BIOMARKER ASSAY TO MEASURE LIPOPROTEIN(a)

Abstract

The present disclosure relates to a method for detecting or measuring Lp(a), a risk factor for cardiovascular disease, in a human sample, as well as Apo(a) antibodies for use in said methods

Description

Ref. No.30972_WO BIOMARKER ASSAY TO MEASURE LIPOPROTEIN(a) FIELD OF THE INVENTION

[0001] The present disclosure is in the field of diagnostic medicine. More particularly the present disclosure relates to compounds, methods, and diagnostics, which include apo(a) antibodies and biomarker assays to measure lipoprotein (a) which is a risk factor for cardiovascular disease. REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30972A_US_PRI Sequence Listing ST26.xml” created March 19, 2025, and is 79.9 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety. No new matter is added herewith. BACKGROUND OF THE INVENTION

[0003] Lipoprotein(a), Lp(a), is a peculiar lipoprotein composed of apolipoprotein(a) [apo(a)] covalently bound to a low-density lipoprotein (LDL)-like particle containing one molecule of apolipoprotein B-100 (Boffa et al. Curr Opin Lipidol.2022;33(3):185-192). Lp(a) is a genetically determined, independent cardiovascular risk factor. Elevated serum Lp(a) levels greater than 50 mg / dL or 125 nmol / L found in ~20% of the population confers at least a 1.6-fold risk of a first cardiovascular event (Kronenberg, F. Clin. Res. Cardiol. Suppl.14, 5-12 (2019)) and a >1.42-fold increase risk of a second event (Madsen, C. M. et al. Arterioscler. Thromb. Vasc. Biol.40, 255-266 (2020)). Lp(a) may exhibit both prothrombotic and antithrombotic properties, and atherogenic and atherothrombotic properties. Lp(a) may inhibit fibrinolysis and accumulate in the vascular wall inducing thrombogenesis and atherosclerotic lesions. Plasma levels of Lp(a) vary substantially among individuals. Unlike the other risk factors, Lp(a) plasma levels do not vary significantly with diet and exercise.

[0004] Lp(a) has been pursued as a therapeutic target to reduce the risks of cardiovascular events (Reyes-Soffer et al. Arterioscler Thromb Vasc Biol.2022;42(1): e48-e60; Nicholls et al. JAMA.2023;330(11):1042-1053). In order to aide in the discovery and / or development of a disease modifying treatment to decrease levels of Lp(a), reliable and sensitive diagnostics for Lp(a) are needed.Ref. No.30972_WO

[0005] There are several approved diagnostic applications in use for Lp(a), e.g., Randox Lp(a) Assay (Randox Laboratories Ltd.). However, one limitation of the commercially available Lp(a) assays is that they only use apo(a) antibodies and so they detect the presence of apo(a), whether present as part of the Lp(a) molecule or in “unbound” form. This may lead to an overestimation of the Lp(a) levels. Marcovina, et al., J. Lipid Res. (2022) 63(8) 100239, disclose a particular assay in which Lp(a) is captured with monoclonal antibody LPA4, which is primarily directed to an epitope in apolipoprotein(a) KIV2, and it is detected with monoclonal antibody LPA-KIV9, directed to a single antigenic site present on KIV9. Both of these antibodies target apo(a). There are Lp(a) assays disclosed in Marcovina, et al., Clin. Chem. (1995) 41 / 2246-255 and Tsimikas et al., Circulation, (2004) 1093164-3170 which employ both an apo(a) antibody and an Apo(B), however, in these assays the apo(a) antibody used is sensitive to the different isoforms of apo(a) and this can result in inaccurate Lp(a) measurements.

[0006] Apo(a) shares high sequence homology with several regions of plasminogen, including the protease domain, and the so-called kringle IV (KIV) and V domains. The KIV domain reveals the complexity of apo(a) and is formed by 10 distinct KIV types numbered from 1 to 10. All KIV types, except KIV type 2 (KIV-2), are present as a single copy, while the KIV-2 repeats vary from 3 to >40 copies, resulting in a large heterogeneity in apo(a) isoform size circulating in plasma. As a result, apo(a) concentration is generally inversely correlated with apo(a) size and varies widely between individuals.

[0007] The heterogeneity of the apo(a) isoforms poses a key challenge in measuring apo(a), which results in the underestimation or overestimation of Lp(a) concentrations. Unless calibrants in tested samples have the same range of isoforms as test samples, those with higher numbers of the KIV-2 domain unit repeats will represent with an overestimation in Lp(a) concentrations, while those with smaller numbers of the KIV-2 domain unit repeats, will represent with an underestimation.

[0008] There remains a need for apo(a) antibodies which bind to KIV domains, other than KIV- 2, and can be used in Lp(a) diagnostic assays that are both sensitive and reliable. Furthermore, there is a need for an Lp(a) diagnostic assay applicable for testing blood, plasma, and serum, which overcomes one or more limitations of the currently available assays. In particular, there is a need for an Lp(a) diagnostic assay which is sensitive, reliable, accurately measures Lp(a) levels irrespective of the isoform of apo(a) present in the Lp(a), eliminates variability between individuals, and / or only measures apo(a) when bound to ApoB in the Lp(a) molecule. MoreRef. No.30972_WO generally, there is a need for a Lp(a) diagnostic assay which can identify and / or differentiate between patients in need of further diagnostic evaluation and / or therapeutic treatment. SUMMARY OF THE DISCLOSURE

[0009] The present disclosure provides an antibody which specifically binds apo(a).

[0010] In particular, the present disclosure provides an antibody which specifically binds apo(a) and comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein the LCDR1 comprises SEQ ID NO:4, the LCDR2 comprises SEQ ID NO:5, the LCDR3 comprises SEQ ID NO:6, the HCDR1 comprises SEQ ID NO:10, the HCDR2 comprises SEQ ID NO:11, and the HCDR3 comprises SEQ ID NO:12.

[0011] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:7 and a VH comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:13.

[0012] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence of SEQ ID NO:7 and a VH comprising an amino acid sequence of SEQ ID NO:13.

[0013] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:8 and the HC comprises an amino acid sequence of SEQ ID NO:14.

[0014] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein the LCDR1 comprises SEQ ID NO:27, the LCDR2 comprises SEQ ID NO:28, the LCDR3 comprises SEQ ID NO:29, the HCDR1 comprises SEQ ID NO:32, the HCDR2 comprises SEQ ID NO:33, and the HCDR3 comprises SEQ ID NO:34.

[0015] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQRef. No.30972_WO ID NO:30 and a VH comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:35.

[0016] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence of SEQ ID NO:30 and a VH comprising an amino acid sequence of SEQ ID NO:35.

[0017] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:31 and the HC comprises an amino acid sequence of SEQ ID NO:36.

[0018] In some embodiments, the antibody specifically binds apo(a), comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein the LCDR1 comprises SEQ ID NO:37, the LCDR2 comprises SEQ ID NO:38, the LCDR3 comprises SEQ ID NO:39, the HCDR1 comprises SEQ ID NO:42, the HCDR2 comprises SEQ ID NO:43, and the HCDR3 comprises SEQ ID NO:44.

[0019] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:40 and a VH comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:45.

[0020] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence of SEQ ID NO:40 and a VH comprising an amino acid sequence of SEQ ID NO:45.

[0021] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:41 and the HC comprises an amino acid sequence of SEQ ID NO:46.

[0022] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein the LCDR1 comprises SEQ ID NO:47, the LCDR2 comprises SEQ ID NO:48, the LCDR3 comprises SEQ ID NO:49, the HCDR1 comprises SEQ ID NO:52, the HCDR2 comprises SEQ ID NO:53, and the HCDR3 comprises SEQ ID NO:54.Ref. No.30972_WO

[0023] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:50 and a VH comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:55.

[0024] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence of SEQ ID NO:50 and a VH comprising an amino acid sequence of SEQ ID NO:55.

[0025] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:51 and the HC comprises an amino acid sequence of SEQ ID NO:56.

[0026] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein the LCDR1 comprises SEQ ID NO:57, the LCDR2 comprises SEQ ID NO:58, the LCDR3 comprises SEQ ID NO:59, the HCDR1 comprises SEQ ID NO:62, the HCDR2 comprises SEQ ID NO:63, and the HCDR3 comprises SEQ ID NO:64.

[0027] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:60 and a VH comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:65.

[0028] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence of SEQ ID NO:60 and a VH comprising an amino acid sequence of SEQ ID NO:65.

[0029] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:61 and the HC comprises an amino acid sequence of SEQ ID NO:66.

[0030] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein the LCDR1 comprises SEQ ID NO:67, the LCDR2 comprises SEQ ID NO:68,Ref. No.30972_WO the LCDR3 comprises SEQ ID NO:69, the HCDR1 comprises SEQ ID NO:72, the HCDR2 comprises SEQ ID NO:73, and the HCDR3 comprises SEQ ID NO:74.

[0031] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:70 and a VH comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:75.

[0032] In some embodiments, the antibody which specifically binds apo(a), comprises a VL comprising an amino acid sequence of SEQ ID NO:70 and a VH comprising an amino acid sequence of SEQ ID NO:75.

[0033] In some embodiments, the antibody which specifically binds apo(a), comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:71 and the HC comprises an amino acid sequence of SEQ ID NO:76.

[0034] In the various aspects and embodiments, the present disclosure provides methods for detecting or measuring Lp(a) in a human sample.

[0035] In particular, the present disclosure provides a method for detecting or measuring Lp(a) in a human sample comprising the steps of: a. contacting the human sample with a capture antibody that is an apo(a) antibody that binds to the Kringle IV type 7 (KIV-7) and Kringle IV type 8 (KIV-8) domains of human apo(a), wherein the human apo(a) is covalently bound to human ApoB forming a capture antibody-apo(a)-ApoB protein complex, b. detecting the capture antibody-apo(a)-ApoB protein complex with a detection antibody that is an ApoB antibody that binds to human ApoB.

[0036] In some embodiments, the detecting step is an indication of the presence, amount, or both of Lp(a) in the human sample.

[0037] In other embodiments, the disclosure provides a kit comprising the antibody which specifically binds apo(a) described herein, the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, and a package insert containing instructions for use of the kit and / or any component thereof, comprising instructions for testing a human sample, wherein the human sample is from a patient with a disease, disorder, or condition associated with Lp(a) expression.

[0038] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a)Ref. No.30972_WO in a human sample, is used in combination with the administration of a treatment to reduce Lp(a) levels in a patient.

[0039] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is used in association with the administration of a treatment to reduce Lp(a) levels in a patient.

[0040] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is used to determine if a patient is eligible to receive treatment to reduce Lp(a) levels.

[0041] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is used to determine if the treatment is working properly to reduce Lp(a) levels in a patient.

[0042] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is administered prior to a patient receiving treatment, during a patient receiving treatment, or after a patient is receiving treatment.

[0043] In some embodiments, the treatment to reduce Lp(a) levels in a patient is selected from the group consisting of Pelacarsen, Olpasiran, Lepodisiran, Zerlasiran, Muvalaplin, HRS-5346, or any other compound that can reduce the amount of Lp(a).

[0044] In some embodiments, the disease, disorder, or condition associated with Lp(a) expression is a cardiometabolic disease, optionally atherosclerosis, NAFLD, NASH, Calcific aortic valve stenosis (CAVS), cardiovascular events including coronary revascularization, CV mortality, cerebrovascular disease, chronic kidney disease, coronary heart disease, dyslipidemia, heart failure, MI and stroke, and / or peripheral vascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 illustrates the Lp (a) structure.

[0046] Figure 2 illustrates the Dual-epitope biomarker assay to measure Lp(a). The abbreviations are as follows: Bt- = biotinylated; Ru- = ruthenium-labeled; MSD = MesoScale Discovery; apo (a) = apolipoprotein (a); apoB = apolipoprotein B; Lp (a) = lipoprotein (a).Ref. No.30972_WO

[0047] Figure 3 illustrates standard curves with plasminogen demonstrating the specificity of the Lp(a) biomarker assay.

