Isoform-independent antibody against lipoprotein (a)

Abstract

This disclosure provides antibodies and antibody fragments that bind to lipoprotein(a) epitopes, as well as methods of use thereof, including the production of transgenic animal models and the use of fragments as therapeutic and diagnostic agents for treating Lp(a)-related diseases and disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 459,010, filed on 13 April 2023, the disclosures of which are incorporated herein by reference.

[0002] Technical field This disclosure provides human antibodies and humanized antibodies that bind to lipoprotein (a) (Lp(a)), single-chain variable regions (scFv), and their binding domains. This disclosure relates to methods and compositions for treating patients using antibodies, scFv, and their binding domains for the treatment of Lp(a)-related diseases and disorders, and for use in diagnosis.

[0003] Incorporating sequence lists by reference This application is accompanied by a sequence listing titled "00015-409WO1.xml", prepared on 12 April 2024, containing 18,698 bytes of data and machine-formatted on the IBM-PC and MS-Windows operating systems. For all purposes, this sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Lipoprotein(a) [Lp(a)] is composed of apolipoprotein(a) covalently bound to apolipoprotein B-100. Apolipoprotein(a) exhibits wide size heterogeneity due to variations in the number of kringle IV type 2 (KIV2) repeats between individuals and populations (see, e.g., Figure 1A). Apolipoprotein(a) consists of 10 unique kringle IV repeats, each present in a single copy, except for KIV2, which is present in a variable number of identical copies (1 to over 40). This also includes one copy of kringle V (KV) and an inactive protease-like domain. Plasma Lp(a) levels are genetically determined by the rate of apolipoprotein(a) production in hepatocytes, with isoforms containing fewer KIV2 repeats being secreted more efficiently, resulting in an inverse correlation between KIV2 repeat count and plasma Lp(a) levels.

[0005] Numerous assays exist for measuring plasma Lp(a), but the assay methods vary by manufacturer and are not globally standardized. Variations in the number of KIV2 repeats create significant methodological constraints in accurately measuring plasma Lp(a) levels. All commercially available assays use polyclonal antibodies, which are mixtures of antibodies that can bind multiple times to KIV2 repeats and other segments of apolipoprotein(a). Furthermore, assay calibrators are derived from pooled plasma from multiple donors and generally cannot represent all known apolipoprotein isoforms of the sample being measured. Compared to calibrators, polyclonal antibodies tend to bind more frequently to larger apolipoprotein(a) isoforms, which is seen in subjects with low plasma Lp(a) levels and therefore tends to overestimate these values. In contrast, such antibodies bind less frequently to smaller isoforms, which is seen in subjects with high plasma Lp(a) levels and therefore tends to underestimate the values. This size-dependent bias can be minimized by using separate pools of calibration materials representing the majority of isoforms, rather than serially diluting them in a single pool, and it also allows for a more accurate reassignment of new values ​​to the calibration materials.

[0006] New guidelines recommending that everyone have their Lp(a) measured at least once in their lifetime, along with the development of new treatments that significantly lower Lp(a), are expected to expand Lp(a) testing. Therefore, the need for accurate methods is stronger than ever. [Overview of the project] [Means for solving the problem]

[0007] This disclosure provides an antibody or antibody fragment that recognizes and binds to lipoprotein (a), wherein the antibody or antibody fragment has a variable heavy chain (V H ) domain and / or variable light chain (V L ) including the domain, (a)V HThe domain comprises an amino acid sequence containing one, two, or three complementarity-determining regions (CDRs) selected from the group consisting of SEQ ID NO: 9 or its variant, SEQ ID NO: 10 or its variant, and SEQ ID NO: 11 or its variant, (b)V L The domain comprises an amino acid sequence containing one, two, or three complementarity-determining regions (CDRs) selected from the group consisting of SEQ ID NO: 12 or its variant, SEQ ID NO: 13 or its variant, and SEQ ID NO: 14 or its variant, wherein an antibody or antibody fragment containing SEQ ID NOs: 9, 10, 11, 12, 13, and 14, and CDRs selected from any of the aforementioned combinations, binds to Lp(a). In one embodiment, V H The domain includes an amino acid sequence comprising CDRs including sequence numbers 9, 10, and 11. In another embodiment, V L The domain comprises an amino acid sequence including a CDR containing SEQ ID NOs. 12, 13, and 14. In another or further embodiment, the antibody or antibody fragment is selected from the group consisting of antibodies or scFv having a heavy-chain domain and a light-chain domain containing the complementarity-determining regions of SEQ ID NOs. 9, 10, 11, 12, 13, and 14. In yet another or further embodiment, the heavy-chain domain and the light-chain domain are linked to an Fc region. In a further embodiment, the Fc region is a human Fc region. In another embodiment, the antibody fragment comprises a single-chain variable fragment ("scFv") that recognizes the KIV4 epitope of lipoprotein (a). In a further embodiment, the epitope comprises or consists of the sequence FVPPNVI (SEQ ID NO: 1). In one embodiment, the epitope comprises SEQ ID NO: 1 and contains 1 to 10 additional amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 1. In yet another or further embodiment, the scFv is soluble under physiological conditions. In yet another or further embodiment, scFv includes a light chain variable region having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 16. In yet another embodiment, scFv includes a heavy chain variable region having a sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 18.

[0008] This disclosure also provides antibodies comprising variable light chains and variable heavy chains as described in the preceding paragraph. In one embodiment, the antibody is humanized. In another embodiment, the antibody is a chimeric antibody.

[0009] This disclosure also provides a pharmaceutical composition comprising the antibody or antibody fragment of this disclosure and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.

[0010] This disclosure also provides an antibody or antibody fragment of this disclosure conjugated to a solid substrate.

[0011] This disclosure also provides an antibody or antibody fragment of this disclosure operably linked to a detectable label.

