JPWO2021092421A5 - - Google Patents

Description

[Cross reference to related applications]
This application is based on U.S. Provisional Application No. 63/104,410 filed on October 22, 2020, U.S. Provisional Application No. 62/940,164 filed on November 25, 2019, and November 2019. Claims priority to U.S. Provisional Application No. 62/932,257, filed on May 7th. The entire disclosures of these US provisional applications are incorporated herein by reference.

1. FIELD The present disclosure relates generally to methods of using antibodies that specifically bind human endothelial lipase (EL) and antigen-binding fragments thereof for the treatment of diseases or disorders, such as cardiovascular diseases and disorders. Advantageous uses are provided.

2. Background Endothelial lipase (EL) is a circulating phospholipase that has been identified as a member of the triglyceride lipase family. EL has both phospholipase and triglyceride lipase activities and hydrolyzes high density lipoprotein (HDL) more efficiently than other lipoproteins. EL is thought to play an important role in regulating plasma HDL cholesterol (HDL-C) levels. EL hydrolyzes HDL phospholipids, causing destabilization of HDL particles and rapid clearance by the kidneys.

Increased plasma EL concentrations are associated not only with a worsening of the lipoprotein-lipid profile in conjunction with increased plasma triglyceride and apolipoprotein B concentrations, but also with smaller low-density lipoprotein particle sizes (Non-Patent Document 1). ). Among individuals with elevated plasma EL concentrations, elevated proinflammatory cytokine concentrations and increased prevalence of metabolic syndrome have also been observed (Non-Patent Document 1). Considering these and other factors, EL is thought to play an important role in cardiovascular diseases (Non-Patent Document 1).

Despite the effectiveness of current therapies for cardiovascular disease, such as high-potency statins, there remains a significant residual risk of major cardiovascular (CV) events in patients with acute coronary syndrome (ACS). Most myocardial infarctions (MIs) occur in patients with normal low-density lipoprotein (LDL) levels, despite treatment with high doses of high-potency statins, PCSK9 inhibitors, and/or ezetimibe post-MI. First, the residual risk of a second CV event remains high. For example, in the IMPROVE-IT study (ezetimibe plus statins), there was a 33% risk of CV events over 7 years of follow-up. Additionally, ODYSSEY (PCSK9 inhibitor + statin) has a residual risk of 9.5% over 2.8 years.

Low HDL-C levels have been identified as a predictor of atherosclerotic CV events and coronary heart disease (CHD) risk factors. It has also been hypothesized that HDL particle size and particle number may be useful clinical markers of HDL and related diseases.

Several attempts have been made to raise HDL levels pharmacologically using various mechanisms of action. In particular, four trials of cholesterol transfer protein (CETP) inhibitors have been completed. CETP inhibitors increase HDL cholesterol, but three out of four trials did not improve CV outcomes, and one trial found that although they reduced CV events, the effect was only a 9% relative risk reduction. It was modest. This approach has been criticized because inhibition of CETP causes a blockade of LDL receptor-mediated reverse cholesterol transport.

Humans with partial and complete loss-of-function mutations in the gene encoding EL exhibit elevated HDL-C, increased cholesterol extraction capacity (CEC), and a tendency toward reduced CV risk. Neutralization of EL therefore represents a promising therapeutic mechanism. However, there are currently no approved therapies that target EL or sufficiently reduce CV risk. Therefore, there is a need for methods of effectively treating diseases and disorders, such as CV diseases and disorders, using anti-EL antibodies and antibody fragments thereof.

Paradis et al., Can J Cardiol 22: 31B-34B (2006)

3. SUMMARY Provided herein are methods of treating cardiovascular disease in a subject. In certain embodiments, the method comprises administering to the subject from about 100 mg to about 350 mg of an antibody that specifically binds human endothelial lipase (EL), or an antigen-binding fragment thereof.

Provided herein are methods of reducing atherosclerosis in a subject. In certain embodiments, the method comprises administering to the subject from about 100 mg to about 350 mg of an antibody or antigen-binding fragment thereof that specifically binds human EL.

Provided herein are methods of treating cardiovascular disease or reducing atherosclerosis in a subject. In certain embodiments, the method comprises administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to EL, wherein administration of the antibody or antigen-binding fragment thereof comprises: (a) (b) increase high-density lipoprotein (HDL) particle number in the subject, (c) increase HDL particle size in the subject, (d) increase high-density lipoprotein (HDL-C) in the subject; (e) increasing ApoA1 in the subject; and/or (f) increasing cholesterol extraction capacity (CEC) in the subject. In certain embodiments, the administration reduces the risk of cardiovascular death, non-fatal myocardial infarction (MI), non-fatal stroke, and/or coronary revascularization in subjects with a history of acute coronary syndrome (ACS). Reduce. In certain embodiments, the administration prevents a secondary cardiovascular event in the subject. In certain embodiments, the administration reduces the risk of major adverse cardiovascular events (MACE) in the subject.

Provided herein are methods for reducing the risk of cardiovascular death, non-fatal myocardial infarction (MI), non-fatal stroke, and/or coronary revascularization in subjects with a history of acute coronary syndrome (ACS). provided. In certain embodiments, the methods include administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human EL.

Provided herein are methods of preventing secondary cardiovascular events in a subject. In certain embodiments, the methods include administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human EL.

Provided herein are methods of reducing the risk of major adverse cardiovascular events (MACE) in a subject. In certain embodiments, the methods include administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human EL.

In certain aspects of the present disclosure, administration of the antibody or antigen-binding fragment thereof (a) increases high-density lipoprotein cholesterol (HDL-C) in the subject; and (b) increases high-density lipoprotein (HDL-C) in the subject. (c) increasing HDL particle size in the subject; (d) increasing HDL phospholipids in the subject; (e) increasing ApoA1 in the subject; and/or (f) increasing HDL particle size in the subject. Increases cholesterol extraction capacity (CEC).

Provided herein are methods of increasing HDL-C, HDL particle number, HDL particle size, HDL phospholipids, ApoA1, and/or CEC in a subject. In certain embodiments, the method comprises administering to the subject an antibody or antigen-binding fragment thereof that specifically binds EL.

Certain embodiments of the disclosure include administering about 100 mg to about 350 mg of the antibody or antigen-binding fragment thereof. Certain aspects of the present disclosure provide about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210mg, about 220mg, about 225mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 275mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 325mg, about 330mg, about 340mg, or about 350 mg of the antibody or antigen-binding fragment thereof. Certain embodiments of the present disclosure involve administering about 125 mg of the antibody or antigen-binding fragment thereof. Certain aspects of the disclosure include administering 125 mg of the antibody or antigen-binding fragment thereof. Certain embodiments of the present disclosure involve administering about 250 mg of the antibody or antigen-binding fragment thereof. Certain aspects of the disclosure include administering 250 mg of the antibody or antigen-binding fragment thereof. Certain embodiments of the present disclosure involve administering about 200 mg of the antibody or antigen-binding fragment thereof. Certain embodiments of the disclosure include administering 200 mg to 250 mg of the antibody or antigen-binding fragment thereof.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is administered once per month.
Administer twice. In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is administered once per month for at least 3 months. In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is administered once a month for at least 12 months or at least 24 months.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is administered parenterally. In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is administered subcutaneously.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is administered via an attached prefilled syringe (APFS) or autoinjector.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof inhibits EL in the subject for 30 days.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL-C in the subject by at least 30%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL-C in the subject by at least 35%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL-C in the subject by at least 40%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL-C in the subject within 30 days of initial administration. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL-C in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases ApoA1 in the subject by at least 30%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases ApoA1 in the subject by at least 35%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases ApoA1 in the subject within 30 days of initial administration. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases ApoA1 in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases non-ABCA1 cholesterol abstraction capacity in the subject by at least 30%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases non-ABCA1 cholesterol abstraction capacity in the subject by at least 35%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases non-ABCA1 cholesterol abstraction capacity in the subject within 30 days of initial administration. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases non-ABCA1 cholesterol abstraction capacity in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases the number of HDL particles in the subject by at least 5% (eg, as measured using NMR). In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases the number of HDL particles in the subject by at least 8% (eg, as measured using NMR). In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases the number of HDL particles in the subject within 30 days of initial administration. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases the number of HDL particles in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL particle size in the subject by at least 3%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL particle size in the subject by at least 5%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL particle size in the subject within 30 days of initial administration. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL particle size in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL phospholipids in the subject by at least 50%. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL phospholipids in the subject within 30 days of initial administration. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases HDL phospholipids in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases plasma phosphatidylinositol (PI) levels in the subject by at least 100% or at least 250%. In certain aspects of the present disclosure, the increase in plasma PI levels includes PI(14:2/20:0) levels, PI(14:2/22:0) levels, PI(14:2/22:1) ) level, PI (14:2/22:2) level, PI (16:0/16:1) level, PI (16:0/18:0) level, PI (16:0/18:2) level , PI (16:0/20:2) level, PI (16:0/20:3) level, PI (16:0/20:4) level, PI (16:0/22:4) level, PI (16:1/18:0) level, PI (16:1/18:1) level, PI (18:0/18:0) level, PI (18:0/18:1) level, PI (18 :0/18:2) level, PI (18:0/18:3) level, PI (18:0/20:2) level, PI (18:0/20:3) level, PI (18:0) /20:4) level, PI (18:0/22:4) level, PI (18:0/22:5) level, PI (18:0/22:6) level, PI (18:1/16) :0) level, PI (18:1/18:1) level, PI (18:1/18:2) level, PI (18:1/20:2) level, PI (18:1/20:3) ) level, PI (18:1/20:4) level, and/or PI (18:2/18:2) level. In certain embodiments of the present disclosure, administration of the antibody or antigen-binding fragment thereof increases plasma phosphatidylinositol (PI) levels in the subject within 90 days of initial administration.

In certain embodiments of the present disclosure, the subject is suffering from cardiovascular disease. In certain aspects of the disclosure, the cardiovascular disease is coronary artery disease, coronary heart disease (CHD), chronic arterial disease, cerebrovascular disease, atherosclerotic cardiovascular disease, or peripheral artery disease. be.

In certain aspects of the present disclosure, the subject has stable coronary artery disease or stable coronary heart disease. In certain aspects of the present disclosure, the subject has a history of acute coronary syndrome (ACS).

In certain embodiments of the present disclosure, the subject is receiving statin therapy. In certain embodiments of the present disclosure, the subject is not receiving statin therapy.

In certain embodiments of the present disclosure, the subject has a triglyceride level of 500 mg/dL or less prior to administration. In certain embodiments of the present disclosure, the subject has an LDL-C of 100 mg/dL or less prior to administration.

In certain embodiments of the present disclosure, the subject is a human.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof neutralizes EL activity.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is associated with reduced effector function. In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof does not have antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In certain embodiments of the present disclosure, the antibody does not have complement dependent cytotoxicity (CDC) activity.

In certain embodiments of the disclosure, the antibody binds to cynomolgus monkey EL.

In certain aspects of the present disclosure, the antibody or antigen-binding fragment thereof has a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8. Competitively inhibits binding. In certain aspects of the present disclosure, the antibody or antigen-binding fragment thereof has the same EL as the antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8. Binds to an epitope.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof has a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, light chain variable region (VL ) CDR1, VL CDR2, and VL CDR3. In certain aspects of the disclosure, the CDRs are CDRs as defined by Kabat, CDRs as defined by Chothia, or CDRs as defined by AbM. In certain aspects of the present disclosure, the antibody or antigen-binding fragment thereof has a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3. , VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

In certain aspects of the present disclosure, the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7, and/or a VL comprising the amino acid sequence set forth in SEQ ID NO:8.

In certain embodiments of the disclosure, the antibody or antigen-binding fragment comprises an IgG heavy chain constant region. In certain aspects of the disclosure, the IgG heavy chain constant region is an IgG4 heavy chain constant region. In certain embodiments of the present disclosure, the IgG4 heavy chain constant region is an IgG4P heavy chain constant region. In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment comprises a heavy chain constant region and/or a light chain constant region. In certain embodiments of the present disclosure, the heavy chain constant region is a human IgG4P heavy chain constant region and/or the light chain constant region is a human IgGκ light chain constant region.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is SEQ ID NO: 1
A heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 2 and/or a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 13.

In certain embodiments of the present disclosure, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is a full-length antibody.

In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In certain aspects of the disclosure, the antigen-binding fragment is a Fab, Fab', F(ab') ₂ , single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2 , minibody, F(ab') ₃ , tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb ² , (scFv) ₂ , or scFv-Fc.

Provided herein are methods of treating cardiovascular disease in a subject. In certain embodiments, the method comprises administering to the subject subcutaneously 250 mg of the antibody or antigen-binding fragment thereof once per month, wherein the antibody or antigen-binding fragment specifically targets human EL. It contains a VH that binds and includes the amino acid sequence shown in SEQ ID NO: 7, and a VL that includes the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the method comprises administering to the subject subcutaneously about 200 mg of the antibody or antigen-binding fragment thereof once per month, wherein the antibody or antigen-binding fragment is specific for human EL. and includes a VH that binds to and includes the amino acid sequence shown in SEQ ID NO: 7, and a VL that includes the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the method comprises administering 200 mg to 250 mg of the antibody or antigen-binding fragment thereof subcutaneously to the subject once per month, wherein the antibody or antigen-binding fragment is specific for human EL. and a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8. Provided herein are methods for reducing the risk of cardiovascular death, non-fatal myocardial infarction (MI), non-fatal stroke, and/or coronary revascularization in subjects with a history of acute coronary syndrome (ACS). provided. In certain embodiments, the method comprises administering to the subject subcutaneously 250 mg of the antibody or antigen-binding fragment thereof once per month, wherein the antibody or antigen-binding fragment specifically targets human EL. It contains a VH that binds and includes the amino acid sequence shown in SEQ ID NO: 7, and a VL that includes the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the method comprises administering to the subject subcutaneously about 200 mg of the antibody or antigen-binding fragment thereof once per month, wherein the antibody or antigen-binding fragment is specific for human EL. and includes a VH that binds to and includes the amino acid sequence shown in SEQ ID NO: 7, and a VL that includes the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the method comprises administering 200 mg to 250 mg of the antibody or antigen-binding fragment thereof subcutaneously to the subject once per month, wherein the antibody or antigen-binding fragment is specific for human EL. and a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8.