[0048] Figure 4 illustrates an assay response to an apo(a) protein spike. DETAILED DESCRIPTION OF THE DISCLOSURE

[0049] The present disclosure provides apo(a) antibodies which specifically binds to the KIV-7 and KIV-8 domains of human apo(a). In an embodiment, the present disclosure provides apo(a) antibodies which specifically bind to the KIV-7 and KIV-8 domains, without binding to KIV-2 domain (SEQ ID NO:3), as well as methods and diagnostic assays using such antibodies. In more specific embodiments, the apo(a) antibodies disclosed herein bind to an epitope region present in the KIV-7-10 domains of hApo(a) (designated as SEQ ID NO:2).

[0050] In further embodiments, the apo(a) antibodies of the present disclosure comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence ofRASQDIGNSLT (SEQ ID NO:4), LCDR2 has the amino acid sequence ofYATSSLDS (SEQ ID NO:5), LCDR3 has the amino acid sequence of LQYASYPFT (SEQ ID NO:6), HCDR1 has the amino acid sequence ofKASGYTFTDYNMD (SEQ ID NO:10), HCDR2 has the amino acid sequence ofNINPNNGGTIYNQRFRG (SEQ ID NO:11), and HCDR3 has the amino acid sequence ofARPPYYDKGFDV (SEQ ID NO:12).

[0051] In various embodiments, the apo(a) antibodies provided by the present disclosure comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 7 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 13.

[0052] In some embodiments, the apo(a) antibodies provided by the disclosure comprise an VL amino acid sequence of SEQ ID NO: 7 and an VH amino acid sequence of SEQ ID NO: 13.

[0053] In some embodiments, the apo(a) antibodies comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence of RASKSISKYLA (SEQ ID NO:27), LCDR2 has the amino acid sequence of SGSTLQS (SEQ ID NO:28), LCDR3 has the amino acid sequence ofQHHYEYPLT (SEQ ID NO:29), HCDR1 has the amino acid sequence ofGYTFTDY (SEQ ID NO:32), HCDR2 has the amino acid sequence of DPETGG (SEQ ID NO:33), and HCDR3 has the amino acid sequence of RAYYSTYGYFDY (SEQ ID NO:34).Ref. No.30972_WO

[0054] In various embodiments, the apo(a) antibodies provided by the present disclosure comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:30 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:35.

[0055] In some embodiments, the apo(a) antibodies provided by the disclosure comprise an VL amino acid sequence of SEQ ID NO:30 and an VH amino acid sequence of SEQ ID NO:35.

[0056] In some embodiments, the apo(a) antibodies comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence ofRASGNIHNYLA (SEQ ID NO:37), LCDR2 has the amino acid sequence of NVKTLAE (SEQ ID NO:38), LCDR3 has the amino acid sequence ofQHHYGTPPT (SEQ ID NO:39), HCDR1 has the amino acid sequence ofGFTFSDY (SEQ ID NO:42), HCDR2 has the amino acid sequence of SSGSST (SEQ ID NO:43), and HCDR3 has the amino acid sequence of PGYYALYFDY (SEQ ID NO:44).

[0057] In various embodiments, the apo(a) antibodies provided by the present disclosure comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:40 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:45.

[0058] In some embodiments, the apo(a) antibodies provided by the disclosure comprise an VL amino acid sequence of SEQ ID NO:40 and an VH amino acid sequence of SEQ ID NO:45.

[0059] In some embodiments, the apo(a) antibodies comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence ofKASQDVGTAVA (SEQ ID NO:47), LCDR2 has the amino acid sequence of WASTRHP (SEQ ID NO:48), LCDR3 has the amino acid sequence ofQQYSTYPLT (SEQ ID NO:49), HCDR1 has the amino acid sequence ofGYTFTDY (SEQ ID NO:52), HCDR2 has the amino acid sequence of NPKNGG (SEQ ID NO:53), and HCDR3 has the amino acid sequence of FDAKGNYYAMDY (SEQ ID NO:54).

[0060] In various embodiments, the apo(a) antibodies provided by the present disclosure comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:50 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:55.Ref. No.30972_WO

[0061] In some embodiments, the apo(a) antibodies provided by the disclosure comprise an VL amino acid sequence of SEQ ID NO:50 and an VH amino acid sequence of SEQ ID NO:55.

[0062] In some embodiments, the apo(a) antibodies comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence ofKSSQSLLDSDGKTYLH (SEQ ID NO:57), LCDR2 has the amino acid sequence of LVSKLAS (SEQ ID NO:58), LCDR3 has the amino acid sequence ofWQGTHFPWT (SEQ ID NO:59), HCDR1 has the amino acid sequence ofGFNIKDD (SEQ ID NO:62), HCDR2 has the amino acid sequence of DPENGY (SEQ ID NO:63), and HCDR3 has the amino acid sequence of TAYDYDY (SEQ ID NO:64).

[0063] In various embodiments, the apo(a) antibodies provided by the present disclosure comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:60 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:65.

[0064] In some embodiments, the apo(a) antibodies provided by the disclosure comprise an VL amino acid sequence of SEQ ID NO:60 and an VH amino acid sequence of SEQ ID NO:65.

[0065] In some embodiments, the apo(a) antibodies comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence ofRASKSISKYLA (SEQ ID NO:67), LCDR2 has the amino acid sequence of SGSTLQS (SEQ ID NO:68), LCDR3 has the amino acid sequence ofQQHNEYPLT (SEQ ID NO:69), HCDR1 has the amino acid sequence ofGYTFTDY (SEQ ID NO:72), HCDR2 has the amino acid sequence of DPETGG (SEQ ID NO:73), and HCDR3 has the amino acid sequence of RAYYSTYGYFDY (SEQ ID NO:74).

[0066] In various embodiments, the apo(a) antibodies provided by the present disclosure comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:70 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:75.

[0067] In some embodiments, the apo(a) antibodies provided by the disclosure comprise an VL amino acid sequence of SEQ ID NO:70 and an VH amino acid sequence of SEQ ID NO:75.

[0068] According to some embodiments, the apo(a) antibodies of the present disclosure may be murine, chimeric, or humanized antibodies. In some embodiments, the apo(a) antibodies of theRef. No.30972_WO present disclosure comprise an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, antibodies of the present disclosure comprise a kappa light chain.

[0069] In some embodiments, the apo(a) antibodies comprise an LC amino acid sequence of SEQ ID NO:8 and an HC amino acid sequence of SEQ ID NO:14.

[0070] In some embodiments, the apo(a) antibodies comprise an LC amino acid sequence of SEQ ID NO:31 and an HC amino acid sequence of SEQ ID NO:36.

[0071] In some embodiments, the apo(a) antibodies comprise an LC amino acid sequence of SEQ ID NO:41 and an HC amino acid sequence of SEQ ID NO:46.

[0072] In some embodiments, the apo(a) antibodies comprise an LC amino acid sequence of SEQ ID NO:51 and an HC amino acid sequence of SEQ ID NO:56.

[0073] In some embodiments, the apo(a) antibodies comprise an LC amino acid sequence of SEQ ID NO:61 and an HC amino acid sequence of SEQ ID NO:66.

[0074] In some embodiments, the apo(a) antibodies comprise an LC amino acid sequence of SEQ ID NO:71 and an HC amino acid sequence of SEQ ID NO:76.

[0075] In various aspects and embodiments, the present disclosure provides a method of detecting or measuring Lp(a) in a human sample. Such methods to detect or measure Lp(a) in a human sample comprise contacting the human sample with a capture antibody, e.g., an apo(a) antibody; and a detection antibody, e.g., an ApoB antibody. In an embodiment, the apo(a) antibody specifically binds to the Kringle IV type 7 (KIV-7) and Kringle IV type 8 (KIV-8) domains of human apo(a). In a further embodiment, the bioassay uses an apo(a) antibody that does not bind to the Kringle IV type 2 (KIV-2) domain repeats, which can vary from 3 to greater than 40 copies in different individuals. In a particular embodiment, such methods comprise the steps of contacting the human sample with an antibody of the present disclosure that specifically binds to the KIV-7 and KIV-8 domains of human apo(a), and a detection antibody with a signal that binds to binds to human ApoB.

[0076] In a particular embodiment, the present disclosure provides a method for detecting Lp(a) in a human sample comprising the steps of: a. contacting the human sample with a capture antibody that is an apo(a) antibody that binds to the KIV-7 and KIV-8 domains of human apo(a), wherein the human apo(a) is covalently bound to human ApoB forming a capture antibody-apo(a)-ApoB protein complex, b. detecting the capture antibody-apo(a)-ApoB protein complex with a detection antibody that is an ApoB antibody that binds to human ApoB.Ref. No.30972_WO

[0077] In an alternate embodiment, the present disclosure provides a method for measuring Lp(a) in a human sample comprising the steps of: a. contacting the human sample with a capture antibody that is an apo(a) antibody that binds to the KIV-7 and KIV-8 domains of human apo(a), wherein the human apo(a) is covalently bound to human ApoB forming a capture antibody-apo(a)-ApoB protein complex, b. detecting the capture antibody-apo(a)-ApoB protein complex with a detection antibody that is an ApoB antibody that binds to human ApoB.

[0078] In a further embodiment, the present disclosure provides a method of identifying individuals with elevated Lp(a) plasma levels comprising: a. contacting a human plasma sample from the individual with a capture antibody that is an apo(a) antibody that binds to the KIV-7 and KIV-8 domains of human apo(a), wherein the human apo(a) is covalently bound to human ApoB forming a capture antibody-apo(a)-ApoB protein complex, b. detecting the capture antibody-apo(a)-ApoB protein complex with a detection antibody that is an ApoB antibody that binds to human ApoB.

[0079] In an embodiment, elevated Lp(a) plasma level refers to a plasma level of Lp(a) that is equal to or above about 50 mg / dL.

[0080] In a further embodiment, the present disclosure provides a method of identifying individuals in need of treatment with Lp(a) lowering treatments comprising: a. contacting a human plasma sample from the individual with a capture antibody that is an apo(a) antibody that binds to the KIV-7 and KIV-8 domains of human apo(a), wherein the human apo(a) is covalently bound to human ApoB forming a capture antibody-apo(a)-ApoB protein complex, b. detecting the capture antibody-apo(a)-ApoB protein complex with a detection antibody that is an ApoB antibody that binds to human ApoB.

[0081] According to some embodiments, the method of detecting or measuring Lp(a) uses an antibody which specifically binds human Apolipoprotein B (human ApoB or hApoB) (SEQ ID NO:16).

[0082] In even more specific embodiments, the ApoB antibodies of the present disclosure comprise a VL and a VH, wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the VH comprises CDRs HCDR1, HCDR2 and HCDR3 wherein LCDR1 has the amino acid sequence ofSASSSVNYMH (SEQ ID NO:17),Ref. No.30972_WO LCDR2 has the amino acid sequence ofYSTSNLPS (SEQ ID NO:18), LCDR3 has the amino acid sequence of QQRSSYPFT (SEQ ID NO:19), HCDR1 has the amino acid sequence of KAAGYTFTDYNIH (SEQ ID NO:22), HCDR2 has the amino acid sequence of HIYPYNGVTSSNQKFKN (SEQ ID NO:23), and HCDR3 has the amino acid sequence of ARPPYYDKGFDV (SEQ ID NO:24).

[0083] In various embodiments, the ApoB antibodies provided by the present disclosure, comprise an VL amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:20 and an VH amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:25.

[0084] In some embodiments, the ApoB antibodies provided by the disclosure, comprise an VL amino acid sequence of SEQ ID NO:21 and an VH amino acid sequence of SEQ ID NO:26.

[0085] According to some embodiments, the ApoB antibodies of the present disclosure may be murine, chimeric, or humanized antibodies. In some embodiments, the ApoB antibodies of the present disclosure comprise an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, antibodies of the present disclosure comprise a kappa light chain.

[0086] In some embodiments, the ApoB antibodies comprise an LC amino acid sequence of SEQ ID NO:21 and an HC amino acid sequence of SEQ ID NO:26.

[0087] Non-limiting sources of a "human sample” for use in the present disclosure include blood, plasma, and serum, preferably a serum sample.