[0012] This disclosure also provides polynucleotides encoding antibodies, antibody fragments, variable light chains, variable heavy chains, or scFvs of the present disclosure. In one embodiment, the polynucleotide comprises SEQ ID NOs. 15 and / or 17, or variants thereof, wherein the polynucleotide encodes a polypeptide that binds to the KIV4 epitope of Lp(a), including SEQ ID NOs. 1.

[0013] This disclosure further includes a vector comprising a polynucleotide encoding the antibody or antibody fragment of this disclosure.

[0014] This disclosure also provides host cells transformed with the polynucleotides of this disclosure.

[0015] This disclosure also provides host cells transformed with the vector of this disclosure.

[0016] The present disclosure also provides a transgenic animal comprising a polynucleotide encoding an antibody or antibody fragment of the present disclosure. In one embodiment, the animal is a mouse. In another or further embodiment, the scFv or antibody of the present disclosure is expressed from hepatocytes and / or macrophages of the transgenic animal. In a further embodiment, the transgenic animal is used to model the effects of coronary artery diseases or disorders such as aortic valve stenosis and / or atherosclerosis.

[0017] The present disclosure also provides a method of treating or preventing coronary artery disease, the method comprising administering an antibody or antibody fragment, or scFv of the present disclosure, to a subject having or at risk of having coronary artery disease, stroke, peripheral artery disease, or calcific aortic valve stenosis.

[0018] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the binding domains of the VH domain and / or VL domain described above and herein, wherein the CAR binds to Lp(a).

[0019] The present disclosure also provides an immunological composition comprising an antigenic peptide comprising the sequence FVPPNVI (SEQ ID NO: 1). In one embodiment, the antigenic peptide comprises SEQ ID NO: 1 and 1 to 10 additional amino acids at the N-terminus and / or C-terminus. In one embodiment, the peptide sequence comprising or consisting of SEQ ID NO: 1 is conjugated to an adjuvant or administered with an adjuvant. The present disclosure also provides an antibody (polyclonal or monoclonal) obtained by immunizing a mammal with the peptide of SEQ ID NO: 1.

[0020] The present disclosure also provides CAR-T cells comprising the chimeric antigen receptor of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [Figure 1A] A schematic diagram of apolipoprotein (a) and the KIV domain is shown. [Figure 1B]A schematic diagram of the methodology for the Lp(a) and total apo(a) assays is shown. The Lp(a) and total apo(a) assays using the capture antibody MB47 and the detection antibody biotin-LPA4 are shown. [Figure 1C] A schematic diagram of the methodology for the Lp(a) and total apo(a) assays is shown. The Lp(a) and total apo(a) assays using LPA4 and the detection antibody biotin-LPA4 are shown. [Figure 1D] A schematic diagram of the methodology for the Lp(a) and total apo(a) assays is shown. The Lp(a) and total apo(a) assays using the capture antibody MB47 and the detection antibody biotin-LPA-KIV4 are shown. [Figure 1E] A schematic diagram of the methodology for the Lp(a) and total apo(a) assays is shown. The Lp(a) and total apo(a) assays using LPA4 and the detection antibody biotin-LPA-KIV4 are shown. [Figure 2] This shows the IgG titer against K8 in immunized mouse plasma. [Figure 3] Eight representative colonies screened against the antigen array are shown. Colony #6 was subcloned in a semi-solid gel to ensure monoclonality. [Figure 4] This indicates that subcolony 1F4 was cultured, and Ab was purified by BioXCell and named LPA-KIV4. [Figure 5] This dot blot shows the reactivity of Ab derived from hybridoma 1F4 to 8K, peptide KIV4, peptide KIV5, plasminogen, and KIV2(3). [Modes for carrying out the invention]

[0022] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to “single-chain variable fragment” or “scFv” includes multiple single-chain variable fragments, and a reference to “lipoprotein(a)” includes one or more lipoprotein(a) molecules and their equivalents known to those skilled in the art.

[0023] All publications referenced herein are incorporated in full by reference for the purpose of explaining and disclosing methodologies described in publications that may be used in connection with the description herein. The publications discussed above and throughout this text are provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the inventors have no prior rights to such disclosures by prior disclosure. Furthermore, if any term presented in one or more publications is similar to or identical to a term expressly defined in this disclosure, the definition of the term expressly provided in this disclosure shall prevail in all respects.

[0024] Furthermore, the use of "and" means "and / or" unless otherwise specified. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be limiting.

[0025] Furthermore, where the term “including” is used in the description of various embodiments, those skilled in the art will understand that in some specific circumstances the embodiments can be described alternatively using the expression “essentially consisting of” or “consisting of.”

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure pertains. In the practice of the disclosed methods and compositions, any methods and reagents similar or equivalent to those described herein may be used, but only illustrative methods and materials are described here.

[0027] Lipoprotein(a) (Lp(a)) is common in the human population. Currently, there is no standardized method or composition for measuring Lp(a) content in biological samples. Current antibodies used to measure Lp(a) bind to kringle repeats (type-2), and therefore the measurement is ambiguous because it depends on the number of repeats present.

[0028] "Lipoprotein(a)" or "Lp(a)" refers to a variant of low-density lipoprotein that contains a protein called apolipoprotein(a) (apo(a)). Genetic and epidemiological studies have revealed that lipoprotein(a) is a risk factor for atherosclerosis and related diseases such as coronary heart disease and stroke. Lipoprotein(a) consists of an LDL-like particle and a specific apolipoprotein(a) covalently bound to apoB contained in the particle's outer shell. The size of the apo(a) protein varies by size polymorphism [KIV-2 VNTR], which is caused by a variable number of kringle IV repeats in the LPA gene. This size variation at the gene level is also expressed at the protein level, resulting in apo(a) proteins with 10 to over 50 kringle IV repeats (each variable kringle IV domain consists of 114 amino acids). These variable apo(a) sizes are known as “apo(a) isoforms.” There is generally an inverse correlation between apo(a) isoform size and Lp(a) plasma concentration. Lp(a) concentrations can vary between individuals from over 1,000 to <0.2 to >200 mg / dL. Mean Lp(a) plasma concentrations in African populations are 2–3 times higher than in Asian, Oceanian, or European populations. A general inverse correlation between apo(a) isoform size and Lp(a) plasma concentration is observed in all populations. A schematic diagram of Lp(a) is provided in Figure 1A.