In certain embodiments of the present disclosure, the antibody comprises a heavy chain constant region amino acid sequence set forth in SEQ ID NO: 9 and a light chain constant region amino acid sequence set forth in SEQ ID NO: 10.

In certain embodiments of the present disclosure, the method further comprises administering an inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9). In certain embodiments, the administration of the antibody or antigen-binding fragment thereof that specifically binds human EL and the administration of the inhibitor of PCSK9 are simultaneous. In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds human EL and the inhibitor of PCSK9 are administered in separate pharmaceutical compositions. In certain embodiments, the administration of the antibody or antigen-binding fragment thereof that specifically binds human EL and the administration of the inhibitor of PCSK9 are sequential.

In certain embodiments, the inhibitor of PCSK9 is an anti-PCSK9 antibody or antigen-binding fragment thereof. In certain embodiments, the inhibitor of PCSK9 is HS9, evolocumab, alirocumab, or bococizumab.

4. Brief description of the drawing

FIG. 3 shows a dose-dependent increase in exposure to MEDI5584 (see Example 4 and Example 5). FIG. 4 shows MEDI5884 dose-dependent target association (inhibition of EL) (see Example 4 and Example 5). FIG. 4 shows MEDI5884 dose-dependent increase in high-density lipoprotein cholesterol (HDL-C) (see Examples 4 and 5). FIG. 3 shows MEDI5884 dose-dependent increase in apolipoprotein A1 (ApoA1) (see Example 4 and Example 5). FIG. 4 shows MEDI5884 dose-dependent increase in high-density lipoprotein phospholipids (HDL-PL) (see Examples 4 and 5). FIG. 4 shows MEDI5884 dose-dependent increase in non-ATP-binding cassette transporter A1 (ABCA1) cholesterol abstraction (see Example 4). FIG. 3 shows the dose-response relationships of HDL-C, ApoA1, and HDL-PL based on area under the curve (AUEC _d60-90 ) between days 60 and 90 (see Example 5). FIG. 3 shows pharmacokinetic (PK) and pharmacodynamic (PD) modeling for MEDI5884. CLd = intercompartmental clearance; CL = apparent systemic clearance; conc = concentration; dHDL(t)/dt = change in HDL over time; HDL = high-density lipoprotein cholesterol; HDL(t) = HDL level at time t ; IC50 = concentration reaching 50% of maximal effect (estimated simultaneously with Km); IH = inhibitory effect from MEDI5884; Imax = maximal inhibitory effect; Ka = absorption rate; Kin = HDL production rate; Km = 50 of Vmax %; Kout = HDL elimination rate; SC = subcutaneous; Vmax = maximum contribution of dose-dependent nonlinear clearance (see Example 5). FIG. 3 shows a PK simulation of MEDI5884 administered at 250 mg monthly (see Example 5). FIG. 2 shows a study design to analyze the effects of combined inhibition of EL and proprotein convertase subtilisin/kexin type 9 (PCSK9) in cynomolgus monkeys. FIG. 3 shows the effect of combined inhibition of EL and PCSK9 on LDL-C and HDL-C levels in cynomolgus monkeys. Data are expressed as mean change from baseline (day 0) measurements±SEM. In each plot, the average baseline value (n=16) is shown (see Example 6). FIG. 3 shows the effect of combined inhibition of EL and PCSK9 on ApoB and ApoA1 levels in cynomolgus monkeys. Data are expressed as mean change from baseline (day 0) measurements±SEM. In each plot, the average baseline value (n=16) is shown (see Example 6). FIG. 6 shows the effect of combined inhibition of EL and PCSK9 on cholesterol withdrawal capacity (global withdrawal and ABCA1 withdrawal) in cynomolgus monkeys (see Example 6). Time course and plasma phosphatidylinositol (PI) species for selected doses in studies using single ascending doses (SAD) and multiple ascending doses (MAD) in healthy patients and patients with coronary artery disease (CAD). FIG. 7 shows a dose-response (see Example 7). Time course and dose of plasma phosphatidylinositol (PI) species for all doses in studies using single ascending doses (SAD) and multiple ascending doses (MAD) in healthy patients and patients with coronary artery disease (CAD). - shows the response (see Example 7). FIG. 7 shows the effect of MEDI5884 on plasma phosphatidylinositol (PI) species in healthy volunteers and CAD patients at day 21 (see Example 7). FIG. 7 shows the effect of MEDI5884 on plasma phosphatidylinositol (PI) species in healthy volunteers and patients with coronary artery disease (CAD) on all days (see Example 7). FIG. 7 shows the percent change from baseline for various plasma phosphatidylinositol (PI) species in coronary artery disease (CAD) patients treated with various doses of MEDI5884 or placebo (see Example 7). FIG. 3 shows the mean percent change from baseline for all measured plasma phosphatidylinositol (PI) species in coronary artery disease (CAD) patients treated with various doses of MEDI5884 or placebo (Example 7). reference). FIG. 7 shows the percent change from baseline for various plasma phosphatidylinositol (PI) species in healthy volunteers treated with various doses of MEDI5884 or placebo (see Example 7).

5. DETAILED DESCRIPTION Provided herein are methods of administering antibodies (eg, monoclonal antibodies) and antigen-binding fragments thereof that specifically bind endothelial lipase (EL, eg, human EL). Anti-EL antibodies and antigen-binding fragments thereof can be administered, for example, to treat cardiovascular disease in a subject. The anti-EL antibody or antigen-binding fragment thereof increases high-density lipoprotein cholesterol (HDL-C), increases HDL particle number, increases HDL particle size, increases HDL phospholipids, and increases ApoA1 in a subject. and/or increase cholesterol withdrawal capacity. In some embodiments of the present disclosure, about 100 mg to about 350 mg (eg, about 250 mg) of the antibody or antigen-binding fragment thereof is administered to the subject, eg, administration occurs about once a month (QM).

5.1 Terminology As used herein, the term "endothelial lipase" or "EL" refers to mammalian lipases, including, but not limited to, naturally occurring EL polypeptides and isoforms of EL polypeptides. Refers to EL polypeptide. "EL" encompasses full-length, unprocessed EL polypeptides as well as forms of EL polypeptide that result from processing within the cell. As used herein, the term "human EL" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:11. "EL polynucleotide,""ELnucleotide," or "EL nucleic acid" refers to a polynucleotide encoding EL.

The term "antibody" refers to an antibody that recognizes a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations thereof, through at least one antigen recognition site within the variable region of an immunoglobulin molecule; Refers to an immunoglobulin molecule that specifically binds to a target. As used herein, the term "antibody" refers to intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibodies so long as the antibody exhibits the desired biological activity. and any other modified immunoglobulin molecules. Antibodies are based on the five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, or on the characteristics of their heavy chain constant domains called alpha, delta, epsilon, gamma, and mu, respectively. It can be any subclass (isotype) (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioactive isotopes, and the like.

The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment,""antigen-bindingdomain," or "antigen-binding region" refers to the portion of an intact antibody that binds an antigen. An antigen-binding fragment can contain the antigen recognition site of an intact antibody (eg, sufficient complementarity determining regions (CDRs) to specifically bind the antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab') ₂ fragments, and Fv fragments, linear antibodies, and single chain antibodies. can be mentioned. Antigen-binding fragments of antibodies may be derived from any animal species, including rodents (eg, mice, rats, or hamsters) and humans, or may be produced artificially.

The terms "anti-EL antibody," "EL antibody," and "antibody that binds to EL" refer to antibodies that bind to EL with sufficient affinity such that they are useful as diagnostic and/or therapeutic agents in targeting EL. Refers to an antibody that can specifically bind. As used herein, the terms "specifically bind," "immunospecifically bind," "immunospecifically recognize," and "specifically recognize" refer to antibodies or Similar terminology in the context of its antigen-binding fragments. These terms indicate that an antibody or antigen-binding fragment thereof binds to an epitope through its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to human EL (SEQ ID NO: 11) may also bind to EL proteins from other species (e.g., cynomolgus monkeys) and/or produced from other human alleles. , the extent of binding to unrelated non-EL proteins (e.g., liver lipase or other lipases such as lipoprotein lipase) is less than about 10% of the binding of the antibody to EL, as determined by, e.g., an in vitro neutralization assay. It is.

A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments that engage in highly specific binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof refers to both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab') ₂ , Fv, etc.), single chain (scFv) variants, Fusion proteins containing antibody portions and any other modified immunoglobulin molecules containing antigen recognition sites are included. Additionally, "monoclonal" antibodies or antigen-binding fragments thereof refer to such antibodies and antigen-binding fragments thereof produced by a number of methods, including, but not limited to, hybridomas, phage selection, recombinant expression, and transgenic animals.

As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region typically comprises a portion of an antibody, generally a light chain or a portion of a heavy chain, typically about the amino terminal 110-120 or 110-125 amino acids of the mature heavy chain and the mature light chain. This refers to about 90 to 115 amino acids of amino acids, which differ in sequence between antibodies and are used in the binding and specificity of a particular antibody for a particular antigen. Sequence variability is concentrated in regions called complementarity determining regions (CDRs), while more highly conserved regions within variable domains are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the light chain and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In some embodiments of this disclosure, the variable regions are human variable regions. In some embodiments of this disclosure, the variable regions include rodent or murine CDRs and human framework regions (FRs). In certain embodiments of the present disclosure, the variable region is a primate (eg, non-human primate) variable region. In some embodiments of the present disclosure, the variable regions include rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

The term "Kabat numbering" and similar terms are art-recognized and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of antibodies, or antigen-binding fragments thereof. In some embodiments, CDRs can be identified according to the Kabat numbering system (e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391, and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule typically include amino acids 35 followed by optionally one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme). It is present at amino acid positions 31 to 35 (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically amino acids 24-34 (CDR1), amino acids 50-56 (CDR2), and amino acids 89-97 (CDR3). ) exists in In some aspects of this disclosure, the CDRs of the antibodies described herein were determined according to the Kabat numbering scheme.

Instead, Chothia refers to the position of a structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Chothia CDR-H1 loop ends when numbered using Kabat numbering conventions differ between H32 and H34 depending on loop length (this is because the Kabat numbering scheme places inserts in H35A and H35B). If neither 35A nor 35B is present, the loop ends at 32; If only 35A is present, the loop ends at 33; If both 35A and 35B are present, the loop ends at 34 do). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software.

As used herein, the terms "constant region" and "constant domain" are used interchangeably and have a common meaning in the art. Constant regions are antibody portions, eg, the carboxyl-terminal portions of light and/or heavy chains, which are not directly involved in the binding of an antibody to an antigen, but which may exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have more conserved amino acid sequences compared to immunoglobulin variable domains.

As used herein, the term "heavy chain" when used in reference to antibodies can be of any of different types, such as IgA, IgD, IgE, IgG and IgM, based on the amino acid sequence of the constant domain. alpha (α), delta ( _δ ), _epsilon (ε _{) ,} gamma ( γ) and mu (μ). Heavy chain amino acid sequences are well known in the art. In some embodiments of this disclosure, the heavy chain is a human heavy chain.

As used herein, the term "light chain" when used in reference to antibodies can be of any different type, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. It may refer to Light chain amino acid sequences are well known in the art. In some embodiments of this disclosure, the light chain is a human light chain.

The term "MEDI5884" refers to an anti-EL antibody comprising a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10. MEDI5884 is also referred to as "S6F1-4P" and is the heavy chain variable region of the h55A1-S6 antibody disclosed in U.S. Patent Application Publication No. 2017/0260290, which is incorporated herein by reference in its entirety. and the light chain variable region of the h55A1-F1 antibody.

The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from more than one species. Typically, both the light and heavy chain variable regions are derived from a mammalian species (e.g., mouse, rat, rabbit, etc.) or antibodies with the desired specificity, affinity, and potency. While corresponding to the variable region of the antigen-binding fragment, the constant region is homologous to the sequence of the antibody or its antigen-binding fragment derived from another species (usually human) to avoid eliciting an immune response in that species. be.

The term "humanized" antibody or antigen-binding fragment thereof refers to specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. ) refers to the form of an antibody or antigen-binding fragment. Typically, a humanized antibody or antigen-binding fragment thereof is derived from a non-human species (e.g., mouse, mouse,
human immunoglobulin that has been replaced (“CDR-grafted”) by the remainder from the CDRs of a rat, rabbit, hamster (Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-327 (1988), Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, certain Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues in an antibody or fragment from a non-human species with the desired specificity, affinity, and potency. The humanized antibody or antigen-binding fragment thereof can be further modified by substitution of additional residues either in the Fv framework regions and/or in the non-human CDR residues to improve the specificity, affinity, or affinity of the antibody or antigen-binding fragment thereof. , and/or capabilities may be refined and optimized. Generally, a humanized antibody or antigen-binding fragment thereof will contain a variable domain that includes all or substantially all of the CDR regions that correspond to a non-human immunoglobulin, but all or substantially all of the FR regions are human. It is a consensus sequence of immunoglobulin. The humanized antibody or antigen-binding fragment thereof may also include at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin constant region or constant domain (Fc). Examples of methods used to generate humanized antibodies include U.S. Pat. No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994). ), and Roguska et al., Protein
Eng. 9(10):895-904 (1996). In some embodiments of the present disclosure, a "humanized antibody" is a resurfaced antibody.