[0088] As used herein, an “antibody” is an immunoglobulin molecule comprising 2 HCs and 2 LCs interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the CDRs contained therein. The CDRs are interspersed with regions that are more conserved, termed framework regions (“FR”). Each VL and VH is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the LC are referred to as “LCDR1, LCDR2, and LCDR3,” and the 3 CDRs of the HC are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues that form specific interactions with the antigen. The functional ability of an antibody to bind a particular antigen is largely influenced by the six CDRs. Assignment of amino acids to CDR domains within the VL and VH regions of the antibodies of the present invention is based on the well-known Kabat numbering convention (Kabat, et al., Ann. NY Acad. Sci.190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIHRef. No.30972_WO Publication No.91-3242 (1991)), and North numbering convention (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)).

[0089] According to some embodiments of the present disclosure, the light chains (LC) are classified as kappa or lambda and are each characterized by a particular constant region as known in the art. In some other embodiments of the disclosure, the heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. According to some embodiments, the antibodies include IgG HC, which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. The carboxy-terminal portion of each HC defines a constant region primarily responsible for effector function. In a particular embodiment, the antibodies of the present disclosure have one or more modifications in the constant region of each HC that reduces effector function.

[0090] In an embodiment, the antibodies of the present disclosure are monoclonal antibodies. Monoclonal antibodies are antibodies derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone, and not the method by which it is produced. Monoclonal antibodies can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such or other technologies known in the art.

[0091] Methods of producing and purifying antibodies are well known in the art and can be found, for example, in Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring harbor, N.Y., chapters 5-8 and 15. For example, mice or rabbits may be immunized with KIV-7-10 domains of human apo(a) (SEQ ID NO:2), and the resulting apo(a) antibodies can be recovered and purified, and sequenced using conventional methods well-known in the art. Embodiments of antibodies of the present disclosure may include antibodies engineered to contain one or more human framework regions surrounding CDRs derived from a non-human antibody. Human framework germline sequences can be obtained, for example, from ImMunoGeneTics (IMGT), or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351.

[0092] In particular embodiments of the present disclosure, the apo(a) and ApoB antibodies, e.g., Antibody H and Antibody M, respectively, or the nucleic acid encoding same, are provided in isolated form. As used herein, the term “isolated” refers to a protein, peptide, or nucleic acid that is free or substantially free from other macromolecular species found in a cellular environment.Ref. No.30972_WO

[0093] As used interchangeably herein, the term “patient,” “subject,” and “individual,” refers to a human. In certain embodiments, the patient is characterized with a disease, disorder, or condition, e.g., increased risk of cardiovascular disease, including acute coronary syndrome, ischemic stroke and aortic stenosis. In some embodiments, the patient is further characterized with increased or elevated levels of Lp(a).

[0094] As used herein, the term “binds” or “specifically binds” refers to an interaction of an antibody. For example, in the context of an apo(a) antibody, e.g., Antibody H, it refers to the interaction with an epitope region of apo(a) protein (SEQ ID NO:1), e.g., an epitope region of KIV-7-10 domains (SEQ ID NO:2). For example, in the context of an Apo(B) antibody, e.g., Antibody M, refers to the interaction with an epitope region of the Apo(B) protein.

[0095] The term “epitope region” as used herein refers to discrete, three-dimensional sites of an antigen that are recognized, either in total or in part, by the antibodies of the present disclosure. The amino acids of an epitope region provide chemically active surface groupings of the apo(a) protein and form a specific three-dimensional structure and may provide specific charge characteristics. Confirmational and non-confirmational / linear epitopes may be distinguished in that the binding to the confirmational epitope region is lost in the presence of denturing solvents whereas linear epitope regions is not.

[0096] “Percent identity” or “percent sequence identity”, as referred to in the present disclosure, in the context of two or more amino acid sequences refers to two or more sequences having a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm (e.g., BLASTP and BLASTN or other algorithms available to persons of skill in the art) or by visual inspection. Depending on the application, the percent identity can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. By way of example, percent identity of a sequence may be compared to a reference sequence. For example, when using a sequence comparison algorithm, test and reference sequences may be input into a computer (and subsequence coordinates may be further designated if desired along with sequence algorithm program parameters). The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence(s), based on the designated program parameters. Exemplary sequence alignment and / or identity algorithms are available through, Smith & Waterman, Adv. Appl. Math.2:482 (1981), Needleman & Wunsch, J. Mol. Biol.48:443 (1970), Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), GAP, BESTFIT, FASTA, and TFASTA.Ref. No.30972_WO One example of an algorithm that is suitable for determining percent sequence identity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).

[0097] The present disclosure also pertains to compositions, bioassays, and methods of clinical diagnosis, prognosis, or theranosis of a subject performed by a medical professional using the methods disclosed herein. The methods, as described herein, can, for example, be performed by an individual, a health professional, or a third party, for example a service provider who interprets information from the subject. As explained herein, a medical professional may initiate or modify treatment after receiving information regarding a diagnostic method of the present disclosure. For example, a medical professional may recommend a therapy, a change in therapy or an additional diagnostic assessment.

[0098] The methods disclosed herein comprise the step of contacting the human sample with a capture antibody that specifically binds apo(a) and then using a second detection antibody, wherein the second detection antibody specifically binds ApoB. In some such methods, the second detection antibody comprises a detectable label, wherein the step of detecting is provided by a detectable label upon formation of a complex comprising the apo(a) antibody, the second antibody, ApoB.

[0099] Accordingly in some embodiments, the apo(a) antibody is immobilized on a substrate, e.g., a streptavidin / biotin system.

[0100] In some embodiments of the methods of the present disclosure, the steps of contacting the human sample with the apo(a) antibody and contacting the human sample with the ApoB antibody occurs simultaneously.

[0101] The apo(a) antibodies of the present disclosure that specifically bind to KIV-7 and KIV- 8 domains can be used to detect or measure apo(a) covalently bound to ApoB, by techniques, such as affinity chromatography, immunoprecipitation, immunohistochemistry or ELISA-based assay. Such assay can be used to detect and measure Lp(a) for diagnostic, prognostic, or theranostic purposes to monitor Lp(a) levels, for example in blood, plasma, or serum as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Furthermore, it is understood from the present disclosure that apo(a) antibodies of the present disclosure may be part of an Lp(a) biomarker assay in which the capture antibody binds to apo(a), and a detection antibody binds to ApoB. In some embodiments, the detection antibody is coupled to a detectable substance or label to facilitate its detection. Examples of detectableRef. No.30972_WO substances or labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, chemiluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotnazinylamine fluorescein, dansyl chloride or phycoerythnn; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, ruthenium and aequorin, and examples of suitable radioactive material include125I,131I,35S or3H. In an exemplary embodiment, the ApoB detection antibody is coupled to ruthenium. Levels or measurements of apo(a), ApoB, and Lp(a), as provided by assays of the present disclosure, may be absolute values (e.g., concentration within a biological sample) or relative values (e.g., concentration compared to a reference). As used herein, Lp(a) is referred to as "increased" in a human sample if the methods for detecting Lp(a) as described herein indicates that the level or concentration of Lp(a) in the human sample is higher than a reference value. Conversely, Lp(a) is referred to as "decreased" in a human sample if the Lp(a) level or concentration of Lp(a) in a human sample is lower than a reference value, or for example, the Lp(a) value measured in a previous human sample.

[0102] A “reference value” as used herein refers to a known, or approximate concentration of apo(a), ApoB, and Lp(a) associated with a specific condition. The concentration levels in a reference value can be an absolute or relative amount, a range of amount, or a minimum amount, a mean amount, and / or a median amount of apo(a), ApoB, and Lp(a). A reference value can also serve as a baseline of apo(a), ApoB, and Lp(a) to which the human sample from a patient is compared.

[0103] As used herein, the term “capture antibody” refers to an antibody that will bind apo(a). In such embodiments, the capture antibody is capable of binding and capturing apo(a) in a human sample under suitable conditions, such that the capture antibody-apo(a) complex can be separated from the rest of the sample. In some embodiments, the capture antibody may be an antibody that specifically binds KIV-7 and KIV-8 domains, without binding to the KIV-2 domain. In some embodiments, the capture antibody is immobilized to a plate through a streptavidin / biotin system. In some embodiments, the capture antibody is immobilized in an immunoassay. In such immunoassays, a “detection antibody” is also utilized. According to some embodiments, a detection or second antibody may bind specifically to ApoB and is labelled with a detectableRef. No.30972_WO label. In some embodiments, the detection antibody specifically binds to ApoB already bound, or captured by the capture antibody, i.e. in the form of a capture antibody-apo(a)-ApoB protein complex. In such embodiments, the detection antibody binds ApoB of the capture antibody- apo(a)-ApoB protein complex, at a second epitope region that does not overlap with epitope region of the capture antibody, and may be labelled with a detectable label. In some such embodiments, the detection antibody is an antibody of the present disclosure that specifically binds an epitope region of ApoB.

[0104] As used herein, a “detectable label” is a moiety, composition, or technique that can be used to measure Lp(a) in a human sample and detect the formation of a complex between antibodies of the present disclosure that specifically bind apo(a) and ApoB of Lp(a) and the Lp(a) molecule. According to some embodiments, the detectable label may be conjugated to the antibody, e.g., either the capture antibody or detection antibody, directly or indirectly. Exemplary embodiments of detectable labels include biotin, radioisotopes fluorophores, or other fluorescent moieties, and enzymatic moieties.

[0105] The term "diagnosis" or “diagnosing”, as used interchangeably herein, refers to methods by those of ordinary skill in the art can estimate and / or determine the probability ("a likelihood") of whether or not a patient is suffering from a given disease or condition. In the case of the present disclosure, “diagnosing” the patient includes using the results of an assay of the present disclosure to identify or diagnose a cardiovascular disease , including acute coronary syndrome, ischemic stroke and aortic stenosis, as well as identify a patient, for example, that is the presence or occurrence of a cardiovascular disease or disorder or syndrome or the need for treatment, or the effectiveness of a treatment against the cardiovascular disease with the patient. A diagnosis may, according to the present disclosure, be based on a combination of other clinical indicia, as understood by the healthcare professional, to arrive at a diagnosis.

[0106] The present disclosure may be better understood with reference to the Examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, bioassays, methods, and diagnostics claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure.

[0107] Certain abbreviations are defined as follows: “BSA” refers to Bovine Serum Albumin; “EDTA” refers to ethylenediamine tetraacetic acid; “EGTA” refers to ethylene glycol tetraacetic acid; “h” refers to hour or hours; “HEPES” refers to 4-(2-hydroxyethyl)-1-Ref. No.30972_WO piperazineethanesulfonic acid; “min” refers to minute or minutes “RT” refers to room temperature; “TBS” refers to Tris-buffered saline.

[0108] In some embodiments, the disclosure provides a kit comprising an antibody which specifically binds apo(a), and instructions for use. In some embodiments, the kit comprises an antibody which specifically binds apo(a) described herein, the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, and a package insert comprising instructions for testing a human sample, wherein the human sample is from a patient with a disease, disorder, or condition associated with Lp(a) expression. In some embodiments, the kit comprises, in a suitable container, an apo(a) antibody described herein, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe or other container means, into which the apo(a) antibody is placed, and in some instances, suitably aliquoted. In some embodiments where an additional component is provided, the kit contains additional containers into which this component is placed. The kits can also include a means for containing the apo(a) antibody described herein, and any other reagent in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and / or kits can include labeling with instructions for use and / or warnings.

[0109] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is used in combination with the administration of a treatment to reduce Lp(a) levels in a patient.

[0110] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is used in association with the administration of a treatment to reduce Lp(a) levels in a patient.

[0111] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is used to determine if a patient is eligible to receive treatment to reduce Lp(a) levels.

[0112] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a)Ref. No.30972_WO in a human sample, is used to determine if the treatment is working properly to reduce Lp(a) levels in a patient.

[0113] In some embodiments, the kit comprising the antibody which specifically binds apo(a) described herein, and the use of the apo(a) antibody in a method for detecting or measuring Lp(a) in a human sample, is administered prior to a patient receiving treatment, during a patient receiving treatment, or after a patient is receiving treatment.