[0029] Lp(a) contributes to the process of atherogenesis. The structure of apolipoprotein(a) is similar to that of plasminogen and tPA (tissue plasminogen activator), competing with plasminogen for its binding site and leading to decreased fibrinolysis. In addition, Lp(a) stimulates the secretion of PAI-1, thus causing thrombus formation. It may also promote coagulation by inhibiting the function of tissue factor pathway inhibitors. Furthermore, Lp(a) carries cholesterol that causes atherosclerosis and binds to atherosclerotic, pro-inflammatory oxidized phospholipids as a preferred carrier of oxidized phospholipids in human plasma, thereby attracting inflammatory cells to the blood vessel wall and causing smooth muscle cell proliferation. Lp(a) is also hypothesized to be involved in wound healing and tissue repair by interacting with components of the blood vessel wall and extracellular matrix. The apo(a) characteristic of Lp(a) particles binds to immobilized fibronectin, thereby conferring serine-proteinase-type proteolytic activity to Lp(a).

[0030] High levels of Lp(a) in the blood are associated with coronary heart disease (CHD), cardiovascular disease (CVD), atherosclerosis, thrombosis, and stroke. High Lp(a) is associated with early atherosclerosis independently of other cardiac risk factors, including LDL. In patients with advanced cardiovascular disease, Lp(a) indicates a coagulation risk for plaque thrombosis. Apo(a) contains a domain very similar to plasminogen (PLG). Lp(a) accumulates in the vascular wall and inhibits the binding of PLG to the cell surface, reducing plasmin production and thereby increasing coagulation. Inhibition of PLG by Lp(a) also promotes the proliferation of smooth muscle cells. These unique characteristics of Lp(a) suggest that Lp(a) causes blood clot formation and atherosclerosis. Numerous studies confirming a strong correlation between elevated Lp(a) levels and heart disease have led to a consensus that Lp(a) is an important independent predictor of cardiovascular disease. Animal studies have shown that Lp(a) may directly contribute to atherosclerotic damage by increasing plaque size, inflammation, instability, and smooth muscle cell growth. Genetic data also support the theory that Lp(a) causes cardiovascular disease. Lp(a) is associated with increased atherogenic capacity, particularly at levels above 30 mg / dl, and has been shown to be an independent predictor of cardiovascular risk (odds ratio -1.5 to -2), especially in younger subjects (under 60 years old) and subjects with high LDL cholesterol levels.

[0031] Lp(a) appears in different isoforms of the apolipoprotein (per clingle repeat), and 40% of the variation in Lp(a) levels when measured in mg / dl may be due to these different isoforms. Lower concentrations of Lp(a) are also associated with disease. Therefore, tests with simple quantitative results may not provide a complete assessment of risk.

[0032] As will be further described above and in the examples, this disclosure provides LPA-KIV4, a novel monoclonal antibody that detects seven unique amino acid epitopes on KIV4 of apolipoprotein(a) that exist in only one copy. LPA-KIV4 successfully quantified a wide range of plasma Lp(a) levels in a chemiluminescent ELISA format and demonstrated staining of human carotid endarterectomy specimens. This antibody can be used in the research setting for immunological detection of apolipoprotein(a) and in the clinical setting for measurement of plasma Lp(a) levels. Because it is a monoclonal antibody that binds to only one unique epitope of apolipoprotein(a), it can overcome many of the current limitations in measuring Lp(a) levels.

[0033] Growing evidence suggests that Lp(a) is an independent genetic and possibly causative risk factor for cardiovascular disease (CVD). Elevated Lp(a) levels are associated with a higher risk of myocardial infarction, stroke, peripheral artery disease, and calcific aortic stenosis. The association between Lp(a) and CVD risk is nearly linear, with the highest Lp(a) levels associated with a 2-4 times higher risk. Lp(a) levels can vary more than 1000-fold between individuals, ranging from <0.1 to >300 mg / dL (>750 nmol / L).

[0034] Elevated Lp(a) levels are fairly common, with an estimated 20% of the population having levels above 50 mg / dL (greater than 125 nmol / L), which is the threshold above which risk arises in statin-treated patients. Furthermore, it is a target for therapies that reduce the risk of cardiovascular disease, and recent studies have shown that antisense oligonucleotides can lower Lp(a) by more than 80%, paving the way for ongoing Phase 3 clinical trials (Lp(a)HORIZON, Assessing the Impact of Lipoprotein(a)Lowering With TQJ230 on Major Cardiovascular Events in Patients With CVD, [https: / / ]clinicaltrials.gov / ct2 / show / NCT04023552). In addition, numerous academic societies, both domestic and international, have proposed measuring Lp(a) in individuals at moderate to high risk of CVD to enhance risk prediction or to consider it as a risk-enhancing factor. The EAS / ESC also recommends measuring Lp(a) at least once in a person's lifetime to assess cardiovascular risk, and there is a strong need to develop more accurate methods for measuring Lp(a) in clinical settings.

[0035] In 2018, the NHLBI Working Group recommended the development of a globally standardized measurement of Lp(a) applicable to commercial laboratories. Current Lp(a) measurement methods are one of the following: 1) assigning a target value of assay calibrator in relation to total Lp(a) mass [apo(a), apoB, lipid and carbohydrate components] and expressing the value in mg / dL. However, these methods have the limitation that there is no traceability of various calibrators to any established reference substance, or 2) assigning a target value to an assay calibrator traceable to the World Health Organization / International Union of Clinical Chemistry and Clinical Laboratory Medicine secondary reference substance PRM-2B and expressing the value in nmol / L (i.e., molar concentration of Lp(a) particles). The NHLBI Group also recommended discontinuing reporting total Lp(a) mass in mg / dL and instead reporting the value in nmol / L traceable to a common reference system. Furthermore, after performing a common calibration based on accuracy, harmonization of results obtained by various methods should be carried out.