The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof that has an amino acid sequence derived from human immunoglobulin loci, where such antibody or antigen-binding fragment is Made using any technique known in the art. This definition of human antibodies or antigen-binding fragments thereof includes intact or full-length antibodies and fragments thereof.

"Binding affinity" generally refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g. an antibody or antigen-binding fragment thereof) and its binding partner (e.g. an antigen). refers to the strength of Unless otherwise indicated, "binding affinity" as used herein reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). , refers to intrinsic binding affinity. The affinity of molecule X for its partner Y may generally be expressed by a dissociation constant (K _D ). Affinity can be measured and/or expressed in many ways known in the art, including, but not limited to, equilibrium dissociation constant (K _D ) and equilibrium association constant (K _A ). obtain. K _D is calculated from the quotient of k _off /k _on , while K _A is calculated from the quotient of k _on /k _off . k _on refers to the association rate constant of an antibody or antigen-binding fragment thereof, eg, to an antigen, and k _off refers to the dissociation of an antibody or antigen-binding fragment thereof, eg, from an antigen. k _on and k _off can be determined by techniques known to those skilled in the art such as BIAcore™ or KinExA.

As used herein, "epitope" is a term in the art that refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (a linear or contiguous epitope), or an epitope may be derived, for example, from two or more non-contiguous regions of a polypeptide(s) together. (conformational, non-linear, discontinuous, or non-contiguous epitopes). In some embodiments of the present disclosure, the epitope to which the antibody or antigen-binding fragment thereof specifically binds is determined by hydrogen/deuterium coupled, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, mass spectrometry. may be identified by exchange (eg, liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenic mapping (eg, site-directed mutagenic mapping). For X-ray crystallography studies, crystallization is performed using methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350, McPherson A (1990 ) Eur J Biochem 189: 1-23, Chayen NE (1997) Structure 5: 1269-1274, McPherson A (1976) J Biol Chem 251: 6300-6303). Antibodies/antigen-binding fragments thereof: Antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff
HW et al., US Patent Application Publication No. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60, Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenic mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including, for example, alanine scanning mutagenesis techniques, see Champe M et al., (1995) J Biol Chem 270: 1388-1394, and Cunningham BC & Wells JA (1989) Science 244: 1081-1085. Please refer to

An antibody that "binds the same epitope" as a reference antibody refers to an antibody that binds to the same amino acid residues as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody is determined by hydrogen/deuterium exchange assays (see Coales et al. Rapid Commun. Mass Spectrom. 2009; 23:639-647) or by X-ray crystallography studies. can be determined.

An antibody "competitively inhibits the binding of a reference antibody to a given epitope" if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks binding of the reference antibody to the epitope to some extent. ” is said. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, an "inhibitor of PCSK9" is an agent that blocks the interaction of PCSK9 with the LDL receptor.

An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form that is not found in nature. An isolated polypeptide, antibody, polynucleotide, vector, cell, or composition includes one that has been purified to the extent that it is no longer in the form in which it is found in nature. In some embodiments of the present disclosure, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" means that a material is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure; or at least 99% pure.

The terms "polypeptide,""peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. Polymers can be linear or branched, can include modified amino acids, and can be interrupted by non-amino acids. The term also includes modifications that occur naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling moiety). It includes amino acid polymers. Also included within the definition are polypeptides containing, for example, one or more analogs of amino acids (including, for example, unnatural amino acids, etc.) and other modifications known in the art. Since the polypeptides of the present disclosure are based on antibodies, it is understood that in some aspects of the present disclosure the polypeptides may exist as single chains or related chains.

As used herein, the term "host cell" can be any type of cell, such as a primary cell, a cell in culture, or a cell derived from a cell line. In some embodiments of this disclosure, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule, and the progeny or potential progeny of such cells. The progeny of such cells may not be identical to the parent cell transfected with the nucleic acid molecule, for example due to mutations or environmental influences that may occur in subsequent generations or integration of the nucleic acid molecule into the host cell genome.

The term "pharmaceutical preparation" refers to a preparation that is in such a form that the biological activity of the active ingredient is effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the preparation is administered. . The formulation may be sterile.

As used herein, terms such as "administering," "administering," "administration" and the like refer to the delivery of a drug, e.g., an anti-EL antibody or antigen-binding fragment thereof, to the desired site of biological action. Refers to methods that may be used to enable delivery, such as intravenous administration. Administration techniques that can be used with the agents and methods described herein include, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Seen by Pa.

As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some embodiments of the present disclosure, the subject is a mammal, such as a non-human animal (eg, a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some embodiments of the present disclosure, the subject is a cynomolgus monkey. In some embodiments of the present disclosure, the subject is a human.

The term "therapeutically effective amount" refers to an amount of a drug, such as an anti-EL antibody or antigen-binding fragment thereof, effective to treat a disease or disorder in a subject. Terms such as "treat," "treatment," "treating," "alleviation," and "alleviation" refer to curing, delaying, or alleviating the symptoms of a pathological condition or disorder. and/or therapeutic measures to halt its progression. Accordingly, subjects in need of treatment include subjects who have already been diagnosed with the disorder or who are suspected of having the disorder. Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concomitant) administration or sequential administration in any order.

As used in this disclosure and the claims, the singular forms "a," "an," and "the" refer to the plural forms "a," "an," and "the" unless the context clearly dictates otherwise. include.

Whenever an aspect of the disclosure is described herein with the word "comprising," analogous aspects described with the word "consisting of" and/or "consisting essentially of" are also provided. be understood.

As used herein, the term "or" is understood to be inclusive unless specifically stated or clear from the context. As used herein in phrases such as "A and/or B," the term "and/or" includes both "A and B,""A or B,""A" and "B." is intended. Similarly, the term "and/or" used in phrases such as "A, B, and/or C" means A, B and C; A, B or C; A or C; A or B; or C; A and C; A and B; B and C; A (alone);
It is intended to include each of embodiments B (alone); and C (alone).

As used herein, the terms "about" and "approximately" when used to modify a value or range of numbers refer to 5% to 10% above that value or range and 5% to 10% above that value or range. A deviation of less than 10% also indicates being within the intended meaning of the specified value or range.

Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

5.2 Methods of treatment using anti-EL antibodies or antigen-binding fragments thereof Anti-EL antibodies or antigen-binding fragments thereof as described herein, or pharmaceutical compositions thereof as described herein A method of administering the same to a subject in need thereof is provided.

As shown herein, administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can treat cardiovascular disease in a subject (eg, a human subject). Cardiovascular disease can be, for example, coronary artery disease, coronary heart disease, cerebrovascular disease, or peripheral artery disease.

Administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can reduce or prevent atherosclerosis in a subject (eg, a human subject).

Administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can prevent or reduce the risk of secondary cardiovascular events in a subject (eg, a human subject).

Administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, may lead to cardiovascular death, non-fatal myocardial infarction (MI), non-fatal stroke, coronary revascularization in a subject (e.g., a human subject). , or any combination thereof. The subject can be, for example, a subject with a history of acute coronary syndrome (ACS).

Administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can prevent or reduce the risk of major adverse cardiovascular events (MACE) in a subject (eg, a human subject).

Administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, (i) increases high-density lipoprotein cholesterol (HDL-C) and (ii) increases the number of high-density lipoprotein (HDL) particles. (iii) increase HDL particle size, (iv) increase HDL phospholipids, (v) increase ApoA1, (vi) increase cholesterol extraction capacity (CEC), or (vii) It can be any combination of them.

Administration of anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can also increase plasma phosphatidylinositol (PI) levels.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof inhibits EL.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases HDL-C in the subject. Administration of an anti-EL antibody or antigen-binding fragment thereof can increase HDL-C by, for example, at least 30%, at least 35%, or at least 40%. Therefore, administration of an anti-EL antibody or antigen-binding fragment thereof can reduce HDL-C by about 30% to about 125%, about 30% to about 100%, about 30% to about 75%, about 30% to about 50%. , about 30% to about 45%, or about 30% to about 40%. The increase in HDL-C can occur within 30 days of the first dose, within 60 days of the first dose, or within 90 days of the first dose.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases the number of HDL particles in the subject. Administration of an anti-EL antibody or antigen-binding fragment thereof can increase the number of HDL particles by, for example, at least 5%, at least 8%, at least 10%, or at least 15% (e.g., as measured using NMR). ). Thus, administration of an anti-EL antibody or antigen-binding fragment thereof can increase the number of HDL particles by about 5% to about 20%, or by about 5% to about 18% (e.g., as measured using NMR). ). Increase in HDL particle number can occur within 30 days of first administration, within 60 days of first administration, or within 90 days of first administration.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases HDL particle size in the subject. Administration of an anti-EL antibody or antigen-binding fragment thereof can increase HDL particle size by, for example, at least 3%, or at least 5%. Thus, administration of an anti-EL antibody or antigen-binding fragment thereof can increase HDL particle size by about 3% to about 10%, or about 3% to about 7%. Increase in HDL particle size may occur within 30 days of first administration, within 60 days of first administration, or within 90 days of first administration.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases HDL phospholipids in the subject. Administration of an anti-EL antibody or antigen-binding fragment thereof can increase HDL phospholipids, eg, by at least 50%. The increase in HDL phospholipids can occur within 30 days of the first dose, within 60 days of the first dose, or within 90 days of the first dose.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases apolipoprotein A1 (apoA1) in the subject. Administration of an anti-EL antibody or antigen-binding fragment thereof can increase apoA1, eg, by at least 30%. Thus, administration of an anti-EL antibody or antigen-binding fragment thereof can increase apoA1 by about 30% to about 40%, or about 30% to about 35%. The increase in apoA1 can occur within 30 days of the first dose, within 60 days of the first dose, or within 90 days of the first dose.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases cholesterol extraction capacity (CEC) in the subject. CEC can be measured, for example, using the method shown in Thacker et al., Journal of Lipid Research 56: 1282-1295 (2015). Administration of an anti-EL antibody or antigen-binding fragment thereof can increase non-ATP-binding cassette transporter A1 (ABCA1) cholesterol abstraction capacity, eg, by at least 30%, or by at least 35%. Thus, administration of an anti-EL antibody or antigen-binding fragment thereof can increase non-ABCA1 cholesterol abstraction capacity by about 30% to about 40%, or about 30% to about 35%.

In some embodiments, administration of an anti-EL antibody or antigen-binding fragment thereof increases plasma phosphatidylinositol (PI) levels in the subject. In some aspects, the increase in PI level includes PI(14:2/20:0) level, PI(14:2/22:0) level, PI(14:2/22:1) level, PI( 14:2/22:2) level, PI (16:0/16:1) level, PI (16:0/18:0) level, PI (16:0/18:2) level, PI (16: 0/20:2) level, PI (16:0/20:3) level, PI (16:0/20:4) level, PI (16:0/22:4) level, PI (16:1/ 18:0) level, PI (16:1/18:1) level, PI (18:0/18:0) level, PI (18:0/18:1) level, PI (18:0/18: 2) Level, PI (18:0/18:3) level, PI (18:0/20:2) level, PI (18:0/20:3) level, PI (18:0/20:4) level, PI (18:0/22:4) level, PI (18:0/22:5) level, PI (18:0/22:6) level, PI (18:1/16:0) level, PI (18:1/18:1) level, PI (18:1/18:2) level, PI (18:1/20:2) level, PI (18:1/20:3) level, PI ( 18:1/20:4) level and/or PI (18:2/18:2) level. In some aspects, the increase in PI level includes PI(14:2/22:2) level, PI(16:0/16:1) level, PI(16:0/18:2) level, PI( 16:0/20:3) level, PI (16:0/20:4) level, PI (18:0/18:1) level, PI (18:0/18:2) level, PI (18: 0/20:2) level, PI (18:0/20:3) level, PI (18:0/20:4) level, PI (18:0/22:6) level, and/or PI (18 :1/16:0) This is an increase in level. Administration of anti-EL antibodies or antigen-binding fragments thereof can increase plasma PI levels, e.g., by at least several hundred percent for more abundant PI species and from baseline for less abundant PI species. It can be increased by a few hundred percent to a few thousand percent change. Thus, administration of anti-EL antibodies or antigen-binding fragments thereof can increase plasma PI by about 100% to 1000% depending on the PI species. In some cases, administration of an anti-EL antibody or antibody binding fragment thereof increases the level of at least 10 plasma PI species, eg, by at least 100% or from 100% to 1000%. In some cases, administration of anti-EL antibodies or antigen-binding fragments thereof is administered to at least 12 plasma PI species (e.g., PI(14:2/22:2), PI(16:0/16:1), PI( 16:0/18:2), PI (16:0/20:3), PI (16:0/20:4), PI (18:0/18:1), PI (18:0/18: 2), PI (18:0/20:2), PI (18:0/20:3), PI (18:0/20:4), PI (18:0/22:6), and PI ( 18:1/16:0)) by at least 100% or from 100% to 1000%. In some cases, administration of anti-EL antibodies or antigen-binding fragments thereof is administered to at least 30 plasma PI species (e.g., PI(14:2/20:0), PI(14:2/22:0), PI( 14:2/22:1), PI (14:2/22:2), PI (16:0/16:1), PI (16:0/18:0), PI (16:0/18: 2), PI (16:0/20:2), PI (16:0/20:3), PI (16:0/20:4), PI (16:0/22:4), PI (16 :1/18:0), PI (16:1/18:1), PI (18:0/18:0), PI (18:0/18:1), PI (18:0/18:2) ), PI (18:0/18:3), PI (18:0/20:2), PI (18:0/20:3), PI (18:0/20:4), PI (18: 0/22:4), PI (18:0/22:5), PI (18:0/22:6), PI (18:1/16:0), PI (18:1/18:1) , PI (18:1/18:2), PI (18:1/20:2), PI (18:1/20:3), PI (18:1/20:4), and PI (18: 2/18:2)), for example by at least 100% or from 100% to 1000%.