[0114] In some embodiments, the treatment to reduce Lp(a) levels in a patient is selected from the group consisting of Pelacarsen (antisense oligonucleotide; Novartis), Olpasiran (siRNA; Amgen), Lepodisiran (siRNA; Eli Lilly and Company), Zerlasiran (siRNA; Silence Therapeutics), Muvalaplin (small molecule inhibitor; Eli Lilly and Company), HRS-5346 (oligonucleotide-based therapy; HRS Pharma), or any other compound that can reduce the amount of Lp(a).

[0115] In some embodiments, the therapy can be selected from a small molecule disclosed in US Patent Publication US / 2025 / 0042847, the contents which are incorporated herein by reference in their entirety.

[0116] In some embodiments, the disease, disorder, or condition associated with Lp(a) expression is a cardiometabolic disease, optionally atherosclerosis, NAFLD, NASH, Calcific aortic valve stenosis (CAVS), cardiovascular events including coronary revascularization, CV mortality, cerebrovascular disease, chronic kidney disease, coronary heart disease, dyslipidemia, heart failure, MI and stroke, and / or peripheral vascular disease. EXAMPLES Example 1: Anti-apo(a) Antibody Engineering

[0117] Murine-derived apo(a) antibodies of the present disclosure were generated by immunizing mice with a recombinant protein composed of KIV-7-10 domains derived from human apo(a) and employing hybridoma methodology, for example, and as described by Kohler et al., Nature, 256:495 (1975).

[0118] Binding specificities of a panel of murine apo(a) antibodies produced by the hybridoma technology were determined by an ELISA assay and the results are shown in Table 1. The apo(a) antibodies were selected for their ability to bind KIV-7-10 domains without binding to KIV type 2 protein.

[0119] Table 1: ELISA assay to test binding specificities of apo(a) antibodiesRef. No.30972_WO Apo(a) antibody Recombinant Proteins clones KIV7-8-9-10 KIV2 Antibody A 1.069 0.098 Antibody B2.207 0.124Antibody C 1.960 0.094 Antibody D1.911 0.112Antibody E1.214 0.078Antibody F1.915 0.086Antibody G2.324 0.098Antibody H 2.405 0.110 Antibody I1.466 0.088Antibody J 1.811 0.094 Antibody K2.041 0.092Antibody L 2.045 0.096 Antibody N1.658 0.065Antibody O1.198 0.076

[0120] Antibodies from hybridoma supernatants were loaded on to anti-mouse IgG (AMC) Octet tips. Biolayer interferometry was used to determine the antibody binding specificity. The observed nm shift after 240 seconds is listed and shown in Table 2. Table 2. Antibody binding specificity from apo(a) antibody clones Apo(a) Recombinant Proteins antibody KIV7 KIV8 KIV7-8 KIV7-8-9-10 KIV2 clones Antibody A 0.0681 0.1045 0.0966 0.2231 0.0009Ref. No.30972_WO Antibody B 0.0544 0.1433 0.2344 0.4944 0.0062 Antibody C 0.1315 0.1595 0.2008 0.4919 0.0066 Antibody D 0.0308 0.1149 0.2267 0.4932 0.0041 Antibody E 0.0775 0.0743 0.2189 0.2258 -0.0004 Antibody F 0.1675 0.0405 0.2058 0.4102 0.0064 Antibody G 0.1046 0.1211 0.1588 0.3143 0.0092 Antibody H 0.1243 0.1551 0.1873 0.3646 0.0078 Antibody I 0.0234 0.0717 0.1476 0.399 0.0134 Antibody J 0.0898 0.056 0.1893 0.3922 0.0207 Antibody K 0.1086 0.0521 0.1824 0.3588 0.0281 Antibody L 0.1091 0.0649 0.2032 0.4151 0.0141

[0121] The hybridomas may be subcloned and grown by standard methods, including as ascites tumors in vivo Goding James W., Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by the hybridomas and / or subclones are purified according to conventional procedures, e.g., affinity chromatography, e.g., protein A or protein G-Sepharose, or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or the like.

[0122] cDNAs encoding antibodies of the present disclosure were sequenced using conventional procedures. The cDNA sequences encoding the heavy and light chains were cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector was then stably transfected into CHO cells. Mammalian expression of antibodies will result in glycosylation, typically at highly conserved N- glycosylation sites in the Fc region. Stable clones were verified for expression of an antibody specifically binding to human Apo(a). Positive clones were expanded into serum-free culture medium for antibody production in bioreactors. Media, into which an antibody has been secreted, was purified by conventional techniques. For example, the medium was applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such asRef. No.30972_WO phosphate buffered saline. The column was washed to remove nonspecific binding components. The bound antibody was then eluted, for example, by pH gradient and antibody fractions are detected, such as by SDS-PAGE, and then pooled. The antibody was then concentrated and / or sterile filtered using common techniques. Soluble aggregate and multimers were effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The resulting product was then immediately frozen, for example at -70°C, or may be lyophilized.

[0123] The variable heavy and light chain CDR sequences of the murine-derived apo(a) antibodies, Antibodies H, D, I, N, O, and B of the present disclosure are provided in Tables 3-14.

[0124] Table 3. Variable Heavy Chain CDRs of Antibody H Start End Feature Sequence 23 35 CDR1KASGYTFTDYNMD (SEQ ID NO:10) 50 66 CDR2NINPNNGGTIYNQRFRG (SEQ ID NO:11) 97 108 CDR3ARPPYYDKGFDV (SEQ ID NO:12)

[0125] Table 4. Variable Light Chain CDRs of Antibody H Start End Feature Sequence 24 34 CDR1RASQDIGNSLT (SEQ ID NO:4)49 56 CDR2YATSSLDS (SEQ ID NO:5)89 97 CDR3LQYASYPFT (SEQ ID NO:6)

[0126] Table 5. Variable Heavy Chain CDRs of Antibody D Start End Feature Sequence 23 35 CDR1GYTFTDY (SEQ ID NO:32)50 66 CDR2DPETGG (SEQ ID NO:33)97 108 CDR3RAYYSTYGYFDY (SEQ ID NO:34)

[0127] Table 6. Variable Light Chain CDRs of Antibody DRef. No.30972_WO Start End Feature Sequence 24 34 CDR1RASKSISKYLA (SEQ ID NO:27)49 56 CDR2SGSTLQS (SEQ ID NO:28)89 97 CDR3QHHYEYPLT (SEQ ID NO:29)

[0128] Table 7. Variable Heavy Chain CDRs of Antibody I Start End Feature Sequence 23 35 CDR1GFTFSDY (SEQ ID NO:42)50 66 CDR2SSGSST (SEQ ID NO:43)97 108 CDR3PGYYALYFDY (SEQ ID NO:44)Table 8. Variable Light Chain CDRs of Antibody I Start End Feature Sequence 24 34 CDR1RASGNIHNYLA (SEQ ID NO:37) 49 56 CDR2NVKTLAE (SEQ ID NO:38)89 97 CDR3QHHYGTPPT (SEQ ID NO:39)Table 9. Variable Heavy Chain CDRs of Antibody N Start End Feature Sequence 23 35 CDR1GYTFTDY (SEQ ID NO:52)50 66 CDR2NPKNGG (SEQ ID NO:53) 97 108 CDR3FDAKGNYYAMDY (SEQ ID NO:54)

[0129] Table 10. Variable Light Chain CDRs of Antibody N Start End Feature Sequence 24 34 CDR1KASQDVGTAVA (SEQ ID NO:47)Ref. No.30972_WO 49 56 CDR2WASTRHP (SEQ ID NO:48)89 97 CDR3QQYSTYPLT (SEQ ID NO:49)

[0130] Table 11. Variable Heavy Chain CDRs of Antibody O Start End Feature Sequence 23 35 CDR1GFNIKDD (SEQ ID NO:62)50 66 CDR2DPENGY (SEQ ID NO:63)97 108 CDR3TAYDYDY (SEQ ID NO:64)

[0131] Table 12. Variable Light Chain CDRs of Antibody O Start End Feature Sequence 24 34 CDR1KSSQSLLDSDGKTYLH (SEQ ID NO:57) 49 56 CDR2LVSKLAS (SEQ ID NO:58)89 97 CDR3WQGTHFPWT (SEQ ID NO:59)

[0132] Table 13. Variable Heavy Chain CDRs of Antibody B Start End Feature Sequence 23 35 CDR1GYTFTDY (SEQ ID NO:72) 50 66 CDR2DPETGG (SEQ ID NO:73)97 108 CDR3RAYYSTYGYFDY (SEQ ID NO:74)

[0133] Table 14. Variable Light Chain CDRs of Antibody B Start End Feature Sequence 24 34 CDR1RASKSISKYLA (SEQ ID NO:67)49 56 CDR2SGSTLQS (SEQ ID NO:68)89 97 CDR3QQHNEYPLT (SEQ ID NO:69)Ref. No.30972_WO Example 2: – Dual-epitope Lp(a) Biomarker Assay Method

[0134] The detection of Lp(a) in serum samples using the assay of the present disclosure was investigated. A streptavidin-coated MSD plate (herein referred to as the assay plate) was washed with wash buffer (TBS-0.05% Tween 20, 1X TBS T), then blocked by adding 200 µL / well of 1X TBS + 1% BSA. The plate was incubated at RT for 60 min with shaking (600 rpm). The plate was then washed with wash buffer and coated with 50 µL / well of a biotinylated apo(a) antibody [Antibody H] diluted to 1 µg / mL in a coating buffer (1X TBS T + 0.1% BSA). The plate was sealed with an aluminum plate sealer and incubated for 60 min at RT with shaking (600 rpm).

[0135] Next, a Lp(a) standard curve was prepared as a 12-step dilution by starting at 0.925 nM (5000 ng Lp(a) / mL) and diluting 1:3 for a total of 11 dilutions with the 12thwell at a 0 Lp(a) point. In a separate plate (herein referred to as the dilution plate), unknown serum samples or controls were diluted 1:2500 in dilution buffer (Dilution A = 10 µL serum sample + 490 µL dilution buffer mixed well; Dilution B = 10 µL Dilution A + 490 µL dilution buffer mixed well). Dilution buffer: 50 mmol / L HEPES, pH 7.40, 150 mmol / L NaCl, 10 mL / L Triton X-100, 5 mmol / L EDTA, 5 mmol / L EGTA supplemented with 1 g / L BSA and 100 µg / mL HBR1.

[0136] The assay plate was washed with wash buffer, then 50 µL / well of Lp(a) standard and unknown samples in duplicate were added from the dilution plate to the assay plate. The assay plate was incubated at RT for 60 min with shaking (600 rpm), washed with wash buffer, and then 50 µL / well of ruthenium-labeled anti-ApoB antibody [Antibody M] diluted to 1 µg / mL in detection buffer (50 mmol / L HEPES, pH 7.40, 150 mmol / L NaCl, 10 mL / L Triton X-100, 5 mmol / L EDTA, 5 mmol / L EGTA supplemented with 1 g / L BSA) was added. The assay plate was incubated at RT for 60 min with shaking (600 rpm), washed with wash buffer, and then 150 µL / well of 2X MSD Read Buffer was added to the plate.

[0137] The electrochemiluminescence in each well of the assay plate was immediately read on an MSD plate reader (MESO QuickPlex SQ 120). Unknowns were calculated using a log-log 4- 5 PL fit on the MSD Discovery Workbench software. Lp(a) in unknown serum or plasma samples were calculated in ng / mL or in nM concentrations. To convert Lp(a) levels calculated in ng / mL to mg / dL, the calculated Lp(a) in ng / mL were divided by 10,000.

[0138] Table 15 shows representative Lp(a) levels in 60 individual normal human serum (NHS) donor samples. Lp(a) levels varied greatly among individuals ranging from 6.84 nM to 236.18 nM.