[0036] The lack of widely available monoclonal antibodies against Lp(a) that do not bind to the KIV2 repeat or cross-react with plasminogen that has substantial homology to apolipoprotein(a) has hindered both the accurate measurement of Lp(a) levels and the international standardization of Lp(a) assays. The difficulty in producing monoclonal antibodies against Lp(a) suggests that only a limited number of antigen sites are available for producing such antibodies. One such monoclonal antibody described, a40, binds to an unknown site on KIV9 and has been used to measure Lp(a) in an ELISA format. An ELISA format employing a capture antibody that detects KIV2 and a second detection antibody that binds to only one unique site on apolipoprotein(a), along with calibrators tracked to the WHO / IFCC, is considered the optimal method for reducing size-dependent bias. This disclosure provides methods and compositions for carrying out such ELISAs and methods.

[0037] Commercial assays typically use polyclonal antibodies, which bind multiple times to the same KIV2 repeat segment, thus exhibiting isoform dependence. Some assays can minimize apolipoprotein (a) size bias to some extent by using independent calibrators, each containing a suitable distribution of apo(a) isoforms and accurately assigned values ​​by separate isoform-independent assays. Nevertheless, most assays remain sensitive to apolipoprotein (a) size because there is no consistent relationship between Lp(a) levels and apo(a) isoform size. Therefore, samples encompassing a relatively wide range of apolipoprotein (a) sizes may show high or low Lp(a) levels. A recent study evaluating Lp(a) levels using six commercial methods to reflect size heterogeneity bias concluded that current commercial immunological assays are calibrated differently, and their biases differ significantly across clinically relevant concentration ranges in a non-linear manner that is not entirely dependent on apolipoprotein (a) phenotype. Based on these findings, it was suggested that international harmonization of methods, along with further improvements in methodological techniques, is necessary.

[0038] This disclosure demonstrates that LPA-KIV4 can be successfully used to measure Lp(a) levels and is comparable to previously validated in-house research assays. Current data also show a significant correlation between the “true Lp(a)” vs. “total apo(a)” assays. In most settings, this type of distinction is not usually necessary because a minimum amount of circulating free apolipoprotein(a) is present. However, in the current era of PCSK9 inhibitors, these assays may provide a way to confirm the nature of circulating Lp(a) / apolipoprotein(a) particles, as suggested. LPA-KIV4 can be used alone or in combination with LPA-KIV9 antibodies.

[0039] This disclosure provides compositions and methods for measuring Lp(a) by binding to non-repeating epitopes, thereby eliminating inaccuracies and creating a standardizable method for measuring Lp(a). This disclosure also provides antibodies and antibody fragments that provide a more accurate measurement of Lp(a) content by binding only to non-repeating epitopes.

[0040] The term “approximately” means, when referring to measurable values ​​such as quantity or temporal duration, to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, for such variations are appropriate for carrying out the disclosed method or for describing the compositions herein. Furthermore, any value or range (e.g., less than 20 or similar terminology) expressly includes any integer between or up to such a value. Thus, for example, “1 to 5 mutations” expressly includes 1, 2, 3, 4, and / or 5 mutations.

[0041] The terms "antibody" and "immunoglobulin" are used interchangeably in their broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies insofar as they exhibit the desired biological activity), and also antibody fragments. Antibodies can be human antibodies, humanized antibodies, and / or affinity-mature antibodies.

[0042] Depending on the amino acid sequence of the constant domain of the antibody heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are publicly known.

[0043] An "antibody fragment" comprises only a portion of an intact antibody, which typically retains at least one, more commonly, or all, of the functions usually associated with that portion of the antibody when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (scFv), and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment contains the antigen-binding site of an intact antibody and therefore retains the ability to bind to its cognitive antigen. Typically, such an antibody fragment has all CDRs. In another embodiment, an antibody fragment, for example, one containing an Fc region, retains at least one of the biological functions usually associated with the Fc region when present in an intact antibody, such as FcR binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody whose in vivo half-life is substantially similar to that of the intact antibody. For example, such an antibody fragment may include an antigen-binding arm linked to an Fc sequence that can confer in vivo stability to the fragment.

[0044] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule, in their naturally occurring conformations, and usually determines the class to which the antibody belongs.

[0045] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules, in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains are the two main antibody light chain isotypes.

[0046] An "antigen" is a target to which an antibody can selectively bind. Target antigens can be polypeptides, carbohydrates, nucleic acids, lipids, haptens, small molecules, or other naturally occurring or synthetic compounds. In one embodiment of this disclosure, the antigen is Lp(a). In another embodiment, the antibody binds to an epitope on the antigen, including LVPPNVI (SEQ ID NO: 1) and CSDAEWTAFVPPNVILAPSLEAFFEQ (SEQ ID NO: 2).

[0047] As used herein, the term “array” generally refers to a binding island, a predetermined spatial arrangement of biomolecules, or a predetermined spatial arrangement of binding islands or biomolecules. An array according to this disclosure, including biomolecules immobilized on a surface, may also be called a “biomolecular array.” An array according to this disclosure, including a surface that has been activated, adapted, prepared, or modified to facilitate the binding of biomolecules to the surface, may also be called a “binding array.” Furthermore, the term “array” may be used herein to refer to multiple arrays arranged on a surface, for example, when the surface carries multiple copies of an array. Such a surface carrying multiple arrays may also be called a “multiple array” or “repeated array.” The use of the term “array” herein may encompass biomolecular arrays, binding arrays, multiple arrays, and any combination thereof, and the appropriate meaning will be clear from the context. A biological sample may include liquid or solid samples from any tissue of the body, including plasma.