In some cases, administration of an anti-EL antibody or antibody binding fragment thereof increases the level of at least 10 plasma PI species by at least 250% or from 250% to 1000%. In some cases, administration of anti-EL antibodies or antigen-binding fragments thereof is administered to at least 12 plasma PI species (e.g., PI(14:2/22:2), PI(16:0/16:1), PI( 16:0/18:2), PI (16:0/20:3), PI (16:0/20:4), PI (18:0/18:1), PI (18:0/18: 2), PI (18:0/20:2), PI (18:0/20:3), PI (18:0/20:4), PI (18:0/22:6), and PI ( 18:1/16:0)) by at least 250% or from 250% to 1000%. In some cases, administration of anti-EL antibodies or antigen-binding fragments thereof is administered to at least 30 plasma PI species (e.g., PI(14:2/20:0), PI(14:2/22:0), PI( 14:2/22:1), PI (14:2/22:2), PI (16:0/16:1), PI (16:0/18:0), PI (16:0/18: 2), PI (16:0/20:2), PI (16:0/20:3), PI (16:0/20:4), PI (16:0/22:4), PI (16 :1/18:0), PI (16:1/18:1), PI (18:0/18:0), PI (18:0/18:1), PI (18:0/18:2) ), PI (18:0/18:3), PI (18:0/20:2), PI (18:0/20:3), PI (18:0/20:4), PI (18: 0/22:4), PI (18:0/22:5), PI (18:0/22:6), PI (18:1/16:0), PI (18:1/18:1) , PI (18:1/18:2), PI (18:1/20:2), PI (18:1/20:3), PI (18:1/20:4), and PI (18: 2/18: Increase the level of 2)) by at least 250% or from 250% to 1000%.

Increases in plasma PI may occur within 90 days of first administration.

In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 100 mg to about 350 mg. In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 100 mg to about 250 mg. In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 100 mg to about 200 mg. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered parenterally, eg, subcutaneously. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered using attached prefilled syringes (APFS) or autoinjectors. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered about once per month.

In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 200 mg to about 350 mg. In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 200 mg to about 300 mg. In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 200 mg to about 250 mg. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered parenterally, eg, subcutaneously. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered using attached prefilled syringes (APFS) or autoinjectors. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered about once per month.

In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 250 mg to about 300 mg. In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 250 mg to about 350 mg. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered parenterally, eg, subcutaneously. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered using attached prefilled syringes (APFS) or autoinjectors. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered about once per month.

In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 100 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 110 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 120 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 125 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 130 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 140 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 150 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 160 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 170 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 175 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 180 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 190 mg. In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered at a dose of about 200 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 210 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 220 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 225 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 230 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 240 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 250 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 260 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 270 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 275 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 280 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 290 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 300 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 310 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 320 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 325 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 330 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 340 mg. In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 350 mg. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered parenterally, eg, subcutaneously. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered using attached prefilled syringes (APFS) or autoinjectors. Doses of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered about once per month.

In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 125 mg or 125 mg. A dose of about 125 mg or 125 mg of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered parenterally, eg, subcutaneously. A dose of about 125 mg or 125 mg of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered using an attached prefilled syringe (APFS) or autoinjector.

In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 250 mg or 250 mg. A dose of about 250 mg or 250 mg of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered parenterally, eg, subcutaneously. Doses of about 250 mg or 250 mg of antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, can be administered using an attached prefilled syringe (APFS) or autoinjector.

In some embodiments, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, are administered about once a month or once a month. Antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, administered about once a month or once a month can be administered parenterally, eg, subcutaneously. Antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, administered about once a month or once a month can be administered using an attached prefilled syringe (APFS) or autoinjector.

In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 125 mg about once a month. A dose of about 125 mg administered about once a month can be administered parenterally, eg, subcutaneously. A dose of about 125 mg administered about once a month can be administered using an attached prefilled syringe (APFS) or an autoinjector.

In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered at a dose of about 250 mg about once a month. A dose of about 250 mg administered about once a month can be administered parenterally, eg, subcutaneously. A dose of about 250 mg administered about once a month can be administered using an attached prefilled syringe (APFS) or an autoinjector.

In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered once a month at a dose of 125 mg. A dose of 125 mg administered once per month can be administered parenterally, eg, subcutaneously. A dose of about 125 mg administered about once a month can be administered using an attached prefilled syringe (APFS) or an autoinjector.

In some embodiments, the anti-EL antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered once a month at a dose of 250 mg. A dose of 250 mg administered once a month can be administered parenterally, eg, subcutaneously. A dose of about 250 mg administered about once a month can be administered using an attached prefilled syringe (APFS) or an autoinjector.

According to the methods provided herein, anti-EL antibodies or antigen-binding fragments thereof, or pharmaceutical compositions comprising anti-EL antibodies or antigen-binding fragments thereof, can be administered parenterally. In some embodiments of the present disclosure, an anti-EL antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-EL antibody or antigen-binding fragment thereof, is administered subcutaneously.

In some embodiments of the disclosure, the anti-EL antibody or antigen-binding fragment thereof is administered for at least 3 months (eg, about once a month). In some embodiments of the disclosure, the anti-EL antibody or antigen-binding fragment thereof is administered for at least 12 months (eg, about once a month). In some embodiments of the disclosure, the anti-EL antibody or antigen-binding fragment thereof is administered for at least 24 months (eg, about once a month).

In some aspects of the present disclosure, the present disclosure provides an anti-EL antibody or anti-EL antibody provided herein for use as a medicament administered at about 100 mg to about 350 mg or about 200 mg to about 350 mg (e.g., 250 mg). The present invention relates to antigen-binding fragments thereof, or pharmaceutical compositions thereof. In some aspects of the present disclosure, the present disclosure provides anti-EL antibodies provided herein for use as a medicament administered at about 100 mg to about 350 mg or about 200 mg to about 350 mg (e.g., about 250 mg). or an antigen-binding fragment thereof, or a pharmaceutical composition.

In some aspects of the disclosure, the disclosure provides herein for use as a medicament administered at about 100 mg to about 350 mg or about 200 mg to about 350 mg (e.g., 250 mg) about once a month. The present invention relates to an anti-EL antibody or an antigen-binding fragment thereof, or a pharmaceutical composition. In some aspects of the present disclosure, the present disclosure provides for use as a medicament administered once a month at about 100 mg to about 350 mg or about 200 mg to about 350 mg (e.g., about 250 mg). The present invention relates to an anti-EL antibody or an antigen-binding fragment thereof, or a pharmaceutical composition.

According to the methods provided herein, an anti-EL antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-EL antibody or antigen-binding fragment thereof, can be administered in combination with an inhibitor of PCSK9. Inhibitors of PCSK9 are described, for example, by Chaudhary et al., World J. Cardiol. 9: 76-91 (2017) and Chodorge et al., Sci. Rep. 8:
17545 (2018), each of which is incorporated herein by reference. An inhibitor of PCSK9 can be, for example, an antibody or antigen-binding fragment that binds to PCSK9. Antibodies or antigen-binding fragments thereof that inhibit PCSK9 include, for example, HS9, alirocumab, evolocumab, and bococizumab.

The HS9 antibody (in the context of a GLP-1 fusion protein called MEDI4166) is described in Chodorge et al., Sci. Rep. 8: 17545 (2018) and PCT International Publication No. WO 2015/127273 (respectively cited (herein incorporated by reference in its entirety). The HS9 antibody contains the following variable heavy and light chain sequences:
HS9 variable heavy chain sequence:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPSGGSTSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS (SEQ ID NO: 14)
HS9 variable light chain sequence:
DIQMTQSPSSLSASVGDRVTITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 15).

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof that inhibits PCSK9 comprises the variable heavy chain sequence of SEQ ID NO: 14. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof that inhibits PCSK9 comprises the variable light chain sequence of SEQ ID NO: 15. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof that inhibits PCSK9 comprises the variable heavy chain sequence of SEQ ID NO: 14 and the variable light chain sequence of SEQ ID NO: 15.

In some embodiments of the disclosure, the antibody that inhibits PCSK9 comprises a human IgG1 heavy chain. In some embodiments of the present disclosure, the antibody that inhibits PCSK9 comprises a human IgG1 heavy chain (“IgG1-TM”) that includes the triple mutation L234F/L235E/P331S. In some embodiments of the disclosure, the antibody that inhibits PCSK9 comprises a human kappa light chain. In some embodiments of the present disclosure, the antibody that inhibits PCSK9 comprises a) an IgG1-TM heavy chain comprising a variable heavy chain sequence of SEQ ID NO: 14; and b) a kappa light chain comprising a variable light chain sequence of SEQ ID NO: 15. including.

In some embodiments of the present disclosure, inhibitors of PCSK9 can promote LDL-C uptake in HepG2 cells treated with recombinant PCSK9 (e.g., Chodorge et al., Sci. Rep. 8: 17545 (2018)).

As provided herein, an inhibitor of PCSK9 can be administered simultaneously (in the same or separate pharmaceutical compositions) or sequentially with an anti-EL antibody or antigen-binding fragment thereof.

5.3 EL Antibodies and Antigen-Binding Fragments Thereof Described herein are methods of treating cardiovascular disease in a subject (e.g., a human subject), comprising: Methods are provided that include administering antibodies (eg, chimeric antibodies, humanized antibodies, or monoclonal antibodies such as human antibodies) and antigen-binding fragments thereof. Exemplary EL antibodies and antigen-binding fragments thereof that can be used in the methods provided herein are known in the art. The amino acid sequence for human EL is known in the art and the mature form of the protein (lacking the leader sequence) is provided herein as the sequence SEQ ID NO: 11.

Mature human EL (lacking leader sequence):
SPVPFGPEGRLEDKLHKPKATQTEVKPSVRFNLRTSKDPEHEGCYLSVGHSQPLEDCSFNMTAKTFFIIHGWTMSGIFENWLHKLVSALHTREKDANVVVVDWLPLAHQLYTDAVNNTRVVGHSIARMLDWLQEKDDFSLGNVHLIGYSLGAHVAGYAGNFVKGTVGRITGLDPAGPMFEGADIHKRLSPDDADFVDVLHTYTRSFGLSI GIQMPVGHIDIYPNGGDFQPGCGLNDVLGSIAYGTITEVVKCEHERAVHLFVDSLVNQDKPSFAFQCTDSNRFKKGICLSCRKNRCNSIGYNAKKMRNKRNSKMYLKTRAGMPFRVYHYQMKIHVFSYKNMGEIEPTFYVTLYGTNADSQTLPLEIVERIEQNATNTFLVYTEEDLGDLLKIQLTWEGASQSWYNLWKEFRSYLSQPRNPGR ELNIRRRIRVKSGETQRKLTFCTEDPENTSISPGRELWFRKCRDGWRMKNETSPTVELP (SEQ ID NO: 11).

In some embodiments of the present disclosure, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human EL. In some aspects of the present disclosure, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human EL and cynomolgus monkey EL.

In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof binds to human EL with an equilibrium dissociation constant (KD) (e.g., measured using surface plasmon resonance) of about 4.06 nM. . In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof binds to cynomolgus monkey EL with a KD (eg, measured using surface plasmon resonance) of about 1.56 nM. In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof binds human EL with a KD constant of about 4.06 nM and has a KD constant of about 1.56 nM (e.g., using surface plasmon resonance). (measured) binds to cynomolgus monkey EL.

In some embodiments of the present disclosure, anti-EL antibodies or antigen-binding fragments thereof are capable of neutralizing or inhibiting EL.

The ability of an anti-EL antibody or antigen-binding fragment thereof to neutralize EL can be determined using the following protocol: conditioned medium was mixed with assay buffer (20 mM Tris-HCl, 150 mM NaCl, 4 mM CaCl2 ₎ . , 0.5% BSA) and HDL (e.g., human HDL) in the presence or absence of an anti-EL antibody or antigen-binding fragment thereof in a half-area 96-well microplate containing concentrations ranging from 1000 nM to 31.6 pM. Incubate for 2 hours at 37°C. Following this incubation, free fatty acid release is measured in each well using the NEFA-HR(2) assay kit (Wako Diagnostics, Mountain View, Calif.). Absorbance at both 550 nm and 660 nm is measured using a SpectraMax M5 plate reader. The value obtained by subtracting the 660 nm reading from the 550 nm reading is used for further analysis. Calculate the percent activity "Ax" at antibody concentration "x" using the following equation:
Ax=[(Ex-V0)/(E0-V0)]×100
(In the formula, "Ex" is the average value of absorbance units in the presence of the enzyme with the inhibitor included, and "E0" and "V0" are the average value of absorbance units in the case with the inhibitor and in the absence of the enzyme. The 50% inhibitory concentration (IC50) is calculated and plotted using GraphPad Prism.

