SEQ ID NO. 24 (L-CDR3). 2. An anti-ANGPT2 antibody or an antigen-binding fragment thereof according to claim 1, wherein said antibody comprises a variable heavy chain and a variable light chain comprising amino acid sequences selected from the group consisting of SEQ ID NO. 3 and SEQ ID NO. 8, respectively; SEQ ID NO. 4 and SEQ ID NO. 9, respectively; SEQ ID NO. 5 and SEQ ID NO. 10, respectively; SEQ ID NO. 6 and SEQ ID NO. 11, respectively; or SEQ ID NO. 7 and SEQ ID NO. 12, respectively. 3. The anti-ANGPT2 antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody comprises a heavy chain and a light chain containing amino acid sequences selected from the group consisting of SEQ ID NO. 31 and SEQ ID NO. 32, respectively; SEQ ID NO. 33 and SEQ ID NO. 34, respectively; SEQ ID NO. 35 and SEQ ID NO. 36; respectively; SEQ ID NO. 37 and SEQ ID NO. 38; respectively; or SEQ ID NO. 39 and SEQ ID NO. 40. 4. A pharmaceutical composition containing an anti-ANGPT2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier. 5. A method of treating diseases or disorders characterized by cells expressing ANGPT2, wherein the method comprises administering to a patient in need thereof a pharmaceutically effective amount of an anti-ANGPT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. 6. The method of claim 5, wherein the disease or disorder is selected from the group consisting of cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury, hypertensive stroke, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, trauma-induced brain disorders, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy-induced hypertension and preeclampsia, sepsis, severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal segmental glomerulosclerosis (FSGS) , nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic renal failure, end-stage renal disease, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF), emphysema, acute injury lung disease (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), hypervascular permeability (and associated disorders), acute kidney injury, renal cell carcinoma, heart failure, lupus nephritis , Raynaud's syndrome, pancreatitis, peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma, retinopathy, diabetic nephropathy, portal hypertension, varicose veins and liver transplantation. 7. The method of claim 6, wherein the disease or disorder is selected from the group consisting of chronic kidney disease, nonalcoholic steatohepatitis (NASH), diabetic nephropathy, sepsis, and hypervascular permeability. 8. The method of claim 6, wherein the disease or disorder is selected from the group consisting of ALI, ARDS, SARS, MERS, and hypervascular permeability and related disorders. 9. The method of claim 5 or 6, which further comprises administering a second therapeutic agent. 10. The method according to claim 5 or 6, wherein said antibody is administered via the parenteral route, intravenous route or subcutaneous route. 11. Use of an anti-ANGPT2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 for the treatment of diseases or disorders characterized by cells expressing ANGPT2. 12. Use according to claim 11, wherein the disease or disorder is selected from the group consisting of cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury, hypertensive stroke, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, trauma-induced brain disorders, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy- and preeclampsia-induced hypertension, sepsis, severe sepsis, septicemia - 40 047046 shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal segmental glomerulosclerosis (FSGS), nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic kidney failure, end-stage renal disease, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF), emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) ), increased vascular permeability (and associated disorders), acute kidney injury, renal cell carcinoma, heart failure, lupus nephritis, Raynaud's syndrome, pancreatitis, peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma, retinopathy, diabetic nephropathy, portal hypertension, varicose veins and liver transplantation. 13. Use according to claim 12, wherein the disease or disorder is selected from the group consisting of chronic kidney disease, non-alcoholic steatohepatitis (NASH), diabetic nephropathy, sepsis and increased vascular permeability. 14. Use according to claim 12, wherein the disease or disorder is selected from the group consisting of ALI, ARDS, SARS, MERS, and hypervascular permeability and related disorders. 15. Use of an anti-ANGPT2 antibody or antigen-binding fragment according to one of claims 1 to 3 for the preparation of a medicament for the treatment of diseases or disorders that can be alleviated by treatment with an anti-ANGPT2 antibody. 16. Use according to claim 15, wherein the disease or disorder is selected from the group consisting of cardiac hypertrophy, myocardial infarction, ischemia, ischemic reperfusion injury, hypertensive stroke, pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, trauma-induced brain disorders, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, pregnancy-induced hypertension and preeclampsia, sepsis, severe sepsis, septic shock, non-alcoholic steatohepatitis (NASH), cirrhosis, minimal change disease, focal segmental glomerulosclerosis (FSGS) , nephrotic syndrome, diabetic kidney disease (DKD), chronic kidney disease (CKD), diabetic renal failure, end-stage renal disease, ischemia or ischemic reperfusion injury, malignancy, hepatocellular carcinoma, idiopathic pulmonary fibrosis (IPF), emphysema, acute injury lung disease (ALI), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), hypervascular permeability (and associated disorders), acute kidney injury, renal cell carcinoma, heart failure, lupus nephritis , Raynaud's syndrome, pancreatitis, peripheral arterial disease, congenital heart disease, Dengue virus, malaria, hantavirus, edema, regeneration, lupus, interstitial lung disease, scleroderma, retinopathy, diabetic nephropathy, portal hypertension, varicose veins and liver transplantation. 17. Use according to claim 16, wherein the disease or disorder is selected from the group consisting of chronic kidney disease, non-alcoholic steatohepatitis (NASH), diabetic nephropathy, sepsis and increased vascular permeability. 18. Use according to claim 16, wherein the disease or disorder is selected from the group consisting of ALI, ARDS, SARS, MERS, and hypervascular permeability and related disorders. 19. A method of blocking human ANGPT2 function in a human patient, which comprises administering to said patient a composition comprising an anti-ANGPT2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in an amount sufficient to block the ANGPT2 mediated response in said human patient. 20. Use of an antibody to ANGPT2 or an antigen-binding fragment thereof according to any one of claims 1 to 3 for blocking the function of human ANGPT2. 21. Use of an anti-ANGPT2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 for the preparation of a medicament for blocking the function of human ANGPT2. 22. An isolated polynucleotide encoding the amino acid sequence of the heavy chain variable region of an anti-ANGPT2 antibody or an antigen binding fragment thereof according to any one of claims 1 to 3, wherein the amino acid sequence of the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 3- 7, SEQ ID NO: 13-17; SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39. 23. An isolated polynucleotide encoding the amino acid sequence of the light chain variable region of an anti-ANGPT2 antibody or an antigen binding fragment thereof according to any one of claims 1 to 3, wherein the amino acid sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 8- 12, SEQ ID NO: 19-26, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40. -