[0139] Table 15: Levels of Lp(a) in Normal Human Serum SamplesRef. No.30972_WO NHS # Lp(a) (nM) NHS # Lp(a) (nM) 1 100.86 31 58.94 2 25.66 32 43.20 3 28.17 33 34.72 4 15.05 34 9.41 5 61.59 35 236.18 6 9.89 36 16.75 7 11.39 37 38.93 8 53.62 38 93.47 9 14.63 39 25.96 10116.92 40 12.73 11 132.29 41 102.29 1242.63 42 235.38 13157.83 43 50.17 1411.36 44 17.71 1560.47 45 57.12 16 25.83 46 94.88 179.64 47 20.52 18 26.20 48 68.68 1987.89 49 34.45 2042.97 50 17.44 21 54.85 51 232.69 226.91 52 6.84 23 55.80 53 106.18Ref. No.30972_WO 24 28.35 54 9.62 2563.18 55 20.54 26 197.38 56 91.28 27101.51 57 196.46 2847.88 58 46.51 29142.48 59 108.02 3034.45 60 92.56 Average 65.79 St. Dev.60.0

[0140] Lower Limit of Quantification (LLOQ) and Limit of Detection (LOD). LLOQ (sensitivity) is the lowest level of Lp(a) that can be measured using the dual-epitope assay described in Example 2 was determined by diluting an Lp(a) standard sample over a range of dilution factors, from neat to 1:2048. Sets of aliquots were analyzed in duplicate on 4 separate plates over 2 days according to the method described above to generate 4 reportable results. The mean back-calculated value, standard deviation, and coefficient of variation (CV) were determined for the 4 reportable results at each dilution.

[0141] The LLOQ is typically assigned by interpolation of the Lp(a) concentration in the detection range of the assay in which the CV reached 30%. However, none of the dilution levels had an overall CV greater than or equal to 30%. As a result, the LLOQ was estimated to be 0.69 nM because it was the lowest available back-calculated value above the LOD in which all of the replicates were in the detectable range of the assay. The LOD, as reported by the MesoScale software, was 0.0000391 nM (averaged from 64 standard curves over 30 days).

[0142] Table 16: Lower Limit of Quantification (LLOQ) Assessment Standard Lp(a) Concentration (nM) Mean StDev CV Replicates Dilution 1 2 3 4 (nM) (nM) (%) in Factor Detection RangeRef. No.30972_WO Neat 34.32 34.83 33.48 33.82 34.12 0.59 1.72 8 / 8 In Detection Range 1:219.70 19.89 18.83 19.17 19.40 0.49 2.51 8 / 8 In Detection Range 1:4 10.26 10.40 9.73 9.81 10.05 0.33 3.25 8 / 8 In Detection Range 1:85.34 5.35 5.25 5.34 5.32 0.05 0.93 8 / 8 In Detection Range 1:16 2.69 2.65 2.54 2.60 2.62 0.07 2.50 8 / 8 In Detection Range 1:321.33 1.35 1.36 1.37 1.35 0.02 1.31 8 / 8 In Detection Range 1:64 0.73 0.71 0.66 0.66 0.69 0.03 5.03 8 / 8 In Detection Range 1:128BDR BDR BDR BDR - - - 0 / 8 In Detection Range Abbreviations: BDR = Below Detectable Range; CV = Coefficient of Variation; LOD = Limit of Detection; Lp(a) = Lipoprotein(a); StDev = Standard Deviation

[0143] This data shows that the assay of Example 2 has good sensitivity.

[0144] Specificity. According to the Bioanalytical Method Validation Guidance for Industry (FDA, 2018), the sponsor should investigate the cross-reactivity of molecules that could potentially interfere with the binding interaction, including molecules structurally orRef. No.30972_WO physiologically related to the drug. Human plasminogen is structurally / physiologically similar to apo(a) which is covalently bound by a disulfide bond to ApoB to form Lp(a). Three-fold serially diluted recombinant human plasminogen starting at 5000 ng / mL was tested in the dual-epitope Lp(a) biomarker assay described in Example 2. The lack of signal when attempting to capture and detect human plasminogen demonstrates the specificity of the dual-epitope Lp(a) biomarker assay as shown in Table 17 and Figure 2. Additionally, Lp(a) specificity was demonstrated because up to 50 µg / mL of recombinant apo(a) spiked into either buffer or normal human serum did not lead to increased detection of Lp(a) in the assay suggesting the dual-epitope biomarker assay cannot detect free apo(a).

[0145] Table 17: Specificity of Lp(a): standard curves Concentration, Lp(a), Plasminogen, ng / mL ECLU signal ECLU signal 5000.0 285604 86 1666.7 141450 68 555.6 55537 59 185.2 20213 58 61.7 6911 59 20.6 2444 59 6.9 857 63 2.3 321 59 0.8 147 61 0.3 92 59 0.1 70 59 0.0 59 62

[0146] Recovery. The objective of recovery experiments was to evaluate the accuracy (recovery) of the dual-epitope Lp(a) biomarker assay described in Example 2 by spiking Lp(a) standard into serum samples and a buffer control (diluent). A 63.1 nM stock Lp(a) standard wasRef. No.30972_WO used to spike into 5 individual human serum samples and a buffer control. The samples were spiked 1:10 with the Lp(a) stock to a concentration of 6.31 nM where the final serum concentration was 90%. A non-spiked sample was included for each sample and buffer control. Each sample was analyzed in duplicate (2 wells) to generate 1 reportable result. The percent recovery relative to the buffer control for each sample was determined by subtracting unspiked results [endogenous Lp(a)] from spiked results and comparing to the buffer control as shown in Table 18. The recovery was considered acceptable because 5 / 5 (100%) of the spiked samples were within ±30% of the calculated buffer control value.

[0147] Table 18: Spike and Recovery Experiment Matrix Lp(a) Spiked Mean Endogenous % Matrix and Concentration Observed Subtracted Recovery and Lp(a) (nM) Concentration Mean Lp(a) spike (nM) Concentration spike (nM) Buffer Spike 6.31 5.89 No spike 0 NHS #1 Spike 6.31 14.00 6.57 111.64 No spike 0 7.43 N / A N / A NHS #2 Spike 6.31 12.65 6.58 111.77 005 No spike 0 6.07 N / A N / A 2=D NHS #3 Spike 6.31 13.69 6.27 106.46 R M No spike 0 7.42 N / A N / A NHS #4 Spike 6.31 12.05 6.12 103.96 No spike 0 5.93 N / A N / A NHS #5 Low 6.31 14.50 6.14 104.40 No spike 0 8.35 N / A N / A = 0 Buffer Spike 6.31 5.76 D 0 R0,01 No spike 0 MRef. No.30972_WO NHS #1 Spike 6.31 14.22 6.79 115.41 No spike 0 7.68 N / A N / A NHS #2 Spike 6.31 12.23 6.16 104.67 No spike 0 6.00 N / A N / A NHS #3 Spike 6.31 13.77 6.34 107.81 No spike 0 7.29 N / A N / A NHS #4 Spike 6.31 11.70 5.78 98.14 No spike 0 5.83 N / A N / A NHS #5 Low 6.31 13.86 5.50 93.52 No spike 0 8.18 N / A N / A Abbreviations: N / A = Not Applicable; NHS = Normal Human Serum

[0148] Dilutional Linearity. An individual NHS sample containing high levels of Lp(a) was used for a dilutional linearity experiment (Table 19). An initial 1:20 dilution of this sample was used followed by 11 subsequent 2-fold serial dilutions. Each serially diluted sample was added directly to the assay plate without undergoing the procedural assay dilution. The dilutions of 1:20, 1:40, 1:80 and 1:160 were above the fit range of the standard curve. Dilutions of 1:320 and 1:640 were considered acceptable but were near the top of the standard curve. Duplicate results for each dilutional linearity sample above the LLOQ had a %CV less than or equal to 25%. This is important because, during validation, %CVs between the established LLOQ and the established upper limit of quantification (ULOQ) (retroactively applied) must be less than or equal to 30% at the LLOQ and ULOQ.

[0149] Table 19: Dilutional Linearity Dilution (1:X) Mean concentration in matrix (nM) 20Above Fit Curve Range 40Above Fit Curve Range 80 Above Fit Curve Range 160Above Fit Curve RangeRef. No.30972_WO 320 218.61 640205.481280 193.99 2560192.135120192.1410240194.8020480189.4940960 187.15

[0150] Short-Term Stability Assessment. High, mid, and low Lp(a) control samples were prepared by aliquoting 3 individual NHS samples containing high, mid and low concentrations of Lp(a). Multiple aliquots of each sample, previously stored frozen at -80 ºC were thawed and then maintained under various conditions prior to evaluation. Sample storage conditions included: 1) RT for 4, 24, and 48 h, 2) refrigerated (2 to 8 ºC) for 4, 24, and 48 h, and 3) 2, 4, 6 and 8 freeze / thaw cycles. A freshly thawed reference sample was included to represent the baseline for each assay control. The results obtained for stored samples were compared with those obtained from the freshly thawed ones. The percent change from baseline was calculated for each condition and each time point. Stability was acceptable, with a mean percent change between each sample and the freshly thawed baseline sample ≤30% (Tables 20-22).

[0151] Table 20: Short-Term Room Temperature Stability Control Hours at RT Mean (nM) Percent change from 0 hr Low4 7.17 7.3 24 7.11 6.6 48 7.66 13.2 0 6.65 0.0 Mid 4 40.48 5.3 24 35.53 -7.9Ref. No.30972_WO 48 32.80 -16.8 0 38.33 0.0 High 4 199.73 1.1 24 192.50 -2.6 48 183.46 -7.6 0 197.49 0.0

[0152] Table 21: Short-Term 2 to 8 °C Stability Control Hours at 4 Mean Percent change ºC (nM) from 0 hr Low 4 7.28 8.6 24 7.19 7.6 48 7.52 11.5 0 6.65 0.0 Mid 4 43.22 11.3 24 41.17 6.9 48 36.11 -6.2 0 38.33 0.0 High 4 199.25 0.9 24 196.74 -0.4 48 181.43 -8.9 0 197.49 0.0Ref. No.30972_WO

[0153] Table 22: Freeze / Thaw Stability Control Freeze / thaw Mean (nM) % Change from no cycle (#) freeze / thaws High 1 191.51 1.2 2 184.24 -2.7 4 179.10 -5.7 6 176.86 -7.0 8 180.21 -5.0 0 189.25 0.0 Mid 1 39.31 -0.8 2 37.89 -4.6 4 40.32 1.7 6 42.60 7.0 8 42.57 6.9 0 39.62 0.0 Low 1 6.99 -3.5 2 6.12 -18.1 4 6.42 -12.5 6 7.08 -2.2 8 6.81 -6.2 0 7.23 0.0

[0154] Assay Precision. Precision is the ability of the assay to produce the same result, within an acceptable amount of variability, when the same sample is tested multiple times by the same analyst. The objective was to evaluate the intra-assay (within-run) and inter-assay (between-run) precision of the assay. To determine the intra-assay precision of the Lp(a) biomarker assay, each Lp(a) assay control was run 12 times in one assay on a single plate. The mean back-calculatedRef. No.30972_WO value, standard deviation, and %CV was determined. Intra-assay precision met validation requirements for because the %CVs were less than or equal to 25% for all controls above the LLOQ and less than or equal to 30% at the LLOQ (Table 23).

[0155] The inter-assay precision was determined from the results of each assay control in 61 runs over 30 days by 2 analysts. The mean back-calculated value, standard deviation, and %CV of each reportable result across the runs were determined. The inter-assay precision met validation requirements because the %CVs were less than or equal to 25% for all controls above the LLOQ and less than or equal to 30% at the LLOQ (Table 24).