The same assay can be performed using VLDL (e.g., human VLDL) instead of HDL (e.g., human HDL) as the substrate to show that anti-EL antibodies do not neutralize hepatic lipase or lipoprotein lipase. can.

In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof has a half-inhibitory concentration ( _IC50 ) against human EL of about 1.3 nM. In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof has an IC ₅₀ against cynomolgus monkey EL of about 1.7 nM. In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof has an IC ₅₀ of about 1.3 nM against human EL and an IC ₅₀ of about 1.7 nM against cynomolgus monkey EL. .

In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof does not inhibit very low density lipoprotein (VLDL) lipolysis mediated by either hepatic lipase or lipoprotein lipase.

In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof does not block the exchange of cholesterol from HDL particles to LDL particles.

In some embodiments of the present disclosure, the anti-EL antibody or antigen-binding fragment thereof increases delivery of low density lipoprotein (LDL) to the low density lipoprotein receptor (LDLR).

In some embodiments of the present disclosure, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind human EL and are listed as shown in Tables 1 and 2. Contains 6 CDRs of the MEDI5884 antibody.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds human EL and comprises the VH of the MEDI5884 antibody listed in Table 3. .

In some aspects of the present disclosure, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds human EL and comprises the VL of the MEDI5884 antibody listed in Table 4. .

In some embodiments of the present disclosure, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human EL and are of the MEDI5884 antibody listed in Tables 3 and 4. Includes VH and VL.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds human EL and has the heavy chain sequence of the MEDI5884 antibody listed in Table 5. including.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds human EL and has the light chain sequence of the MEDI5884 antibody listed in Table 6. including.

In some embodiments of the present disclosure, the antibodies or antigen-binding fragments used in the methods described herein specifically bind to human EL and are Contains chain and light chain sequences.

In some aspects of the present disclosure, the antibodies or antigen-binding fragments thereof used in the methods described herein are isolated by their VL domain alone or by their VH domain alone, or by their three VL CDRs alone or by their three described by two VH CDRs alone. For example, a murine anti-αvβ3 antibody can be humanized by identifying complementary light or heavy chains from a human light or heavy chain library, respectively, thereby achieving an affinity similar to that of the original antibody or Rader C et al., (1998) PNAS 95: 8910-8915 (incorporated herein in its entirety by reference), which describes obtaining humanized antibody variants with higher affinities. See eggplant). Describes a method of generating antibodies that specifically bind to a particular antigen by using a particular VL domain (or VH domain) and screening a library for complementary VH domains or (VL domains). See also Clackson T et al., (1991) Nature 352: 624-628, incorporated herein by reference in its entirety. The screen generated 14 new partners for the specific VH domain and 13 new partners for the specific VL domain, which were strong binders as determined by ELISA. Kim describes a method for generating antibodies that specifically bind to a particular antigen by using a particular VH domain and screening a library (e.g., a human VL library) for complementary VL domains. See also SJ & Hong HJ, (2007) J Microbiol 45: 572-577 (incorporated in its entirety by reference); using the selected VL domain, now adding was able to guide the selection of a complementary (eg, human) VH domain.

In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the position of structural loops in immunoglobulins (e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917, Al-Lazik
ani B et al., (1997) J Mol Biol 273: 927-948, Chothia C et al., (1992) J Mol Biol 227: 799-817, Tramontano A et al., (1990) J Mol Biol 215( 1): 175-82 and U.S. Pat. No. 7,709,226). Typically, when using Kabat numbering rules, the Chothia CDR-H1 loop is located at amino acids 26 to 32, amino acids 33, or amino acids 34 of the heavy chain, and the Chothia CDR-H2 loop is located at amino acids 52 to 34 of the heavy chain. ~ present at amino acid 56, and the Chothia CDR-H3 loop is present from amino acid 95 to amino acid 102 of the heavy chain, while the Chothia CDR-L1 loop is present from amino acid 24 to amino acid 34 of the light chain, and the Chothia CDR-L2 loop is present from amino acid 24 to amino acid 34 of the light chain. The loop is present from amino acid 50 to amino acid 56 of the light chain, and the Chothia CDR-L3 loop is present from amino acid 89 to amino acid 97 of the light chain. The Chothia CDR-H1 loop ends when numbered using the Kabat numbering convention differ between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places inserts in H35A and H35B). If neither 35A nor 35B is present, the loop ends at 32; If only 35A is present, the loop ends at 33; If both 35A and 35B are present, the loop ends at 32; 34).

In some embodiments, antibodies and antigen-binding fragments thereof that specifically bind to EL (e.g., human EL) and include the Chothia VH and VL CDRs of the MEDI5884 antibody listed in Table 3 and Table 4 are administered. A method is provided herein. In some embodiments of the present disclosure, an antibody or antigen-binding fragment thereof that specifically binds to EL (e.g., human EL) and comprises one or more CDRs in which the Chothia CDR and the Kabat CDR have the same amino acid sequence. Provided herein are methods of administering. In some embodiments of the present disclosure, provided herein are methods of administering antibodies and antigen-binding fragments thereof that specifically bind to EL (e.g., human EL) and include a combination of Kabat and Chothia CDRs. Ru.

In some embodiments of the present disclosure, the CDRs of an antibody or antigen-binding fragment thereof are derived from Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. can be determined according to the IMGT numbering system described in . According to the IMGT numbering scheme, VH-CDR1 is 26th to 35th, VH-CDR2 is 51st to 57th, VH-CDR3 is 93rd to 102nd, VL-CDR1 is 27th to 32nd, VL-CDR2 is is in the 50th to 52nd place, and VL-CDR3 is in the 89th to 97th place. In some embodiments of the present disclosure, compounds that specifically bind to EL (e.g., human EL) and are listed in Tables 3 and 4, such as Lefranc M-P (1999), supra, and Lefranc M-P (1999), supra. Provided herein are methods of administering antibodies and antigen-binding fragments thereof comprising the IMGT VH and VL CDRs of the MEDI5884 antibody described in US Pat.

In some embodiments, the CDRs of the antibody or antigen-binding fragment thereof are
et al., (1996) J Mol Biol 262: 732-745. See, for example, "Protein Sequence and Structural Analysis of Antibody Variable Domains" by Martin A. in Antibody Engineering, edited by Kontermann and Duebel, Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). and Structure Analysis of Antibody Variable Domains). In some embodiments of the present disclosure, the VH CDRs and VH CDRs of the MEDI5884 antibody listed in Tables 3 and 4 that specifically bind to EL (e.g., human EL) and are determined by the method in MacCallum RM et al. Provided herein are methods of administering an antibody or antigen-binding fragment thereof that includes a VL CDR.

In some embodiments, the CDRs of the antibody or antigen-binding fragment thereof correspond to intermediates between Kabat CDRs and Chothia structural loops and
It can be determined according to the AbM numbering scheme that refers to AbM hypervariable regions used by the bM antibody modeling software (Oxford Molecular Group, Inc.). In some embodiments of the present disclosure, the VH CDR and VL CDR of the MEDI5884 antibody that specifically binds EL (e.g., human EL) and are listed in Table 3 and Table 4 as determined by the AbM numbering scheme Provided herein are methods of administering antibodies or antigen-binding fragments thereof.

In some embodiments of the disclosure, provided herein are methods of administering antibodies that include heavy and light chains.

Regarding heavy chains, in some embodiments of the present disclosure, the heavy chain is a gamma heavy chain. The constant region of a human IgG4P heavy chain may include the following amino acid sequence:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12).

In some embodiments of the present disclosure, the antibodies that immunospecifically bind to EL (e.g., human EL) used in the methods described herein have VH domain amino acid sequences shown in Table 1. A heavy chain comprising an amino acid sequence, wherein the heavy chain constant region comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, eg, human IgG4P.

In some embodiments of the present disclosure, the antibodies that immunospecifically bind to EL (e.g., human EL) used in the methods described herein have the amino acid sequence of the VH domain set forth in Table 3. A heavy chain comprising a sequence, wherein the heavy chain constant region comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, eg, human IgG4P.

Regarding light chains, in some embodiments of the present disclosure, the light chains of the antibodies described herein are kappa light chains. The constant region of human Ckappa light chain may include the following amino acid sequence:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13).

In some embodiments of the present disclosure, the antibodies that immunospecifically bind to EL (e.g., human EL) used in the methods described herein have VL domain amino acid sequences set forth in Table 2. A light chain comprising an amino acid sequence, wherein the light chain constant region comprises the amino acid sequence of a human Ckappa light chain constant region.

In some embodiments of the present disclosure, the antibodies that immunospecifically bind to EL (e.g., human EL) used in the methods described herein have the amino acid sequence of the VL domain set forth in Table 4. wherein the light chain constant region comprises the amino acid sequence of a human Ckappa light chain constant region.

In some aspects of the present disclosure, antibodies that immunospecifically bind to EL (e.g., human EL) used in the methods described herein include any VH domain described herein and The constant region comprises the amino acid sequence of a constant region of an IgG (eg, human IgG) immunoglobulin molecule. In some aspects of the present disclosure, antibodies that immunospecifically bind to EL (e.g., human EL) used in the methods described herein include any VH domain described herein and The constant region comprises the amino acid sequence of a constant region of an IgG4P kappa (eg, human IgG4P kappa) immunoglobulin molecule.

As shown herein, the antibodies or antigen-binding fragments thereof that immunospecifically bind to EL (e.g., human EL) used in the methods described herein, e.g. may have reduced effector function compared to antibodies or antigen-binding fragments that have Reduced effector function can be due, for example, to the sequence of the constant region of the antibody or antigen-binding fragment thereof.

As shown herein, an antibody or antigen-binding fragment thereof that immunospecifically binds to EL (e.g., human EL) used in the methods described herein may have a constant region sequence of, e.g. As a result, CDC activity and/or ADCC activity may be lacking.

In some embodiments of the disclosure, the antibodies described herein or antigen-binding fragments thereof that immunospecifically bind to EL (e.g., human EL) include a heavy chain and a light chain, wherein: (i) the heavy chain comprises a VH domain comprising the amino acid sequences of the VH CDR1, VL CDR2, and VL CDR3 of the MEDI5884 antibody listed in Table 1; and (ii) the light chain comprises the VH CDR1, VL CDR2, and VL CDR3 amino acid sequences of the MEDI5884 antibody listed in Table 2. VL CDR1, VH CDR2, and VH
(iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of a constant domain of a human IgG4P heavy chain; and (iv) the light chain comprises a VL domain comprising an amino acid sequence of a human kappa light chain. Further comprising a constant light chain domain comprising the amino acid sequence of a constant domain.

In some embodiments of the disclosure, the antibodies described herein or antigen-binding fragments thereof that immunospecifically bind to EL (e.g., human EL) include a heavy chain and a light chain, wherein: (i) the heavy chain comprises a VH domain comprising the amino acid sequence of the VH domain of the MEDI5884 antibody listed in Table 3, and (ii) the light chain comprises the amino acid sequence of the VL domain of the MEDI5884 antibody listed in Table 4. (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG4P heavy chain, and (iv) the light chain comprises the amino acid sequence of the constant domain of a human kappa light chain. further comprising a constant light chain domain comprising a constant light chain domain.

In certain embodiments, the antigen-binding fragments described herein that immunospecifically bind to EL (e.g., human EL) are from the group consisting of Fab, Fab', F(ab') ₂ , and scFv. selected, where Fab, Fab', F(ab') ₂ , or scFv comprises the heavy chain variable region sequence and light chain variable region sequence of an anti-EL antibody or antigen-binding fragment thereof described herein. include. Fab, Fab', F(ab') ₂ or scFv can be produced by any technique known to those skilled in the art. In some embodiments of the present disclosure, the Fab, Fab', F(ab') ₂ , or scFv further comprises a moiety that extends the half-life of the antibody in vivo. This part is also called the "half-life extension part." Any moiety known to those skilled in the art that increases the half-life of a Fab, Fab', F(ab') ₂ , or scFv in vivo can be used. For example, a half-life extending moiety can include an Fc region, a polymer, albumin, or an albumin binding protein or compound. Examples of the polymer include natural or synthetic optionally substituted linear or branched polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, Mention may be made of amylose, dextran, glycogen or derivatives thereof. Substituents can include one or more hydroxy, methyl, or methoxy groups. In some embodiments of the present disclosure, a Fab, Fab', F(ab') ₂ , or scFv can be modified by adding one or more C-terminal amino acids to attach a half-life extending moiety. In some embodiments of the present disclosure, the half-life extending moiety is polyethylene glycol or human serum albumin. In some aspects of this disclosure, a Fab, Fab', F(ab') ₂ or scFv is fused to an Fc region.

5.4 Pharmaceutical Compositions As used herein, an anti-inflammatory drug of desired purity may be prepared in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Methods of administering compositions comprising EL antibodies or antigen-binding fragments thereof are provided. An acceptable carrier, excipient, or stabilizer is nontoxic to recipients at the dosages and concentrations employed (e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th edition (2003), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical
(See Excipients, 3rd edition, Pharmaceutical Press (2000)). Compositions used for in vivo administration can be sterile. This is easily accomplished, for example, by filtration through a sterile filter membrane.

In some embodiments of the present disclosure, (i) (a) heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable of SEQ ID NO: 1 to SEQ ID NO: 6, respectively; Region (VL) sequence of CDR1, CDR2, and CDR3, (b) variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 7, and/or variable light chain region comprising the amino acid sequence of SEQ ID NO: 8, or (c) sequence an isolated antibody or antigen-binding fragment thereof that specifically binds to human EL comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and/or a light chain comprising the amino acid sequence of SEQ ID NO: 10; and an acceptable excipient are provided.