[0048] The arrays of this disclosure include substrates. In this specification, “substrate” or “solid support” or other grammatical synonyms mean any material suitable for binding biomolecules and corresponding to at least one detection method. As will be understood by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, plastics, ceramics, and various other polymers. Furthermore, as is known in the art, substrates may be coated with any number of materials, including polymers such as dextran, acrylamide, gelatin, or agarose. Such coatings can facilitate the use of the array with biological samples derived from serum. In one embodiment, the biomolecule is an antibody or antibody fragment or derivative of this disclosure.

[0049] The planar arrays of this disclosure generally include addressable locations (e.g., “pads,” “addresses,” or “microlocations”) of biomolecules in an array format. The size of the array depends on the array's configuration and end-use. Arrays can be prepared containing from about two different biomolecules to thousands of biomolecules. In some embodiments, the compositions of this disclosure may not be in array format; that is, in some embodiments, compositions containing a single biomolecule can also be prepared. Furthermore, in some arrays, multiple substrates may be used, either of different or identical compositions. Thus, for example, a large planar array may contain multiple smaller substrates. In one embodiment, the biomolecule is an antibody or antibody fragment or derivative of this disclosure.

[0050] As an alternative to planar arrays, bead-based assays combined with flow cytometry have been developed to perform multiparametric immunoassays. In a bead-based assay system, biomolecules can be immobilized on addressable microspheres. Each biomolecule for each individual immunoassay is bound to a different type of microsphere (i.e., "microbeads"), and the immunoassay reaction occurs on the surface of the microsphere. Stained microspheres with individual fluorescence intensities are separately filled with the appropriate biomolecules. Different bead sets carrying different capture probes can be pooled as needed to generate custom bead arrays. The bead arrays are then incubated with the sample in a single reaction vessel to perform the immunoassay. In one embodiment, the biomolecule is the antibody or antibody fragment or derivative of the present disclosure.

[0051] The terms “anti-Lp(a) antibody” or “antibody that binds to Lp(a)” refer to an antibody that can bind to Lp(a) with sufficient affinity to be useful as a diagnostic and / or therapeutic agent that targets Lp(a). In some embodiments of this disclosure, the anti-Lp(a) antibody specifically binds to KIV4 without binding to KIV9 or KIV2 or plasminogen. In specific embodiments, the antibody or antibody fragment binds to an epitope containing or including LVPPNVI (SEQ ID NO: 1).

[0052] As used herein, “LPA-KIV9 antibody” means the monoclonal antibody and fragments thereof disclosed in PCT / US2021 / 029307 (International Publication / 2021 / 222181), which is incorporated herein by reference for all purposes. In particular, the LPA-KIV9 antibody binds to an epitope comprising or consisting of LETPTVV (SEQ ID NO: 7) or CSETESGVLETPTVVPVPSMEAH (SEQ ID NO: 8).

[0053] A "blocking" antibody or "antagonist" antibody inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of an antigen. In one embodiment, a blocking antibody binds to its antigen but does not bind to FcR or induce any ADCC. Therefore, in one embodiment, an antibody or antibody fragment binds to its antigen and prevents or inhibits its biological activity, but the antibody or antibody fragment itself does not induce the immune system or has a limited effect on the immune system.

[0054] "Binding affinity" generally refers to the sum of the non-covalent interactions between the binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule "X" for its partner "Y" is generally expressed by the dissociation constant (K). d Affinity can be expressed by the following: Affinity can be measured by common methods well known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure.

[0055] "To bind to the same epitope as ~" means the ability of an antibody, scFv, or other antigen-binding domain to bind to a target antigen that has the same epitope as the exemplified antibody, scFv, or other antigen-binding domain. For example, the epitopes of the exemplified antibody, scFv, or other binder and other antibodies can be determined using standard epitope mapping techniques. Epitope mapping techniques well known in the art include Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (edited by Glenn E. Morris, 1996), Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined, for example, by simultaneously synthesizing a number of peptides corresponding to parts of a protein molecule on a solid support and reacting the antibody with the peptides while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871, Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002, Geysen et al., (1985) Proc. Natl. Acad. Sci. USA 82:78-182, and Geysen et al., (1986) Mol. lmmunol. 23:709-715. The epitopes to which the antibodies or antibody fragments of this disclosure are bound can be determined by an epitope binning assay. Epitope binning is a competitive immunoassay used to characterize and then sort a library of monoclonal antibodies against a target protein. Antibodies against similar targets are tested in a pairing manner against all other antibodies in the library to determine whether the antibodies block the binding of each other to the antigen's epitopes. After each antibody has a profile created against all other antibodies in the library, a competitive blockage profile is created for each antibody against the others in the library. Closely related binning profiles indicate that antibodies have the same or closely related epitopes and are "binned" together.Similarly, conformational epitopes can be readily identified by determining the spatial conformation of amino acids, for example, by hydrogen / deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. Antigenic regions of proteins can also be identified using standard antigenicity and hydroxylogram plots, such as those calculated using the Omiga version 1.0 software program available from Oxford Molecular Group. This computer program employs the Hopp / Woods method (Hopp et al. (1981) Proc. Natl. Acad. Sci USA 78:3824-3828) for determining antigenicity profiles, and the Kyte-Doolittle technique (Kyte et al. (1982) J. Mol. Bioi. 157:105-132) for hydropathic plots. To determine whether a selected monoclonal antibody against a target (e.g., Lp(a)) binds to a unique epitope, each antibody can be biotinylated using commercially available reagents. Competitive studies using unlabeled and biotinylated monoclonal antibodies can be performed using Lp(a) coated ELISA plates. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.

[0056] The term "biological sample" encompasses a variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. This definition includes blood, plasma, serum, sputum, cerebrospinal fluid, urine, and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells obtained therefrom, and their offspring. This definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or concentration of certain components such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes. Sources of biological samples may include solid tissues from fresh, frozen, and / or preserved organ or tissue samples or biopsies or aspirates, blood or any blood component, bodily fluids such as cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid, or cells from any point in time of the subject's pregnancy or development. In some embodiments, biological samples are obtained from a site of inflammation, a site of tumor, or a site of coronary artery disease or vascular disease. Biological samples may contain compounds that are not naturally mixed with natural tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics.