[0156] Table 23: Intra-Assay Precision 1 Replicate Number Observed Concentration (nM) (n=2) High Positive Mid-Positive LPC Control (HPC) Control (MPC) 1 186.91 39.56 7.01 2 180.77 37.15 6.53 3 182.67 37.93 6.45 4 179.80 38.03 6.62 5 188.78 38.49 7.15 6 186.00 39.05 7.38 7 180.93 38.51 6.94 8 183.48 38.27 6.50 9 188.15 38.68 7.32 10 170.00 36.67 6.62 11176.16 37.70 6.63 12179.50 36.91 6.53 Mean Replicate181.93 38.08 6.81Std Dev5.4 0.9 0.3CV (%)3.0 2.3 4.9Ref. No.30972_WO

[0157] Table 24: Inter-Assay Precision 2 Observed Concentration (nM) Run High Control Mid Control Low Control 1 198.14 39.85 6.67 2 211.99 42.58 7.12 3 198.87 40.03 6.88 4 199.76 41.66 7.77 5 204.87 44.07 8.03 6 206.06 41.11 7.30 7 203.52 38.45 6.31 8 204.32 38.62 6.32 9 205.83 39.23 6.87 10202.44 39.53 7.17 11190.66 38.26 6.23 12187.53 38.64 6.36 13185.68 38.01 6.31 14190.54 38.77 6.13 15204.29 39.30 6.29 16 208.69 39.52 6.37 17205.35 39.50 6.37 18 209.83 39.23 6.11 19184.01 37.56 6.15 20215.95 41.05 6.35 21 195.51 44.50 7.60 22197.72 45.67 7.89Ref. No.30972_WO 188.90 46.57 7.05 181.74 44.45 7.07 189.61 38.63 6.14 184.91 38.02 6.02 205.64 39.00 6.28 204.28 39.23 6.70 217.87 44.63 7.17 176.92 36.45 5.67 170.34 35.42 5.87 183.53 40.39 6.77 181.93 38.08 6.81 212.88 44.41 7.00 206.12 43.19 7.05 211.22 43.59 7.09 208.65 44.33 7.31 212.22 46.79 7.12 206.39 46.17 7.83 203.81 47.06 8.08 173.48 40.09 6.26 175.20 40.96 6.57 160.24 35.56 6.73 163.38 36.42 7.14 200.32 38.35 6.64 197.36 37.47 6.11Ref. No.30972_WO 47 197.49 38.33 6.65 48178.32 36.41 5.93 49 193.75 41.09 7.12 50182.15 38.16 6.83 51170.78 41.74 6.97 52170.13 40.42 6.96 53178.50 40.64 7.14 54 170.74 41.48 6.75 55180.00 44.35 7.06 56 180.33 45.57 7.39 57184.73 44.76 7.33 58 155.89 36.85 7.42 59158.43 36.39 6.90 60196.19 40.59 5.90 61193.86 41.81 7.08 Mean Replicate192.13 40.64 6.79Std Dev 15.5 3.1 0.6 CV (%)8.1 7.6 8.3

[0158] Dual-epitope biomarker assay vs. Randox assay – Apo(a) spike experiment.10x spikes of recombinant apo(a) were prepared by diluting recombinant apo(a) in TBS buffer. Five µL of 10x apo(a) spike was mixed with 45 µL NHS to prepare to 1x apo(a) spike samples at each concentration. These apo(a) spike samples were analyzed using the dual-epitope biomarker assay described above in Example 2 and the Randox assay. The data in Figure 4 and Table 25 show that in contrast to the Randox assay, the assay of Example 2 is specific to Lp(a) and will not be affected by varying levels of apo(a) in serum.