In some embodiments of the present disclosure, the pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that specifically binds human EL also comprises an inhibitor of PCSK9. In some embodiments of the present disclosure, a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that specifically binds human EL is administered in combination with an inhibitor of PCSK9.

5.5 Antibody Production and Polynucleotides Antibodies and antigen-binding fragments thereof that immunospecifically bind to EL (e.g., human EL) are produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments thereof. For example, it can be produced by chemical synthesis or recombinant expression techniques. Unless otherwise indicated, the methods described herein apply to molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and using conventional techniques in the relevant field within the skill in the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. For example, Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel FM et al., Current Protocols in Molecular
Biology, John Wiley & Sons (1987 and annual updates), Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates), Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press , Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press, Birren B et al., (ed.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In some embodiments, a method of administering an anti-EL antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising such an antibody or fragment, wherein the antibody or fragment is a polynucleotide comprising a nucleotide sequence in a host cell. Provided herein are methods of producing a protein by recombinant expression.

In some embodiments, the anti-EL antibody or antigen-binding fragment administered according to the methods provided herein is optimized, e.g., by codon/RNA optimization, replacement with a heterologous signal sequence, and removal of mRNA instability elements. The antibody is encoded by a polynucleotide encoding an anti-EL antibody or an antigen-binding fragment thereof or a domain thereof. Anti-EL antibodies optimized for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and/or by removing inhibitory regions within the mRNA. or a nucleic acid encoding an antigen-binding fragment thereof or a domain thereof (e.g., a heavy chain, light chain, VH domain, or VL domain), as described, for example, in U.S. Pat. No. 5,965,726, U.S. Pat. , 174,666, U.S. Pat. No. 6,291,664, U.S. Pat. No. 6,414,132, and U.S. Pat. No. 6,794,498. It can be carried out by

Polynucleotides can be in the form of RNA or DNA, for example. DNA includes cDNA, genomic DNA, and synthetic DNA. DNA can be double-stranded or single-stranded. When single-stranded, the DNA can be the coding strand or the non-coding (antisense) strand. In some embodiments of the present disclosure, the polynucleotide is a cDNA or a DNA lacking one or more introns. In some embodiments of this disclosure, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments of this disclosure, polynucleotides are recombinantly produced. In some embodiments of this disclosure, polynucleotides are isolated. In some embodiments of this disclosure, the polynucleotide is substantially pure. In some embodiments of the present disclosure, polynucleotides are purified from natural components.

In some embodiments, vectors (eg, expression vectors) include nucleotide sequences encoding anti-EL antibodies and antigen-binding fragments or domains thereof for recombinant expression in host cells, preferably mammalian cells. In some embodiments, the cell, e.g., a host cell, recombinantly expresses an anti-EL antibody or antigen-binding fragment thereof (e.g., a human or humanized antibody or antigen-binding fragment thereof) described herein. including such vectors. Accordingly, methods of producing antibodies or antigen-binding fragments thereof described herein may include expressing such antibodies or antigen-binding fragments thereof in a host cell.

The expression vector can be introduced into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the antibodies or antigen-binding fragments thereof described herein. (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of MEDI5884, VH, VL, VH and VL, heavy chain, light chain, or heavy and light chains) or a domain thereof (e.g., VH, VL, VH and VL of MEDI5884, VH and VL, heavy chains, or light chains).

In some embodiments of the present disclosure, an anti-EL antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof comprising the CDRs of MEDI5884) is administered according to the methods provided herein and is produced in a host cell. be done. In some embodiments of this disclosure, the host cell is a CHO cell.

In some embodiments of this disclosure, the antibody or antigen-binding fragment thereof administered according to the methods provided herein is isolated or purified. Generally, an isolated antibody or antigen-binding fragment thereof will be substantially free of other antibodies or antigen-binding fragments thereof that have different antigen specificities than the isolated antibody or antigen-binding fragment. For example, in some embodiments of the present disclosure, preparations of antibodies or antigen-binding fragments thereof described herein are substantially free of cellular material and/or chemical precursors.

The following examples are offered by way of illustration and not by way of limitation.

6. EXAMPLES The examples in this section (i.e., Section 6) are provided by way of illustration and not by way of limitation.

6.1 Example 1: Nonclinical Pharmacology of MEDI5884 From nonclinical in vivo pharmacology studies, single subcutaneous (SC) doses of MEDI5884 (0.5 mg/kg, 6 mg/kg, or 30 mg/kg), plasma HDL-C was revealed to increase in a dose-dependent manner. The increase in HDL-C from baseline to maximal effect for doses of 0.5 mg/kg, 6 mg/kg, and 30 mg/kg was from 63 ± 14 mg/dL to 96 ± 29 mg/dL, 60 ± 3.8 mg, respectively. /dL to 111±5.6 mg/dL, and 54±7.4 mg/dL to 122±17 mg/dL. Smaller increases in total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and non-HDL-C (TC minus HDL-C) were also observed.

Apolipoprotein A1 (ApoA1), the major lipoprotein component of HDL, was also increased by 75±5.5% at the 30 mg/kg dose. A dose-dependent increase in serum phospholipids was observed, indicating that MEDI5884 inhibited the hydrolysis of phospholipids contained in HDL particles.

Increases in ATP-binding cassette transporter A1 (ABCA1), cholesterol abstraction capacity (CEC), global cholesterol abstraction, and total number of HDL particles were observed at the highest dose (30 mg/kg). An increase in the average number of total and large low density lipoprotein (LDL) particles was also observed. Obtained from animals in the 0.5 mg/kg and 30 mg/kg MEDI5884 treatment groups at 0 days post-dose, 0.5 days post-dose, 1 day post-dose, 2 days post-dose, 3 days post-dose, 7 days post-dose, and 14 days post-dose. HDL was used to measure cholesterol withdrawal. ABCA1 withdrawal was temporarily decreased below baseline (days 0.5 and 1) after treatment with 30 mg/kg MEDI5884, but decreased on days 3, 7, and 14. exceeded baseline levels by 6%, 26%, and 114%, respectively. Similarly, overall withdrawal decreased temporarily below baseline (-6%) on day 0.5 and by 6% on days 2, 3, 7, and 14, respectively. %, 5%, 41%, and 53%.

HDL particles were characterized using NMR spectroscopy in samples obtained from cynomolgus monkeys treated with MEDI5884. Treatment with 30 mg/kg MEDI5884 increased the total number of HDL particles by up to 49±6.3%, and the number of both small and large particles by up to 182±85% and up to 104±10%, respectively. Moderate HDL particles decreased by up to 48 ± 14% on day 2 and returned to baseline by day 49, indicating HDL particle speciation or Mutual conversion is suggested. HDL size decreased from 10.0 ± 0.033 nm to 10.9 ± 0.17 nm and 10 ± 0.0 nm from baseline to plateau level for doses of 0.5 mg/kg, 6 mg/kg, and 30 mg/kg, respectively. It increased from 23nm to 10.7±0.12nm and from 9.9±0.033nm to 10.7±0.13nm.

These data support the use of MEDI5884 in human patients, for example, for the prevention of secondary cardiovascular events.

6.2 Example 2: Nonclinical Pharmacokinetics and Safety of MEDI 5884 MEDI 5884 was tested in a single-dose Good Laboratory Practice (GLP) PK/Pharmacodynamics (PD) study and a two-dose GLP toxicity study. was administered to cynomolgus monkeys. SC administration of MEDI5884 in single doses up to 30 mg/kg was well tolerated. There were no unplanned deaths, adverse clinical findings, injection site reactions, or adverse effects on body weight. After repeated SC administration of MEDI 5884 at 10 mg/kg, 30 mg/kg, or 100 mg/kg per dose for 1 or 6 months (one dose every 2 weeks), all animals were treated until scheduled sacrifice. Survived. No MEDI5884-related changes in clinical observations, ophthalmological evaluation, body weight, behavior, neurophysiology, respiratory rate, injection site irritation scoring, heart rate, electrocardiogram (ECG), or blood pressure were observed. Treatment-related findings were limited to minimal to moderate pharmacologically mediated increases in TC, HDL-C, LDL-C, and phospholipids at all dose levels. By microscopy, the presence of minimal to moderate perivascular lymphocytic/mixed leukocyte infiltration was observed at the SC injection site in a small number of MEDI5884-treated animals, with treatment-related histopathological findings observed in other tissues. was not observed at all. Based on these results, the no observed adverse effect level (NOAEL) was determined to be 100 mg/kg per dose.

6.3 Example 3: Phase 1 Clinical Evaluation of MEDI 5884 MEDI 5884 was evaluated in a Phase 1, first-in-human, blinded, placebo-controlled, single dose escalation (SDE) study in patients not receiving statin therapy. The safety, PK, and PD of MEDI5884 administered subcutaneously (SC) in healthy subjects was evaluated. Subjects were randomized in a 3:1 ratio to receive 30 mg, 100 mg, 300 mg, or 600 mg of MEDI5884 or placebo. The initial cohort was 6 MEDI5884 subjects and 2 placebo subjects per dose level. These cohorts were then replicated using subjects of Japanese ancestry to provide data to support the conduct of clinical studies in Japan. A total of 64 subjects were enrolled at one site in the United States (US). The follow-up period varied by cohort from 28 days post-dose to 90 days post-dose.

Subjects received a single injection of MEDI5884 (or placebo) for the 30 mg and 100 mg doses, three SC injections (100 mg each) for the 300 mg dose, or three SC injections (100 mg each) for the 600 mg dose. Six SC injections (100 mg each) were administered.

Subjects The median (SD) age of enrolled subjects was 35.9 (9) years, 93.9% were male, 53.1% were Asian, and 28.1% were Caucasian. , 14.1% were Black or African American, and 4.7% reported multiple ethnicities, and 90.6% were of non-Hispanic ethnicity.

Pharmacokinetics After single SC administration of MEDI5884 at doses of 30 mg, 100 mg, 300 mg, and 600 mg, MEDI5884 exhibited nonlinear PK, likely due to target-mediated drug elimination, with Cmax and AUC above dose proportionality observed. Ta. The PK parameters are summarized in Table 7.

The average PK profiles observed between the Japanese American population and the general US population (Western cohort) were nearly overlapping. Higher exposure was observed in Japanese American subjects at the 300 mg dose, possibly due to differences in body weight between the Japanese American population and the general US population. A moderate effect of body weight on PK profiles was observed in both Western and Japanese American subjects. The CL/F for the 600 mg dose was 0.378 L/day and 0.254 L/day in the general US population (Western cohort) and Japanese American population. The data suggest that there are no substantial ethnic differences in PK of MEDI5884.

Safety and Immunogenicity Safety data were presented in 4 dose cohorts of 12 subjects each [30 mg, 100 mg, 300 mg, or 600 mg]; 24 were of Japanese ancestry. Sixty-four subjects were evaluated, including those who received placebo (four dose cohorts of four subjects each; eight were of Japanese ancestry). There were no related treatment-emergent adverse events (TEAEs), treatment-emergent serious adverse events (TESAEs), or deaths leading to withdrawal from the study. TEAEs occurred at a similar rate in subjects receiving MEDI5884 (16/48, 33.3%) or placebo (5/16, 31.3%).

Although no subjects in the general US population had positive ADA results, positive anti-drug antibody (ADA) responses were detected in 6 of 32 Japanese American subjects, including 1 placebo recipient. . No AEs were reported to be associated with ADA. Lower exposure was observed in some ADA-positive Japanese American subjects compared to other Japanese American subjects without ADA. Exposures in these ADA-positive subjects were within the range of exposures of subjects at the same dose in the pooled population, and no effects on PD or safety were observed. In summary, ADAs were rare, were not associated with adverse events (AEs) or effects on PD, and had no clinically relevant effects on PK.

Pharmacodynamics Overall baseline lipid levels were similar between placebo and MEDI5884-treated subjects.

In these healthy subjects not receiving statin therapy, a significant increase in HDL-C was observed after MEDI5884 administration. The mean (SD) percent change from baseline in HDL-C at day 28 was 15.9% (16.8 ), 4.1% (17.4), 42.0% (26.9), 39.7% (22.5), and 49.8% (17.3).

An increase in apoA1 was also observed. Small increases in LDL-C and apoB were observed in the pooled population. These increases did not appear to be dose-related and occurred primarily late after administration. No clear effect on triglyceride levels was observed.

6.4 Example 4: Phase 2a Clinical Evaluation of MEDI5884 MEDI5884 was also evaluated in a Phase 2a, randomized, double-blind, placebo-controlled, parallel design study in patients receiving concomitant high-intensity statin therapy. We evaluated the safety, PK, PD, and immunogenicity of MEDI5884 in subjects with stable coronary heart disease (CHD) and triglyceride levels below 500 mg/dL and LDL-C below 100 mg/dL. evaluated.

A total of 132 subjects received 3 monthly SC doses of placebo or MEDI5884 at doses of 50 mg, 100 mg, 200 mg, 350 mg, and 500 mg.

Subjects This study enrolled primarily Caucasian (90.8%) male (87.0%) subjects, with a median age of 67 years at enrollment. Overall baseline characteristics were similar between placebo and MEDI5884-treated subjects. Subjects who received a total of three doses of study drug were: 22 of 23 (95.7%) in the placebo group, and MEDI5584 50 mg group, MEDI5584 100 mg group, MEDI5584 200 mg group , MEDI5584 350mg group, and MEDI5584 500mg group, 18 out of 20 (90%), 22 out of 24 (91.7%), 20 out of 22 (90.9%), and 21 out of 21, respectively. 20 people (95.2%), and 21 out of 22 people (95.5%). One subject in the MEDI5884 50 mg group was included as completing treatment even though he had only received two doses because he missed the dose 2 visit.