[0057] "Conservative substitution" or "conservative array modification" refers to amino acid modifications that do not significantly affect or change the binding properties or functions of proteins such as antibodies or antibody fragments. For example, "conservative array modification" refers to amino acid modifications that do not significantly affect or change the binding properties or functions of antibodies, antibody fragments, or non-immunoglobulin binding domains. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies or antibody fragments, non-immunoglobulin binding domains, or other proteins or polypeptides of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the anti-Lp(a) antibodies or fragments of the present disclosure can be replaced with other amino acid residues from the same side chain family, and the modified antibodies or antibody fragments can be tested using the binding and / or functional assays described herein.

[0058] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment has a heavy chain variable domain (V L ) connected to a light chain variable domain (V H ) on the same polypeptide chain (V H -V L) are included in ). By using a linker that is too short to allow pairing between two domains on the same strand, these domains are forced to pair with complementary domains on another strand, forming two antigen-binding sites. Diabodies are described in more detail, for example, European Patent No. 404,097, International Publication No. 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0059] "Disorder" or "disease" is any condition for which one would benefit from treatment or diagnosis by the substance / molecule or method of the Disclosure. This includes chronic and acute disorders or diseases, including their pathological conditions that make mammals susceptible to the disorder in question. Non-limiting examples of disorders treated herein include cardiovascular diseases, stenosis, liver diseases (e.g., NASH or NALFD), Kawasaki disease, age-related diseases and disorders, and aging or inflammatory diseases resulting from Lp(a).

[0060] Where used herein, "derived from" indicates a relationship between a first molecule and a second molecule. This generally refers to a structural similarity between the first and second molecules and does not imply or include any limitations on the process or source of the first molecule derived from the second molecule. For example, in the case of an antibody fragment derived from an antibody molecule, the antibody fragment retains sufficient antibody structure to have the necessary function, namely, the ability to bind to an antigen. This does not imply or include any specific process that produces the antibody fragment.

[0061] Antibody "effector function" refers to the biological activity resulting from the Fc region of an antibody, and varies depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0062] As used herein, “epitope” is defined as a portion of an antigen capable of inducing an immune response, or a portion of an antigen that binds to an antibody or antibody fragment. An epitope may be a protein sequence or a subsequence. In one embodiment, the epitope of this disclosure is a peptide comprising or consisting of SEQ ID NO: 1.

[0063] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by a host cell or in an in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0064] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, FcR is a natural human FcR. In some embodiments, FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors (including allelic variants and alternative splicing forms of these receptors). FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, primarily differing in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are outlined, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed herein by the term “FcR”.

[0065] The Fc receptor is also involved in the regulation of homeostasis of the neonatal receptor FcRn (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)), which is responsible for the transfer of maternal IgG to the fetus, and immunoglobulins. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997), Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997), Hinton et al., J.Biol.Chem. 279(8):6213-6216 (2004), International Publication No. 2004 / 92219 (Hinton et al.)).

[0066] In vivo binding to human FcRn and the serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered with polypeptides containing the variant Fc region. International Publication No. 2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001) (each of these publications is incorporated herein by reference in whole).

[0067] As used herein, the term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions.

[0068] A "functional Fc region" possesses the "effector function" of a natural sequence Fc region. Such effector function generally requires the Fc region to be combined with a domain (e.g., an antibody-variable domain) and can be evaluated using various assays (as described, for example, in the references cited herein).

[0069] "Natural sequence Fc region" refers to a region containing amino acid sequences identical to those found in nature. Natural sequence human Fc regions include natural sequence human IgG1 Fc regions (non-A and A allotypes), natural sequence human IgG2 Fc regions, natural sequence human IgG3 Fc regions, and natural sequence human IgG4 Fc regions, as well as their naturally occurring variants.

[0070] "Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen-binding site. In double-stranded Fv species, this region consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly and non-covalently associated. In single-stranded Fv (scFv) species, the one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that of double-stranded Fv species. In this configuration, the three hypervariable regions (sometimes called HVR or CDR) of each variable domain interact to form V H -V L The antigen-binding site is defined on the surface of the dimer. Collectively, six HVRs (or CDRs) confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three antigen-specific HVRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0071] A “Fab fragment” refers to an antibody fragment containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. A Fab' fragment differs from a Fab fragment by adding a few residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the heretical designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. F(ab')2 antibody fragments were originally constructed as pairs of Fab' fragments with hinge cysteines in between. Other chemical bindings of antibody fragments are also known.

[0072] Papain digestion of the antibody produces two identical antigen-binding Fab fragments, each with a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites that can still crosslink antigens.

[0073] The “framework” or “FR” residues are the variable domain residues other than the HVR (or CDR) residues as defined herein. Framework residues are typically variable without affecting the binding specificity of the antibody. Conservative amino acid substitutions can be readily performed in the framework region of an antibody without affecting the binding specificity of the antibody. Therefore, this disclosure includes antibodies or antibody fragments in which the framework region can be altered by only 80%, 90%, 95%, 98%, or 99% identity from the reference sequence, provided that the CDR remains substantially unchanged, or as long as the CDR remains unchanged.

[0074] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing the smallest sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's HVR(CDR) are replaced by residues derived from the HVR(CDR) of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications may be made to further improve antibody performance. Generally, the humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the immunoglobulin constant region (Fc) of human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patents No. 6,982,321 and 7,087,409.