[0159] Table 25. Assay response to apo(a) protein spikeRef. No.30972_WO Measured conc. by assay, nM Apo(a) spike Dual- Randox conc. nM Epitope assay 0 125.7 118.6 3.5 121.2 122 7 135.3 135.4 13.9 126.1 147.9 27.8 127.4 164.3 55.7 132.9 194.7 111.4 127.2 237.4 SEQUENCES >Human_Apolipoprotein (a) | P08519 MEHKEVVLLLLLFLKSAAPEQSHVVQDCYHGDGQSYRGTYSTTVTGRTCQAWSSMTPHQHNRTTE NYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQ APTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPD AVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGN GQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRW EYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRT CQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAV APPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTP EYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSE QAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNP DAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHG NGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVR WEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGR TCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDPVAAPYCYTRDPSVRWEYCNLTQCSDAEGTA VAPPTITPIPSLEAPSEQAPTEQRPGVQECYHGNGQSYQGTYFITVTGRTCQAWSSMTPHSHSRT PAYYPNAGLIKNYCRNPDPVAAPWCYTTDPSVRWEYCNLTRCSDAEWTAFVPPNVILAPSLEAFF EQALTEETPGVQDCYYHYGQSYRGTYSTTVTGRTCQAWSSMTPHQHSRTPENYPNAGLTRNYCRN PDAEIRPWCYTMDPSVRWEYCNLTQCLVTESSVLATLTVVPDPSTEASSEEAPTEQSPGVQDCYH GDGQSYRGSFSTTVTGRTCQSWSSMTPHWHQRTTEYYPNGGLTRNYCRNPDAEISPWCYTMDPNV RWEYCNLTQCPVTESSVLATSTAVSEQAPTEQSPTVQDCYHGDGQSYRGSFSTTVTGRTCQSWSS MTPHWHQRTTEYYPNGGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQCPVMESTLLTTPTVVRef. No.30972_WO PVPSTELPSEEAPTENSTGVQDCYRGDGQSYRGTLSTTITGRTCQSWSSMTPHWHRRIPLYYPNA GLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTRCPVTESSVLTTPTVAPVPSTEAPSEQAPPEK SPVVQDCYHGDGRSYRGISSTTVTGRTCQSWSSMIPHWHQRTPENYPNAGLTENYCRNPDSGKQP WCYTTDPCVRWEYCNLTQCSETESGVLETPTVVPVPSMEAHSEAAPTEQTPVVRQCYHGNGQSYR GTFSTTVTGRTCQSWSSMTPHRHQRTPENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNL TRCSDTEGTVVAPPTVIQVPSLGPPSEQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTF IPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCASSSFDCGKPQVEPKKCPGSI VGGCVAHPHSWPWQVSLRTRFGKHFCGGTLISPEWVLTAAHCLKKSSRPSSYKVILGAHQEVNLE SHVQEIEVSRLFLEPTQADIALLKLSRPAVITDKVMPACLPSPDYMVTARTECYITGWGETQGTF GTGLLKEAQLLVIENEVCNHYKYICAEHLARGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCA RPNKPGVYARVSRFVTWIEGMMRNN (SEQ ID NO:1) >Kringle IV type 7-10 (KIV-7-10) domains APTEQSPTVQDCYHGDGQSYRGSFSTTVTGRTCQSWSSMTPHWHQRTTEYYPNGGLTRNYCRNPD AEIRPWCYTMDPSVRWEYCNLTQCPVMESTLLTTPTVVPVPSTELPSEEAPTENSTGVQDCYRGD GQSYRGTLSTTITGRTCQSWSSMTPHWHRRIPLYYPNAGLTRNYCRNPDAEIRPWCYTMDPSVRW EYCNLTRCPVTESSVLTTPTVAPVPSTEAPSEQAPPEKSPVVQDCYHGDGRSYRGISSTTVTGRT CQSWSSMIPHWHQRTPENYPNAGLTENYCRNPDSGKQPWCYTTDPCVRWEYCNLTQCSETESGVL ETPTVVPVPSMEAHSEAAPTEQTPVVRQCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTP ENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRCSDTEGT (SEQ ID NO:2) >Kringle IV type 2 (KIV-2) domain APTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPD AVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQ (SEQ ID NO:3) >Antibody H _ LCDR1 RASQDIGNSLT (SEQ ID NO:4) >Antibody H _ LCDR2 YATSSLDS (SEQ ID NO:5) >Antibody H _ LCDR3 LQYASYPFT (SEQ ID NO:6) >Antibody H _VL_Pro DIQMTQSPSSLSASLGERVSLSCRASQDIGNSLTWLQQEPDGTIKRLIYATSSLDSGVPKRFSGS RSGSDYSLTISSLESEDFVDYYCLQYASYPFT (SEQ ID NO:7) >Antibody H _LC_Pro DIQMTQSPSSLSASLGERVSLSCRASQDIGNSLTWLQQEPDGTIKRLIYATSSLDSGVPKRFSGS RSGSDYSLTISSLESEDFVDYYCLQYASYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGA SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC (SEQ ID NO:8) >Antibody H _LC_NA GATATCCAGATGACCCAGAGCCCCTCTTCGCTGTCGGCGTCCCTCGGCGAACGGGTGTCACTGTC ATGCCGGGCTAGCCAGGACATCGGAAACTCGCTGACCTGGCTGCAACAGGAACCTGATGGCACCA TTAAGCGCCTTATCTACGCCACTTCGTCATTGGACTCCGGAGTGCCGAAAAGGTTTAGCGGGAGCRef. No.30972_WO AGAAGCGGCTCCGACTACTCCCTGACCATTTCCTCCCTGGAATCCGAGGACTTCGTCGACTACTA CTGCCTGCAATACGCCTCCTATCCATTCACCTTCGGTTCCGGAACTAAGCTCGAGATCAAGCGCG CTGATGCGGCGCCCACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCT AGCGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGG CAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACA GCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAG GCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO:9) >Antibody H _HCDR1 KASGYTFTDYNMD (SEQ ID NO:10) >Antibody H _HCDR2 NINPNNGGTIYNQRFRG (SEQ ID NO:11) >Antibody H _HCDR3 ARPPYYDKGFDV (SEQ ID NO:12) >Antibody H _VH_Pro EVQLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGNINPNNGGTIYNQRFR GKATLTVDKSSSTAYMELRSLTSEDTAVYYCARPPYYDKGFDVWGTGTTVTVSS (SEQ ID NO:13) >Antibody H _HC_Pro EVQLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGNINPNNGGTIYNQRFR GKATLTVDKSSSTAYMELRSLTSEDTAVYYCARPPYYDKGFDVWGTGTTVTVSSAKTTPPSVYPL APGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWP SETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVV DISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFP APIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNT QPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:14) >Antibody H_HC_NA GAAGTGCAGCTGCAGCAGTCCGGTCCAGAACTTGTGAAGCCTGGAGCGAGCGTGAAGATCCCGTG CAAAGCCTCGGGCTACACCTTTACCGACTATAACATGGACTGGGTCAAGCAGTCGCACGGAAAGT CCCTGGAGTGGATCGGGAACATTAATCCCAACAACGGCGGGACTATCTACAACCAACGGTTCAGA GGAAAGGCTACTCTGACGGTCGACAAGTCCAGCTCCACCGCATACATGGAACTCCGGTCACTGAC CTCCGAGGATACCGCCGTGTACTACTGCGCCCGCCCGCCCTACTACGACAAGGGATTCGATGTCT GGGGCACCGGTACTACCGTGACTGTGTCCAGCGCCAAAACGACACCCCCATCTGTCTATCCGCTA GCCCCTGGATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCTGTCTGGTGAAGGGCTACTT CCCTGAGCCTGTGACAGTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTGCACACATTCCCTG CCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAGCGTGACCGTGCCTAGCAGCACATGGCCT AGCGAGACAGTGACATGCAACGTGGCCCACCCTGCCTCTTCTACCAAGGTGGACAAGAAGATCGT GCCCAGAGACTGCGGCTGCAAGCCTTGCATCTGCACCGTGCCTGAGGTGAGCAGCGTGTTCATCT TCCCACCCAAGCCCAAGGACGTGCTCACCATCACCCTCACCCCCAAGGTCACGTGTGTTGTGGTA GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACAC AGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCARef. No.30972_WO TCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCT GCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCAT TCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCT TCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACT CAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAA CTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTG AGAAGAGCCTCTCCCACTCTCCTGGTAAA (SEQ ID NO:15) >Human_ApoB-100 | 04114 MDPPRPALLALLALPALLLLLLAGARAEEEMLENVSLVCPKDATRFKHLRKYTYNYEAESSSGVP GTADSRSATRINCKVELEVPQLCSFILKTSQCTLKEVYGFNPEGKALLKKTKNSEEFAAAMSRYE LKLAIPEGKQVFLYPEKDEPTYILNIKRGIISALLVPPETEEAKQVLFLDTVYGNCSTHFTVKTR KGNVATEISTERDLGQCDRFKPIRTGISPLALIKGMTRPLSTLISSSQSCQYTLDAKRKHVAEAI CKEQHLFLPFSYKNKYGMVAQVTQTLKLEDTPKINSRFFGEGTKKMGLAFESTKSTSPPKQAEAV LKTLQELKKLTISEQNIQRANLFNKLVTELRGLSDEAVTSLLPQLIEVSSPITLQALVQCGQPQC STHILQWLKRVHANPLLIDVVTYLVALIPEPSAQQLREIFNMARDQRSRATLYALSHAVNNYHKT NPTGTQELLDIANYLMEQIQDDCTGDEDYTYLILRVIGNMGQTMEQLTPELKSSILKCVQSTKPS LMIQKAAIQALRKMEPKDKDQEVLLQTFLDDASPGDKRLAAYLMLMRSPSQADINKIVQILPWEQ NEQVKNFVASHIANILNSEELDIQDLKKLVKEALKESQLPTVMDFRKFSRNYQLYKSVSLPSLDP ASAKIEGNLIFDPNNYLPKESMLKTTLTAFGFASADLIEIGLEGKGFEPTLEALFGKQGFFPDSV NKALYWVNGQVPDGVSKVLVDHFGYTKDDKHEQDMVNGIMLSVEKLIKDLKSKEVPEARAYLRIL GEELGFASLHDLQLLGKLLLMGARTLQGIPQMIGEVIRKGSKNDFFLHYIFMENAFELPTGAGLQ LQISSSGVIAPGAKAGVKLEVANMQAELVAKPSVSVEFVTNMGIIIPDFARSGVQMNTNFFHESG LEAHVALKAGKLKFIIPSPKRPVKLLSGGNTLHLVSTTKTEVIPPLIENRQSWSVCKQVFPGLNY CTSGAYSNASSTDSASYYPLTGDTRLELELRPTGEIEQYSVSATYELQREDRALVDTLKFVTQAE GAKQTEATMTFKYNRQSMTLSSEVQIPDFDVDLGTILRVNDESTEGKTSYRLTLDIQNKKITEVA LMGHLSCDTKEERKIKGVISIPRLQAEARSEILAHWSPAKLLLQMDSSATAYGSTVSKRVAWHYD EEKIEFEWNTGTNVDTKKMTSNFPVDLSDYPKSLHMYANRLLDHRVPQTDMTFRHVGSKLIVAMS SWLQKASGSLPYTQTLQDHLNSLKEFNLQNMGLPDFHIPENLFLKSDGRVKYTLNKNSLKIEIPL PFGGKSSRDLKMLETVRTPALHFKSVGFHLPSREFQVPTFTIPKLYQLQVPLLGVLDLSTNVYSN LYNWSASYSGGNTSTDHFSLRARYHMKADSVVDLLSYNVQGSGETTYDHKNTFTLSCDGSLRHKF LDSNIKFSHVEKLGNNPVSKGLLIFDASSSWGPQMSASVHLDSKKKQHLFVKEVKIDGQFRVSSF YAKGTYGLSCQRDPNTGRLNGESNLRFNSSYLQGTNQITGRYEDGTLSLTSTSDLQSGIIKNTAS LKYENYELTLKSDTNGKYKNFATSNKMDMTFSKQNALLRSEYQADYESLRFFSLLSGSLNSHGLE LNADILGTDKINSGAHKATLRIGQDGISTSATTNLKCSLLVLENELNAELGLSGASMKLTTNGRF REHNAKFSLDGKAALTELSLGSAYQAMILGVDSKNIFNFKVSQEGLKLSNDMMGSYAEMKFDHTN SLNIAGLSLDFSSKLDNIYSSDKFYKQTVNLQLQPYSLVTTLNSDLKYNALDLTNNGKLRLEPLK LHVAGNLKGAYQNNEIKHIYAISSAALSASYKADTVAKVQGVEFSHRLNTDIAGLASAIDMSTNY NSDSLHFSNVFRSVMAPFTMTIDAHTNGNGKLALWGEHTGQLYSKFLLKAEPLAFTFSHDYKGST SHHLVSRKSISAALEHKVSALLTPAEQTGTWKLKTQFNNNEYSQDLDAYNTKDKIGVELTGRTLA DLTLLDSPIKVPLLLSEPINIIDALEMRDAVEKPQEFTIVAFVKYDKNQDVHSINLPFFETLQEY FERNRQTIIVVLENVQRNLKHINIDQFVRKYRAALGKLPQQANDYLNSFNWERQVSHAKEKLTAL TKKYRITENDIQIALDDAKINFNEKLSQLQTYMIQFDQYIKDSYDLHDLKIAIANIIDEIIEKLK SLDEHYHIRVNLVKTIHDLHLFIENIDFNKSGSSTASWIQNVDTKYQIRIQIQEKLQQLKRHIQN IDIQHLAGKLKQHIEAIDVRVLLDQLGTTISFERINDILEHVKHFVINLIGDFEVAEKINAFRAK VHELIERYEVDQQIQVLMDKLVELAHQYKLKETIQKLSNVLQQVKIKDYFEKLVGFIDDAVKKLN ELSFKTFIEDVNKFLDMLIKKLKSFDYHQFVDETNDKIREVTQRLNGEIQALELPQKAEALKLFL EETKATVAVYLESLQDTKITLIINWLQEALSSASLAHMKAKFRETLEDTRDRMYQMDIQQELQRY LSLVGQVYSTLVTYISDWWTLAAKNLTDFAEQYSIQDWAKRMKALVEQGFTVPEIKTILGTMPAFRef. No.30972_WO EVSLQALQKATFQTPDFIVPLTDLRIPSVQINFKDLKNIKIPSRFSTPEFTILNTFHIPSFTIDF VEMKVKIIRTIDQMLNSELQWPVPDIYLRDLKVEDIPLARITLPDFRLPEIAIPEFIIPTLNLND FQVPDLHIPEFQLPHISHTIEVPTFGKLYSILKIQSPLFTLDANADIGNGTTSANEAGIAASITA KGESKLEVLNFDFQANAQLSNPKINPLALKESVKFSSKYLRTEHGSEMLFFGNAIEGKSNTVASL HTEKNTLELSNGVIVKINNQLTLDSNTKYFHKLNIPKLDFSSQADLRNEIKTLLKAGHIAWTSSG KGSWKWACPRFSDEGTHESQISFTIEGPLTSFGLSNKINSKHLRVNQNLVYESGSLNFSKLEIQS QVDSQHVGHSVLTAKGMALFGEGKAEFTGRHDAHLNGKVIGTLKNSLFFSAQPFEITASTNNEGN LKVRFPLRLTGKIDFLNNYALFLSPSAQQASWQVSARFNQYKYNQNFSAGNNENIMEAHVGINGE ANLDFLNIPLTIPEMRLPYTIITTPPLKDFSLWEKTGLKEFLKTTKQSFDLSVKAQYKKNKHRHS ITNPLAVLCEFISQSIKSFDRHFEKNRNNALDFVTKSYNETKIKFDKYKAEKSHDELPRTFQIPG YTVPVVNVEVSPFTIEMSAFGYVFPKAVSMPSFSILGSDVRVPSYTLILPSLELPVLHVPRNLKL SLPDFKELCTISHIFIPAMGNITYDFSFKSSVITLNTNAELFNQSDIVAHLLSSSSSVIDALQYK LEGTTRLTRKRGLKLATALSLSNKFVEGSHNSTVSLTTKNMEVSVATTTKAQIPILRMNFKQELN GNTKSKPTVSSSMEFKYDFNSSMLYSTAKGAVDHKLSLESLTSYFSIESSTKGDVKGSVLSREYS GTIASEANTYLNSKSTRSSVKLQGTSKIDDIWNLEVKENFAGEATLQRIYSLWEHSTKNHLQLEG LFFTNGEHTSKATLELSPWQMSALVQVHASQPSSFHDFPDLGQEVALNANTKNQKIRWKNEVRIH SGSFQSQVELSNDQEKAHLDIAGSLEGHLRFLKNIILPVYDKSLWDFLKLDVTTSIGRRQHLRVS TAFVYTKNPNGYSFSIPVKVLADKFIIPGLKLNDLNSVLVMPTFHVPFTDLQVPSCKLDFREIQI YKKLRTSSFALNLPTLPEVKFPEVDVLTKYSQPEDSLIPFFEITVPESQLTVSQFTLPKSVSDGI AALDLNAVANKIADFELPTIIVPEQTIEIPSIKFSVPAGIVIPSFQALTARFEVDSPVYNATWSA SLKNKADYVETVLDSTCSSTVQFLEYELNVLGTHKIEDGTLASKTKGTFAHRDFSAEYEEDGKYE GLQEWEGKAHLNIKSPAFTDLHLRYQKDKKGISTSAASPAVGTVGMDMDEDDDFSKWNFYYSPQS SPDKKLTIFKTELRVRESDEETQIKVNWEEEAASGLLTSLKDNVPKATGVLYDYVNKYHWEHTGL TLREVSSKLRRNLQNNAEWVYQGAIRQIDDIDVRFQKAASGTTGTYQEWKDKAQNLYQELLTQEG QASFQGLKDNVFDGLVRVTQEFHMKVKHLIDSLIDFLNFPRFQFPGKPGIYTREELCTMFIREVG TVLSQVYSKVHNGSEILFSYFQDLVITLPFELRKHKLIDVISMYRELLKDLSKEAQEVFKAIQSL KTTEVLRNLQDLLQFIFQLIEDNIKQLKEMKFTYLINYIQDEINTIFSDYIPYVFKLLKENLCLN LHKFNEFIQNELQEASQELQQIHQYIMALREEYFDPSIVGWTVKYYELEEKIVSLIKNLLVALKD FHSEYIVSASNFTSQLSSQVEQFLHRNIQEYLSILTDPDGKGKEKIAELSATAQEIIKSQAIATK KIISDYHQQFRYKLQDFSDQLSDYYEKFIAESKRLIDLSIQNYHTFLIYITELLKKLQSTTVMNP YMKLAPGELTIIL (SEQ ID NO:16) >Antibody M_LCDR1 SASSSVNYMH (SEQ ID NO:17) >Antibody M_LCDR2 YSTSNLPS (SEQ ID NO:18) >Antibody M_LCDR3 QQRSSYPFT (SEQ ID NO:19) >Antibody M_VL QIVLTQSPAIMSASPGEKVTITCSASSSVNYMHWFQQKPDTSPKLWIYSTSNLPSGVPARFSGSG SGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTNLEIK (SEQ ID NO:20) >Antibody M_LC QIVLTQSPAIMSASPGEKVTITCSASSSVNYMHWFQQKPDTSPKLWIYSTSNLPSGVPARFSGSG SGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTNLEIKRADAAPTVSIFPPSSEQLTSGGASRef. No.30972_WO VVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNEC (SEQ ID NO:21) >Antibody M_HCDR1 KAAGYTFTDYNIH (SEQ ID NO:22) >Antibody M_HCDR2 HIYPYNGVTSSNQKFKN (SEQ ID NO:23) >Antibody M_HCDR3 ARSDYYFDY (SEQ ID NO:24) >Antibody M_VH EVQLQQSGPEVVKPGASVKMSCKAAGYTFTDYNIHWVKQSHGKSLEWVGHIYPYNGVTSSNQKFK NKATLTVDISSSTAYMELRSLTSEDSAVYYCARSDYYFDYWGQGTTLTVSS (SEQ ID NO:25) >Antibody M_HC EVQLQQSGPEVVKPGASVKMSCKAAGYTFTDYNIHWVKQSHGKSLEWVGHIYPYNGVTSSNQKFK NKATLTVDISSSTAYMELRSLTSEDSAVYYCARSDYYFDYWGQGTTLTVSSAKTTPPSVYPLAPG SAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDIS KDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPI EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPI MDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:26) >Antibody D_LCDR1 RASKSISKYLA (SEQ ID NO:27) >Antibody D_LCDR2 SGSTLQS (SEQ ID NO:28) >Antibody D_LCDR3 QHHYEYPLT (SEQ ID NO:29) >Antibody D_VL DVQILQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDF TLTISSLEPEDFAMYYCQHHYEYPLTFGAGTKLELK (SEQ ID NO:30) >Antibody D_LC DVQILQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDF TLTISSLEPEDFAMYYCQHHYEYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE C (SEQ ID NO:31) >Antibody D_HCDR1 GYTFTDY (SEQ ID NO:32) >Antibody D_HCDR2Ref. No.30972_WO DPETGG (SEQ ID NO:33) >Antibody D_HCDR3 RAYYSTYGYFDY (SEQ ID NO:34) >Antibody D_VH QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVIQTPVHGLEWIGSIDPETGGTAYNQKFKGKVILT ADKSSSTAYMELRSLTSEDSAVYYCTRRAYYSTYGYFDYWGQGTTLTVSS (SEQ ID NO:35) >Antibody D_HC QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVIQTPVHGLEWIGSIDPETGGTAYNQKFK GKVILTADKSSSTAYMELRSLTSEDSAVYYCTRRAYYSTYGYFDYWGQGTTLTVSSAKTTPPSVY PLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSST WPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCV VVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAA FPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYK NTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:36) >Antibody I_LCDR1 RASGNIHNYLA (SEQ ID NO:37) >Antibody I_LCDR2 NVKTLAE (SEQ ID NO:38) >Antibody I_LCDR3 QHHYGTPPT (SEQ ID NO:39) >Antibody I_VL DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNVKTLAEGVPSRFSGSGSDTQF SLKINSLQPEDFGIYYCQHHYGTPPTFGSGTKLEIK (SEQ ID NO:40) >Antibody I_LC DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNVKTLAEGVPSRFSGSGSDTQF SLKINSLQPEDFGIYYCQHHYGTPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE C (SEQ ID NO:41) >Antibody I_HCDR1 GFTFSDY (SEQ ID NO:42) >Antibody I_HCDR2 SSGSST (SEQ ID NO:43) >Antibody I_HCDR3 PGYYALYFDY (SEQ ID NO:44) >Antibody I_VHRef. No.30972_WO EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTIS RDNAKNTLFLQMTSLRSEDTAMYYCARPGYYALYFDYWGQGTTLTVSS (SEQ ID NO:45) >Antibody I_HC EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTIS RDNAKNTLFLQMTSLRSEDTAMYYCARPGYYALYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQ TQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMA KDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHE GLHNHHTEKSLSHSPGK (SEQ ID NO:46) >Antibody N_LCDR1 KASQDVGTAVA (SEQ ID NO:47) >Antibody N_LCDR2 WASTRHP (SEQ ID NO:48) >Antibody N_LCDR3 QQYSTYPLT (SEQ ID NO:49) >Antibody N_VL DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQLKPGQSPKVLLYWASTRHPGVPDRFTGSGSGTDF TLTISNVQSEDLADYFCQQYSTYPLTFGAGTKLELR (SEQ ID NO:50) >Antibody N_LC DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQLKPGQSPKVLLYWASTRHPGVPDRFTGSGSGTDF TLTISNVQSEDLADYFCQQYSTYPLTFGAGTKLELRRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE C (SEQ ID NO:51) >Antibody N_HCDR1 GYTFTDY (SEQ ID NO:52) >Antibody N_HCDR2 NPKNGG (SEQ ID NO:53) >Antibody N_HCDR3 FDAKGNYYAMDY (SEQ ID NO:54) >Antibody N_VH EVQLQQSGPELVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPKNGGTIYKQKFKGKAILT VDKSSSTAYMEFRSLTSEDTAVYYCARFDAKGNYYAMDYWGQGTSVTVSS (SEQ ID NO:55) >Antibody N_HC EVQLQQSGPELVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPKNGGTIYKQKFKGKAILT VDKSSSTAYMEFRSLTSEDTAVYYCARFDAKGNYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNS MVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTK VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQRef. No.30972_WO MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVL HEGLHNHHTEKSLSHSPGK (SEQ ID NO:56) >Antibody O_LCDR1 RASGNIHNYLA (SEQ ID NO:57) >Antibody O_LCDR2 NVKTLAE (SEQ ID NO:58) >Antibody O_LCDR3 QHHYGTPPT (SEQ ID NO:59) >Antibody O_VL DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNVKTLAEGVPSRFSGSGSDTQF SLKINSLQPEDFGIYYCQHHYGTPPTFGSGTKLEIK (SEQ ID NO:60) >Antibody O_LC DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNVKTLAEGVPSRFSGSGSDTQF SLKINSLQPEDFGIYYCQHHYGTPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE C (SEQ ID NO:61) >Antibody O_HCDR1 GFTFSDY (SEQ ID NO:62) >Antibody O_HCDR2 SSGSST (SEQ ID NO:63) >Antibody O_HCDR3 PGYYALYFDY (SEQ ID NO:64) >Antibody O_VH EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTIS RDNAKNTLFLQMTSLRSEDTAMYYCARPGYYALYFDYWGQGTTLTVSS (SEQ ID NO:65) >Antibody O_HC EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTIS RDNAKNTLFLQMTSLRSEDTAMYYCARPGYYALYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQ TQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMA KDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHE GLHNHHTEKSLSHSPGK (SEQ ID NO:66) >Antibody B_LCDR1 RASKSISKYLA (SEQ ID NO:67) >Antibody B_LCDR2 SGSTLQS (SEQ ID NO:68)Ref. No.30972_WO >Antibody B_LCDR3 QQHNEYPLT (SEQ ID NO:69) >Antibody B_VL DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGS GSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK (SEQ ID NO:70) >Antibody B_LC DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGS GSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGA SVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC (SEQ ID NO:71) >Antibody B_HCDR1 GYTFTDY (SEQ ID NO:72) >Antibody B_HCDR2 DPETGG (SEQ ID NO:73) >Antibody B_HCDR3 RAYYSTYGYFDY (SEQ ID NO:74) >Antibody B_VH QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGAAYNQKVK GKAIMTADKSSSTAYMELRSLTSEDSAVYYCTRRAYYSTYGYFDYWGQGTTLTVSS (SEQ ID NO:75) >Antibody B_HC QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGAAYNQKVK GKAIMTADKSSSTAYMELRSLTSEDSAVYYCTRRAYYSTYGYFDYWGQGTTLTVSSAKTTPPSVY PLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSST WPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCV VVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAA FPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYK NTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:76)