Pharmacokinetics An interim PK analysis was performed based on data up to day 111. The average MEDI5884 concentration-time profile after three monthly SC doses of MEDI5884 is shown in Figure 1A for each dose cohort. PK parameters based on non-compartmental analysis are summarized in Table 8.

Although MEDI5884 exhibited non-linear PK, likely due to target-mediated drug elimination, PK in the linear range was observed for MEDI5884 doses of 350 mg and 500 mg after 30 days post-dose. Cmax and AUC were nearly dose proportional. Large inter-subject variability and small drug accumulation were observed. The average estimated CL/F at 500 mg was 0.389 L/day.

Safety and Immunogenicity There were no deaths or related TESAEs, and AEs occurred in the MEDI5884 treatment group (59 of 109 [54.1%]) and the placebo group (17 of 23 [73.9%]). The results were approximately balanced between MEDI5884 doses, were independent of the dose of MEDI5884, and were representative of events expected to occur in the enrolled population. Self-reported injection site reactions occurred in 15 of 109 (14%) of MEDI5884-treated subjects and 3 of 23 (13%) of placebo-treated subjects. Investigator-reported injection site reactions occurred in 9 of 109 (8%) of MEDI5884-treated subjects and 3 of 23 (13%) of placebo-treated subjects. These reactions were mild to moderate in severity. Eight subjects discontinued treatment (one placebo recipient and one MEDI5884 recipient withdrew consent for treatment, and six others withdrew for protocol-mandated laboratory observations [ Some were recorded as AEs, 4 had elevated apoB, and 2 had elevated triglycerides]).

ADA was rare, had low potency, was not associated with AEs, and had no effect on PK. ADA was similar in placebo and MEDI5884 recipients. Therefore, the observed ADA may represent a false positive.

Pharmacodynamic Effects Overall baseline lipid levels were similar between placebo and MEDI5884-treated subjects.

Target association of MEDI5884, inhibition of EL, was found to be dose-dependent (Fig. 1B). Specifically, the amount of hEL bound by MEDI5884 in human plasma was measured using an immunoassay platform based on Meso Scale Diagnostics (MSD). Briefly, wells on a 96-well plate were coated with MEDI5884 and incubated overnight at 4°C. The next day, wells were washed with PBS containing 0.05% Tween 20 wash buffer and blocked with I-Block buffer (Applied Biosystems) for 1 hour at room temperature. Washed the plate. Recombinant EL protein standards (Origene Technologies) and human plasma samples were then added to the corresponding wells and incubated for 1 hour at room temperature. After washing, biotinylated EL detection antibody (Origene Technologies) was added to the corresponding wells and incubated for 1 hour at room temperature. After washing the plates, streptavidin-sulfo-TAG antibody (MSD) was added to the corresponding wells and incubated for 1 hour at room temperature. After washing, the wells were incubated with reading buffer (MSD). Plates were read using a MESO Sector S 600 plate reader and data were analyzed using the MSD Discovery Workbench software analysis program.

In these patients, a dose-dependent increase in HDL-C from baseline was observed in the MEDI5884 treatment group. Mean (SD) percent change from baseline in HDL-C at day 91 (using last observation carried forward (LOCF) imputation method) for MEDI5884 administered at 50 mg, 100 mg, 200 mg, 350 mg, or 500 mg, respectively. in subjects, 2.88% (14.86), 21.82% (30.66), 34.41% (34.89) versus -3.01% (13.60) for placebo; They were 43.29% (31.09) and 48.31% (25.63). Figure 2 and Tables 9A and 9B show the observed (non-LOCF) percent change from baseline in HDL-C over a 90-day period and the observed (non-LOCF) percent change from baseline in HDL-C at day 91 ( non-LOCF) changes.

A dose-dependent increase from baseline in HDL particle number and HDL particle size (Table 10) was also observed in the MEDI5884 treatment group versus the placebo group.

Dose-dependent increases from baseline in apoA1 (Figure 3 and Tables 11A and 11B) and high-density lipoprotein phospholipids (HDL-PL) (Figure 4 and Table 12) were also observed in the MEDI5884 treatment group versus the placebo group. It was observed that In particular, Figure 3 and Tables 11A and 11B show the observed (non-LOCF) percent change from baseline in ApoA1 over a 90-day period and the observed (non-LOCF) percent change from baseline in ApoA1 at day 91. ) indicates a change. The mean (SD) percent change from baseline in ApoA1 at day 91 (using the LOCF imputation method) was 1 for placebo in subjects receiving MEDI5884 at 50 mg, 100 mg, 200 mg, 350 mg, or 500 mg, respectively. .42% (11.20) vs. 1.32% (14.81), 15.88% (19.66), 24.82% (21.92), 36.26% (27.36) , and 36.85% (18.03).

Dose-dependent increases from baseline in ABCA1-mediated withdrawal and global withdrawal were also observed in the MEDI5884 treatment group versus the placebo group. The effect of MEDI5884 on non-ABCA1 cholesterol withdrawal is shown in Figure 5.

Slight increases in triglycerides, LDL-C, and apoB were also observed in the MEDI5884 treatment group. In the lower dose group, there was no clear dose-dependent relationship in the increases in triglycerides, LDL-C, and apoB in the MEDI5884 treatment group. Changes in ApoB were significant only at the 500 mg dose. The dose-specific ApoB levels observed on day 91 are shown in Table 13.

In three subjects treated with MEDI5884, triglycerides increased near or above 1000 mg/dL. These subjects had additional risk factors for hypertriglyceridemia. No dose-dependent effect of MEDI5884 on triglyceride levels was observed.

MEDI5884 at a dose of 200 mg resulted in a decrease in placebo (-1.6 [-3.0] mg/dL for HDL-C and -2.6 [-1.0 for LDL-C) on day 91. ]mg/dL, and −0.5 [0.0] mg/dL for apoB), 15.2 (11.0) mg/dL in HDL-C, LDL-C (direct) This caused a mean (median) change from baseline of 6.1 (5.0) mg/dL in apoB and 2 (-1.0) mg/dL in apoB.

The estimated parameters of the main PK/PD models are summarized in Table 14.

6.5 Example 5: Phase 2B clinical evaluation of MEDI5884 Analysis of the data discussed above was performed and a monthly MEDI5884 dose of 250 mg SC was selected for further evaluation.

More specifically, close inspection of the data showed that administration of five doses of MEDI5884 resulted in a clear dose-dependent increase in exposure in patients (Figure 1A), which led to dose-dependent target association (i.e. It was found that this resulted in inhibition of EL levels (Fig. 1B). The EL levels of the 200 mg dose were not consistently inhibited over approximately 30 days, whereas the EL levels of the 350 mg dose maintained complete inhibition during the 30 day dosing interval. Considering the monthly dosing interval, the optimal dose is the studied dose of 200 mg and 35
It appeared to be between 0 mg.

Biomarkers such as HDL-C, ApoA1, and HDL-PL increased in a dose-dependent manner after inhibiting EL activity (Figures 2-4). The time course of the biomarkers indicated that the effective dose should be greater than 200 mg. Safety biomarkers within the pathway, such as LDL-C, ApoB, and TG, increased with less clear dose dependence compared to efficacy biomarkers. Since increases in ApoB and TG at the 500 mg dose were identified as a concern, this indicates that doses below the 500 mg dose should be selected as the optimal dose.

Area under the effect curve of HDL-C, ApoA1, HDL-PL, LDL, ApoB, and TG against dose during the period from day 60 to day 90 by applying the multiple comparison procedural modeling (MCP Mod) method. (AUEC) was evaluated. The desired biomarkers of HDL-C levels, ApoA1 levels, and HDL-PL levels reach a maximum with increasing dose (Figures 2-4), thereby achieving a dose that achieves 90% of maximal biomarker levels (ED ₉₀ ) can be estimated. Estimated _ED90s were 205 mg, 270 mg, and 265 mg, respectively (Figure 6). The undesirable biomarkers of LDL, ApoB, and TG did not reach a plateau, but showed the following trends: (1) LDL levels continued to increase within the range of 50 mg to 500 mg; (2) ApoB levels continued to increase within the range of 50 mg to 500 mg; (3) TG values were constant regardless of the dose administered. Therefore, the results from the MCP Mod approach indicate that a monthly dosing of 250 mg is likely to achieve levels of 90% of maximal potency for the desired biomarkers without causing high levels of undesirable biomarkers. It is supported.

A mathematical model was developed to explain the pharmacokinetics (PK) of MEDI5884 and the biomarker profile of HDL and ApoA1 after administration of MEDI5884. The PK model part used a two-compartment PK model with parallel linear and nonlinear elimination pathways, whereas the PD model part for biomarker modeling analyzed each biomarker that resulted in an increase in biomarker levels after administration. A typical indirect response model with inhibition of the elimination pathway was followed (Fig. 7). This model does not include HDL-P, but changes in HDL-P are indirectly addressed by evaluating HDL-C and ApoA1. An increase in HDL-C without an increase in ApoA1 means larger HDL particles, but not a higher number of particles.

When PK data from a wide dose range (50 mg to 500 mg) were simultaneously analyzed, MEDI5884 exhibited non-linear PK with target-mediated drug elimination likely to saturate at low doses. Therefore, for the simulation of the PK profile at 250 mg, which is an interpolation between the PK profile observed after the 200 mg dose and the PK profile observed after the 350 mg dose, we assume that the PK is linear at high doses. is reasonable.

Observed PK/PD data from a phase 2a study of MEDI5884 in subjects with CHD receiving high-intensity statin therapy were successfully characterized by this model. Although large intersubject variability was observed in PK, HDL, and ApoA1 post-dose, there was a clear relationship between MEDI5884 exposure and corresponding increases in HDL-C and ApoA1. The model's estimated 50% inhibitory concentration (IC ₅₀ ) values were used to calculate the IC ₉₀ (ie, 3.03 μg/mL and 3.43 μg/mL for HDL-C and ApoA1, respectively). The estimated median trough concentration after QM at 250 mg is approximately 3.46 μg/mL based on the simulations below, which is close to the target exposure of IC ₉₀ for HDL-C and ApoA1. MEDI5884 at a monthly dose of 250mg
Figure 8 shows the estimated time course of MEDI5884, HDL, and ApoA1 after administration.

Collectively, these data suggest that the 250 mg monthly dose of MEDI5884 exhibits: (i) linear PK and target association (EL suppression) over 30 days post-dose; (ii) PK /median trough levels above IC90 for maximal HDL-C and ApoA1 increases based on PD modeling and (iii) minimal undesired LDL-C and apoB increases based on a multiple comparison procedural modeling approach.

Therefore, these data support the evaluation of MEDI5884 in a phase 2b, randomized, double-blind, placebo-controlled study in adults with a history of myocardial infarction (MI) receiving high-intensity statin therapy. It is. In these studies, MEDI5884 (250mg) or placebo administered at a dose of 250mg once a month for 24 months showed that MEDI5884 reduced rates of cardiovascular death, MI, stroke, and coronary revascularization. It can be demonstrated that

6.6 Example 6: Inhibition of EL and PCSK9 in Cynomolgus Monkeys A combination pharmacology study was conducted to assess the effect of inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) on lipoprotein metabolism following MEDI5884 treatment. The study protocol is shown in Figure 9. To better mimic a hypothetical patient population already on LDL-C lowering drugs, healthy cynomolgus monkeys were first treated with a PCSK9-neutralizing monoclonal antibody (mAb) (10 mg/kg, n=8) or vehicle. A low LDL-C baseline was established with weekly subcutaneous injections starting on day 0 and treated for 4 weeks. The PCSK9 monoclonal antibody used was HS9 (containing the VH and VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15, respectively). The HS9 antibody (in the context of a GLP-1 fusion protein called MEDI4166) is described in Chodorge et al., Sci. Rep. 8: 17545 (2018) and PCT International Publication No. WO 2015/127273 (respectively cited (herein incorporated by reference in its entirety).

Four animals from each group then received subcutaneous doses of MEDI5884 (10 mg/kg, n=4) or vehicle (n=4) on days 28 (vertical dashed lines in Figures 10 and 11) and 42. ) was administered. LDL-C, HDL-C, ApoB, and ApoA1 were measured in plasma samples collected at the indicated time points. Overall pullout and ABCA1 pullout were also evaluated.

An increase in LDL-C was observed after EL neutralization. Compared to vehicle-treated animals, PCSK9 inhibition reduced LDL-C by 75% (Fig. 10, left graph) and maintained that level of reduction during the 4-week run-in phase, but showed no significant effect on HDL-C. It had no significant effect (Fig. 10, right graph). In vehicle-treated animals during the run-in phase, MEDI5884 caused an increase in both HDL-C and LDL-C. When added in addition to the PCSK9 inhibitor during the second 4-week period, MEDI5884 maintained the ability to raise HDL-C to a similar extent, but the magnitude of the LDL-C increase was significantly blunted. , indicating that LDL particles continued to be taken up by LDL receptors (Figure 10). The patterns for LDL-C and HDL-C were comparable to the respective changes in ApoB and ApoA1 (Figure 11). The effect on overall withdrawal and ABCA1 withdrawal is shown in FIG. In particular, the observed increase in withdrawal associated with administration of MEDI5884 (including administration of MEDI5884+HS9) generally reflects the observed increase in HDL-C.