[0075] A “human antibody” is defined as an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody prepared using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. (See Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991), Marks et al., J.Mol.Biol., 222:581 (1991) (the entire text is incorporated herein by reference)). Methods for preparing human monoclonal antibodies are also available, as described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985), and Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001) (each of those references is incorporated herein by reference in its entirety). Human antibodies can be prepared by administering an antigen to transgenic animals, such as immunized xenomouses, which have been modified to produce such antibodies in response to antigen challenge but whose endogenous gene locus has been deactivated (see, for example, U.S. Patents 6,075,181 and 6,150,584). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) on human antibodies produced by human B-cell hybridoma technology (each of those references is incorporated herein by reference in its entirety).

[0076] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector functions. In certain embodiments, the cell expresses at least FcγRIII and performs ADCC effector functions (may be multiple). Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources (e.g., blood).

[0077] The terms “hypervariable region,” “HVR,” or “HV” (sometimes called “complementarity-determining region” (“CDR”)) as used herein refer to a region of the antibody variable domain whose sequence is hypervariable and / or which forms a structurally defined loop. Generally, antibodies have six HVRs (CDRs), V H There are three (H1, H2, H3), and V L It contains three (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the most diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000) and Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0078] "Isolated" antibodies, antibody fragments, etc., refer to antibodies, antibody fragments, or derivatives identified, isolated, and / or recovered from components of their natural environment. Contaminating components of the natural environment may include materials that interfere with the diagnostic or therapeutic use of the antibody or antibody fragment, and may include enzymes, hormones, lipids, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody or antibody fragment is purified (1) to over 95% by weight, typically over 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; and / or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies or antibody fragments include antibodies or antibody fragments in situ within recombinant cells because at least one component of the antibody's natural environment is absent. However, isolated antibodies or antibody fragments are typically prepared by at least one purification step.

[0079] An "isolated" nucleic acid molecule is a nucleic acid molecule identified and isolated from at least one contaminating nucleic acid molecule that normally associates in the natural source of antibody nucleic acids. An isolated nucleic acid molecule is in a form or setting other than that which is found in nature. Therefore, isolated nucleic acid molecules are distinguished from nucleic acid molecules present in natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained within cells that normally express antibodies, for example, such nucleic acid molecules are located in chromosomal positions different from those in natural cells.

[0080] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent, such as a nucleic acid probe or antibody or antibody fragment, to facilitate the detection of the reagent to which it is conjugated or fused. The label may be detectable in itself (e.g., radioisotope labeling, luminescence or fluorescence labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0081] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for any mutations that may exist in small amounts, such as naturally occurring mutations. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is not a mixture of distinct antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody containing a polypeptide sequence that binds to a target, and the target-binding polypeptide sequence is obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may involve selecting a unique clone from a plurality of clones, such as a hybridoma clone, a phage clone, or a pool of recombinant DNA clones. It should be understood that the selected target-binding sequence may be further modified, for example, to improve affinity for a target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce a multispecific antibody, and that an antibody containing a modified target-binding sequence is also a monoclonal antibody for the purposes of this disclosure. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are typically advantageous in that they are not contaminated by other immunoglobulins.

[0082] The modifier "monoclonal" indicates a characteristic of antibodies that they are obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody production by any specific method. For example, monoclonal antibodies used in accordance with this disclosure include, for example, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975), Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (e.g., see U.S. Patent No. 4,816,567), phage display technology (e.g., see Clackson et al., Nature, 352:624-628 (1991)), Marks et al. al., J.Mol.Biol.222:581-597(1992), Sidhu et al., J.Mol.Biol.338(2):299-310(2004), Lee et al. al., J.Mol.Biol.340(5):1073-1093(2004), Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004), and Lee et al.,J.Immunol.Methods 284(1-2):119-132(2004), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (e.g., International Publication No. 1998 / 24893, International Publication No. 1996 / 34096, International Publication No. 1996 / 33735, International Publication No. 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551(1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al., Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al., Nature It can be prepared by various techniques, including those described in Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0083] The term "monoclonal antibody" as used herein includes, in particular, "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include antibodies in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing a macaque monkey with the antigen of interest.

[0084] As used herein, “oligocyte” refers to a short, typically single-stranded polynucleotide, generally less than approximately 200 nucleotides in length, but not necessarily so. The terms “oligocyte” and “polynucleotide” are not mutually exclusive. The descriptions of polynucleotides herein are equally and fully applicable to oligonucleotides.

[0085] The term "operably linked" means a functional linkage or association between a first component and a second component such that each component can be functional. For example, being operably linked includes the expression of a regulatory sequence as a result of association with a heterogeneous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In the context of two operably linked polypeptides, the first polypeptide functions in a manner independent of any linkage, and the second polypeptide functions as if there were no linkage between the two.

[0086] In the context of two or more nucleic acid or polypeptide sequences, "percent identity" refers to two or more sequences that share a certain degree of similarity. Two sequences are "substantially identical" if, when compared and aligned to the maximum correspondence across a comparison window or specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the two sequences have a certain percentage of identical amino acid residues or nucleotides (for example, 60% identity across a specified region or, if not specified, across the entire sequence; optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity). Selectively, identity exists over a region of at least approximately 30 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0087] For sequence comparison, typically one sequence acts as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, sub-sequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence compared to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics, Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0088] Two examples of algorithms that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0089] The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17), which is incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:444-453), which is incorporated into the GAP program of the GCG software package (available at [www.]gcg.com), using either the Blossom 62 matrix or the PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0090] As used herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, where present, may be conjugated before or after the assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after synthesis, such as by conjugation with labels. Other types of modifications include, for example, "caps," substitutions of one or more naturally occurring nucleotides by analogues, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant portions such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those containing modified bonds (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Furthermore, any of the hydroxyl groups normally present in sugars can be substituted with, for example, phosphonic acid groups, phosphate groups, protected with standard protecting groups, activated to prepare additional binding to additional nucleotides, or conjugated into a solid or semi-solid state. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups having 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides may also include analogs of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methylribose, 2'-O-allylribose, 2'-fluororibose or 2'-azidoribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be substituted with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is substituted with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal") (where each R or R' is independently a substituted or unsubstituted alkyl (1-20C) optionally containing H, or an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl). Not all linkings in the polynucleotide need to be identical. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0091] The terms “protein” or “polypeptide,” as used interchangeably in this specification, comprise one or more chains of chemical building blocks called amino acids, linked together by chemical bonds called peptide bonds.