Claims

Ref. No.30972_WO CLAIMS We claim:

1. An antibody which specifically binds apo(a) comprising: a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein a. LCDR1 comprises SEQ ID NO:4, b. LCDR2 comprises SEQ ID NO:5, c. LCDR3 comprises SEQ ID NO:6, d. HCDR1 comprises SEQ ID NO:10, e. HCDR2 comprises SEQ ID NO:11, and f. HCDR3 comprises SEQ ID NO:

12.

2. The antibody of claim 1, wherein the VL comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:7 and the VH comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:

13.

3. The antibody of claim 2, wherein the VL comprises an amino acid sequence of SEQ ID NO:7 and the VH comprises an amino acid sequence of SEQ ID NO:

13.

4. The antibody of any one of claims 1 to 3, wherein the antibody comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:8 and the HC comprises an amino acid sequence of SEQ ID NO:

14.

5. A kit comprising the antibody of any one of claims 1 to 4 and a detection antibody that binds to that binds to human ApoB.

6. Use of an antibody according to any one of claims 1 to 4 in a method for detecting or measuring Lp(a) in a human sample.

7. A method for detecting or measuring Lp(a) in a human sample comprising the steps of: a. contacting the human sample with a capture antibody that is an apo(a) antibody that binds to the Kringle IV type 7 (KIV-7) and Kringle IV type 8 (KIV-8) domains of human apo(a), wherein the human apo(a) is covalently bound to human ApoB forming a capture antibody-apo(a)-ApoB protein complex,Ref. No.30972_WO b. detecting the capture antibody-apo(a)-ApoB protein complex with a detection antibody that is an ApoB antibody that binds to human ApoB.

8. The method of claim 7, wherein the capture antibody does not bind to the Kringle IV type 2 (KIV-2) domain.

9. The method of claim 7, wherein the capture antibody shows binding at KIV 7 and KIV 8 which is at least 2-fold greater than the binding at KIV-2.

10. The method of any one of claims 7 to 9, wherein the apo(a) antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein a. the LCDR1 comprises SEQ ID NO:4, b. the LCDR2 comprises SEQ ID NO:5, c. the LCDR3 comprises SEQ ID NO:6, d. the HCDR1 comprises SEQ ID NO:10, e. the HCDR2 comprises SEQ ID NO:11, and f. the HCDR3 comprises SEQ ID NO:

12.

11. The method of claim 10, wherein the VL comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:7 and the VH comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:

13.

12. The method of claim 11, wherein the VL comprises an amino acid sequence of SEQ ID NO:7 and the VH comprises an amino acid sequence of SEQ ID NO:

13.

13. The method of any one of claims 7 to 12, wherein the ApoB antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises light chain complementarity determining regions (LCDR) LCDR1, LCDR2, and LCDR3, and the VH comprises light chain complementarity determining regions (HCDR) HCDR1, HCDR2, and HCDR3, wherein a. LCDR1 comprises SEQ ID NO:17, b. LCDR2 comprises SEQ ID NO:18, c. LCDR3 comprises SEQ ID NO:19, d. HCDR1 comprises SEQ ID NO:22, e. HCDR2 comprises SEQ ID NO:23, andRef. No.30972_WO f. HCDR3 comprises SEQ ID NO:

24.

14. The method of claim 13, wherein the VL comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:20 and the VH comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:

25.

15. The method of claim 14, wherein the VL comprises an amino acid sequence of SEQ ID NO:20 and the VH comprises an amino acid sequence of SEQ ID NO:

25.

16. The method of any one of claims 7 to 15, wherein the wherein the ApoB antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

17. The method of any one of claims 7 to 16, wherein the apo(a) antibody comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an amino acid sequence of SEQ ID NO:8 and the HC comprises an amino acid sequence of SEQ ID NO:

14.

18. The method of any one of claims 7 to 17 wherein the human sample is blood, plasma or serum.

19. The method of claim 18, wherein the human sample is serum.

20. The method of any one of claims 7 to 19, wherein binding of the detection antibody to ApoB is detected by a label selected from the group consisting of an enzymatic label, a fluorescent label, a chemiluminescent label, a radioactive label, and a dye label.

21. The method of claim 20, wherein the chemiluminescent label is ruthenium.

22. The method of any one of claims 7 to 21, wherein detecting or measuring Lp(a) is by an immunoassay.

23. The method of claim 22, wherein the immunoassay is an enzyme linked immunosorbent assay or radioimmunoassay.

24. The method of claim 23, wherein the immunoassay comprises immunoblotting, immunodiffusion, immunoelectrophoresis, or immunoprecipitation.

25. The method of any one of claims 7 to 24, wherein the capture antibody is attached to a solid support.

26. The method of claim 25, wherein the solid support is a microtiter plate, a bead, or a slide.

27. The method of any one of claims 7 to 26, wherein the capture antibody and detection antibody is present within a kit, wherein the kit comprises a container, a label on the container, and a composition contained within the container.Ref. No.30972_WO 28. The method of claim 27, wherein the kit is used in combination or association with the administration of a treatment to reduce Lp(a) levels in a patient with a disease, disorder, or condition associated with Lp(a) expression.

29. The method of claim 27, wherein the kit is used to determine if a patient is eligible to receive treatment to reduce Lp(a) levels.

30. The method of claim 27, wherein the kit is used to determine if the treatment is working properly to reduce Lp(a) levels in a patient.

31. The method of claim 27, wherein the kit is used to prior to a patient receiving treatment, during a patient receiving treatment, or after a patient is receiving treatment 32. The method of any one of claims 28 to 31, wherein the treatment for reducing Lp(a) levels in a patient is selected from the group consisting of Pelacarsen, Olpasiran, Lepodisiran, Zerlasiran, Muvalaplin, HRS-5346, or any other compound that can reduce the amount of Lp(a).

33. The method of any one of claims 28 to 32, wherein disease, disorder, or condition associated with Lp(a) expression is a cardiometabolic disease, optionally atherosclerosis, NAFLD, NASH, Calcific aortic valve stenosis (CAVS), cardiovascular events including coronary revascularization, CV mortality, cerebrovascular disease, chronic kidney disease, coronary heart disease, dyslipidemia, heart failure, MI and stroke, and / or peripheral vascular disease.

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