These results provide evidence of cholesterol uptake by LDL receptors and indicate that the increase in LDL-C observed with MEDI5884 treatment in monkeys may be alleviated by a mechanism that upregulates LDL receptors. These results further demonstrate that MEDI5884 can be administered in combination with PCSK9 inhibitors. This combination therapy can exploit the combined action of two complementary mechanisms that both target different aspects of reverse cholesterol transport.

6.7 Example 7: Effect of increasing doses of MEDI 5884 on plasma phosphatidylinositol (PI) levels in subjects with stable coronary heart disease Plasma phosphatidylinositol (PI) levels in patients with stable coronary heart disease We quantified the effect of MEDI5884 on. A high-throughput multiplex method combining hydrophilic interaction chromatography (HILIC) separation with multiple reaction monitoring (MRM) in negative mode was used for quantification. A total of 31 endogenous PI species were monitored.

Reagents All lipid standards were purchased from Avanti Polar Lipids (Alabaster, AL). Three PI standards were used in this study: PI (12:0/13:0) was used as internal standard (IS), PI (17:0/14:1) and PI (21:0/22 :6) was used as a surrogate analyte. High performance liquid chromatography (HPLC) grade water was purchased from Honeywell (Charlotte, NC). HPLC grade isopropanol (IPA), acetonitrile (ACN), and ammonia were all purchased from Sigma-Aldrich (St. Louis, MO). Ammonium acetate and bovine serum albumin (BSA) were purchased from MilliporeSigma (Burlington, MA). PBS was purchased from Lonza BioWhittaker (Morristown, NJ). Special glass-coated 96-well extraction plates and glass vials with PTFE (polytetrafluoroethylene) linings were obtained from Thermo-Fisher (Waltham, MA). Human plasma (pooled and individual plasma) was purchased from BioIVT (Westbury, NY).

Extraction Procedure for Plasma Samples Internal standard (IS) was spiked into isopropanol (IPA) at 60 nM (final concentration) to make an IS-IPA solution. One pooled lot of human plasma was used as quality control (QC), with low QC (LQC) (8-fold diluted in 40 mg/mL bovine serum albumin (BSA) in PBS) and high QC (HQC) ( Two levels of undiluted plasma) were prepared. All test samples were diluted 8 times with 40 mg/mL BSA. Eight-fold dilutions of both HQC and test samples were prepared using an automated liquid handling platform, an Agilent Bravo automated liquid handling system (Santa Clara, CA) equipped with a Series III 96 LT disposable tip head. According to the pre-specified plate map, 20 μL of LQC, HQC, or test samples were transferred to another extraction plate and then precipitated with 180 μL of IS-IPA using the automated device described above. Samples were shaken vigorously (900 rpm to 1200 rpm) for approximately 10 minutes on an IKA MTS 2/4 digital microtiter shaker (Wilmington, NC).

The samples were then centrifuged at 2500g for 5 minutes. Using automated liquid handling again, 20 μL of plasma extracted with IS-IPA was combined with 140 μL of reconstitution solution (90.25% (vol/vol) acetonitrile (ACN), 9.75% (vol/vol) water, 10 mM ammonium acetate). The samples were then shaken at 600 rpm for 5 minutes.

Hydrophilic interaction chromatography separation Chromatographic separation was performed using an Acquity ultra-performance liquid chromatography (UPLC) BEH (ethylene bridged hybrid) hydrophilic interaction chromatography (HILIC) column (130 Å, 1.7 μm, 2.1 mm x 100 mm). , Waters, Milford, MA) on a Nexera X2 UHPLC system (Shimadzu Corporation, Kyoto, Kyoto, Japan). The mobile phases used were mobile phase A (MPA) (5% water, 95% ACN, vol/vol) and mobile phase B (MPB), both containing 10 mM ammonium acetate (pH 8.0-8.5). ) (50% water, 50% ACN, vol/vol). For each sample or QC, 10 μL of reconstituted IPA extract was injected onto the column. Separation was performed at 37° C. with a flow rate of 0.5 mL/min. The separation gradient was 5%→13% MPA over 4 minutes, followed by a 2 minute wash period and a 4 minute equilibration period.

Mass spectrometry Mass spectrometry detection was performed using a 6500+ quadrupole ion trap (QTRAP) mass spectrometer (Sciex, Framingham, MA) operating in negative electrospray ionization (ESI) multiple reaction monitoring (MRM) mode. achieved. The synthetic reference standard PI species listed above were used to adjust MS conditions and define retention times. The structurally characteristic MRM transition with the highest signal intensity in negative ESI mode, which allows identification of the two acyl chains, was selected during the preparation of the synthetic reference standards (surrogate analyte and IS). Structurally similar MRMs allowing identification of the two acyl chains of each endogenous PI species were then predicted using LipidView (Sciex, Redwood Shores, CA) and are shown in Table 15. The same source parameters were used for all PI species after adjusting the synthetic reference standards. Details about the acquisition method can be found in Table 16.

Data Collection, Analysis, and Reporting After acquisition, samples from each acquisition batch were analyzed in MultiQuant software according to the defined quantification method (.qmethod). Details of the quantification method can be found in Table 17 for components and outlier settings. Integration and regression settings were optimized for each individual batch based on the peaks of interest generated. However, the same integration and regression parameters were applied to all samples within the same batch. The peak areas of internal standards and endogenous PI species were calculated for QCs within batches and unknown samples. Peak area ratios of endogenous PI species were calculated based on peak area integrals in MultiQuant. Next, save the MultiQuant quantification result file (.qsession). txt file and further using Excel (Microsoft Office 2016) and Spotfire (TIBCO(TM) Spotfire(TM) Analyst 7.9.2 HF-011 Build Version 7.9.2.0.12) Data was analyzed. To assess the linearity and measurement accuracy of the instrument response for each endogenous PI species, the following were evaluated: the ratio of HQC/LQC, the % difference in the HQC/LQC ratio from the nominal value, and the %CV. Most species had %CV values below 30% for HQC and LQC values, and the % difference in HQC/LQC ratio from the nominal value was within 70% to 130%.

Results A total of 978 plasma samples from subjects with stable coronary heart disease were tested as described above. Samples were analyzed in a total of 15 assays. All 15 assays met acceptance criteria for all PI species, except for PI (16:0/16:0), which had consistently unacceptable variability and HQC/LQC ratios. No assays were considered invalid.

Clinical samples were also obtained and analyzed from healthy volunteers administered MEDI5884. Some variation between batches was observed. To correct for batch-to-batch variability and explore the potential for data bridging between clinical studies on CAD patients and healthy volunteers, the SERRF normalization algorithm (Fan S., et al. Anal Chem Mar 5;91 5:3590-6 (2019)) and found that it outperformed other normalization methods evaluated on this dataset. The results shown in FIGS. 13A-13C, 14A-14C, 15, and 16 show that PI levels increased across PI species in healthy subjects compared to MEDI5884 untreated CAD patients. It has been proven that this was significantly higher. This difference was determined to be statistically significant by a two-tailed unequal variance t-test (p<0.0005 for 12 PI species). In the placebo arm of both clinical studies, PI levels remained relatively stable over a period of several months, with lower levels in CAD patients versus healthy volunteers across the 12 PI species tested. .

Levels of various PI species over time versus visit date in healthy patients and patients with CAD are shown in FIGS. 13A-13C and 14A-14C. A comparison of median PI species levels in healthy volunteers and CAD patients on day 21 is shown in FIG. 15, and a comparison of median PI species levels across all days is shown in FIG. 16. As shown in FIGS. 13A-13C, 14A-14C, 15, and 16, most plasma PI species were dose-dependent relative to placebo after three monthly subcutaneous (SC) administrations of MEDI5884. increased. The duration of increase in plasma PI appeared to be correlated with MEDI5884 exposure. For most PI species, increases in PI levels reached saturation at the 350 mg dose level of MEDI5884, and no further increase in PI over baseline occurred at the 500 mg dose level of MEDI5884. However, at the 200 mg dose level of MEDI5884, at day 91, the increase in PI approached the saturation level observed in the higher dose cohort. The percent change from baseline for each PI species ranges from approximately 1000% for less abundant species to approximately 100% for more abundant species for both CAD and healthy volunteer subjects. 17A-17E, FIG. 18, and FIG. 19A-19E; Table 18, which summarizes data obtained across all PI species at the indicated doses and visit dates, and Tables 19A to 19D showing the data obtained for the individual PI species). The mean change across PI species for CAD patients reached a maximum increase of approximately 250%-300% for doses of 200 mg and above.

The invention is not limited in scope by the particular embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing detailed description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

All references (e.g., publications, or patents, or patent applications) cited in this specification are cited as if each individual reference (e.g., publication, or patent, or patent application) were specific and individually cited. The entire contents of the specification are hereby incorporated by reference for all purposes to the same extent as if by reference they were incorporated by reference into the specification in their entirety for all purposes. form part of

Other embodiments are within the scope of the following claims.

Claims (22)

A composition for treating cardiovascular disease or reducing atherosclerosis in a subject, the composition comprising:
The composition comprises VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and VH CDR1 comprising the amino acid sequence of SEQ ID NO: 4. an antibody or antigen-binding fragment thereof comprising a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6;
The composition includes:
(a) increasing high density lipoprotein cholesterol (HDL-C) in the subject;
(b) increasing the number of high density lipoprotein (HDL) particles in the subject;
(c) increasing HDL particle size in said subject;
(d) increasing HDL phospholipids in the subject;
(e) increasing apolipoprotein A1 (ApoA1) in the subject ;
(f) increasing cholesterol extraction capacity (CEC) in said subject ; and/or
(g) increasing plasma phosphatidylinositol (PI) in said subject;
composition . The composition may reduce cardiovascular death, non-fatal myocardial infarction (MI), non-fatal stroke, peripheral artery disease, atherosclerosis, and/or coronary artery disease in subjects with a history of acute coronary syndrome (ACS). 2. The composition of claim 1 , which reduces the risk of arterial revascularization. 3. The composition of claim 1 or 2, wherein the composition prevents secondary cardiovascular events in the subject. 4. The composition of any one of claims 1-3 , wherein the composition reduces the risk of major adverse cardiovascular events (MACE) in the subject. A composition according to any one of claims 1 to 4 , wherein the composition is administered once a month. A method according to any one of claims 1 to 5 , wherein the composition is administered parenterally. A method according to any of claims 1 to 6 , wherein the composition is administered subcutaneously. 8. A method according to any one of claims 1 to 7 , wherein the composition is administered via an attached prefilled syringe (APFS) or an autoinjector. 9. A composition according to any preceding claim, wherein the composition increases HDL-C in the subject by at least 30%. A composition according to any one of claims 1 to 9 , wherein the composition increases ApoA1 in the subject by at least 30%. 11. A composition according to any preceding claim, wherein the composition increases the number of HDL particles in the subject by at least 5%. 12. A composition according to any preceding claim, wherein the composition increases HDL particle size in the subject by at least 3%. 13. The composition of any one of claims 1-12 , wherein the composition increases HDL phospholipids in the subject by at least 50%. 14. The composition of any one of claims 1-13, wherein administration of the composition increases non-ABCA1 cholesterol abstraction capacity in the subject by at least 35%. The composition increases plasma phosphatidylinositol (PI) levels in the subject, and the increase in plasma PI levels increases PI(14:2/20:0) levels, PI(14:2/22:0) levels. , PI (14:2/22:1) level, PI (14:2/22:2) level, PI (16:0/16:1) level, PI (16:0/18:0) level, PI (16:0/18:2) level, PI (16:0/20:2) level, PI (16:0/20:3) level, PI (16:0/20:4) level, PI (16 :0/22:4) level, PI (16:1/18:0) level, PI (16:1/18:1) level, PI (18:0/18:0) level, PI (18:0) level /18:1) level, PI (18:0/18:2) level, PI (18:0/18:3) level, PI (18:0/20:2) level, PI (18:0/20) :3) Level, PI (18:0/20:4) level, PI (18:0/22:4) level, PI (18:0/22:5) level, PI (18:0/22:6) ) level, PI (18:1/16:0) level, PI (18:1/18:1) level, PI (18:1/18:2) level, PI (18:1/20:2) level , an increase in PI (18:1/20:3) level, PI (18:1/20:4) level, and/ or PI (18:2/18:2) level. Composition according to any one of the above . 16. The cardiovascular disease of claims 1 to 15 , wherein the cardiovascular disease is selected from coronary artery disease, coronary heart disease, chronic arterial disease, cerebrovascular disease, atherosclerotic cardiovascular disease, and peripheral artery disease. Composition according to any one of the above . A composition according to any one of claims 1 to 16 for treating peripheral artery disease. 18. The composition of any one of claims 1-17 , wherein the subject is receiving statin therapy. 19. The composition of any one of claims 1-18, wherein the subject has a triglyceride level of 500 mg/dL or less before the administration. 1 . The antibody or antigen-binding fragment thereof binds to the same endothelial lipase epitope as the antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and/or a VL comprising the amino acid sequence set forth in SEQ ID NO: 8. The composition according to any one of - 19 . 21. A composition according to any one of claims 1 to 20 , further characterized in that the composition and the inhibitor of PCSK9 are administered sequentially, either simultaneously or sequentially . 22. The composition of claim 21 , wherein the inhibitor of PCSK9 is an anti-PCSK9 antibody or antigen-binding fragment thereof, and the anti- PCSK9 antibody or antigen-binding fragment thereof is selected from HS9, evolocumab, alirocumab, and bococizumab. Things .

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