[0092] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are sequentially linked via, for example, a synthetic linker, for example, a short, flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv may have a vL variable region and a vH variable region in either order with respect to, for example, the N-terminus and C-terminus of the polypeptide, and the scFv may comprise a vL-linker-vH or a vH-linker-vL.

[0093] As used herein, the terms “substantially similar” or “substantially identical” mean that a person skilled in the art will know that the difference between two values ​​is such that the difference between those values ​​(e.g., K d This means a sufficiently high degree of similarity between two numerical values ​​(e.g., one relating to the antibody of this disclosure and the other relating to the reference antibody / comparative antibody) to such an extent that it is considered biologically and / or statistically insignificant or not at all within the context of the biological characteristics measured by the value. The difference between the two aforementioned values ​​is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10% as a function of the reference / comparative value.

[0094] As used herein, the phrases “substantially decreased,” “substantially increased,” or “substantially different” mean that a person skilled in the art will know that the difference between two values ​​is such that the difference between those values ​​(e.g., K d The difference between two numerical values ​​(generally one related to a molecule and the other to a reference / comparison molecule) is considered statistically significant within the context of the biological characteristics measured by the values. The difference between the two aforementioned values ​​is, for example, greater than 10%, greater than 20%, greater than 30%, greater than 40%, and / or greater than 50% as a function of the reference / comparison molecule values.

[0095] The term "variable" refers to the fact that certain portions of the variable domain differ significantly in sequence between antibodies, and these differences are used for the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions or hypervariable regions (CDRs or HVRs, used interchangeably herein) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The natural heavy-chain and light-chain variable domains each contain four FRs, primarily employing a three-sheet configuration, connected by three HVRs, forming loops that connect the three-sheet structures and, in some cases, form part of the three-sheet structure. The HVRs of each chain are held together in close proximity by the FRs, and together with the HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0096] A “variant Fc region” contains an amino acid sequence that differs from that of the natural sequence Fc region by at least one amino acid modification, typically one or more amino acid substitutions. Typically, a variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, typically about 1 to about 5 amino acid substitutions, compared to the natural sequence Fc region or the parent polypeptide Fc region. The variant Fc regions of the disclosure have at least about 80% homology, at least about 90% homology, and typically at least about 95% homology with the natural sequence Fc region and / or the parent polypeptide Fc region.

[0097] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector to which an additional DNA segment can be ligated into a viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors, which have bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell and can replicate together with the host genome. Furthermore, certain vectors can direct the expression of a gene to which they are operably ligated. Such vectors are referred herein as “expression vectors.” Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

[0098] Calcific aortic stenosis (CAVS) is a common form of acquired valvular heart disease, affecting 3% of the population over 75 years of age. While risk factors are similar for CAVS and atherosclerosis, over 50% of CAVS patients do not have clinically significant coronary artery disease, suggesting a related but unique pathophysiology. Surgical aortic valve replacement (SAVR) remains the gold standard treatment for most patients, but at least one-third of symptomatic CAVS patients may not undergo SAVR. To meet this clinical need, transcatheter aortic valve replacement (TAVR) is increasingly being used, but overall survival remains moderate due to advanced age and other comorbidities. With the aging population, the prevalence of CAVS is rapidly increasing, foreshadowing medical, financial, and ethical burdens on healthcare systems worldwide. Therefore, identifying the causal pathways mediating CAVS could provide novel targets for early treatment before end-stage disease. One of these pathways involves lipoprotein-associated phospholipase A2 (Lp-PLA2) and the lipoprotein(a) (Lp(a)) axis.

[0099] This disclosure provides antibodies, antibody fragments, and humanized antibodies that bind to Lp(a) with isoform-independent selectivity. Antibody fragments can be produced by conventional means such as enzymatic digestion or by recombinant techniques. In certain situations, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, which may lead to improved access to tumors, plaques, and diseased tissue. For a review of a particular antibody fragment, see Hudson et al. (2003) Nat. Med. 9:129-134, which is incorporated herein by reference in its entirety.

[0100] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were induced by the proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments are all expressed in and can be secreted from E. coli, enabling the easy production of large quantities of these fragments. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically conjugated to form F(ab')2 fragments (see, Carter et al., Bio / Technology 10:163-167 (1992) (the entire work is incorporated herein by reference)). According to another approach, the F(ab')2 fragment is isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with extended in vivo half-lives, containing salvage receptor-binding epitope residues, are described in U.S. Patent No. 5,869,046, which is incorporated herein by reference in its entirety. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv). See International Publication No. 93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458 (each of these publications is incorporated herein by reference in its entirety). Fv and scFv are the only species with intact binding sites lacking a constant region, and therefore they may be suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to result in the fusion of an effector protein at either the amino or carboxyl terminus of the scFv. See Borrebaeck, ed., “Antibody Engineering” (cited above). Antibody fragments can also be “linear antibodies,” as described, for example, in U.S. Patent No. 5,641,870.Such linear antibodies may have monospecificity or bispecificity.

[0101] Furthermore, by knowing the sequences of antibodies and antibody fragments, it is possible to develop the generation of chimeric antigen receptors (CARs) and derivatives (e.g., first-generation, second-generation, and third-generation CARs). Methods for cloning and generating CARs are known in the art. For example, this disclosure provides the nucleic acid and polypeptide sequences of monoclonal antibodies, as well as the respective CDR / HVR of the antibodies. Those skilled in the art can easily clone the binding domain into the transmembrane domain (with or without a linker) of a chimeric antigen receptor to generate a CAR with selectivity for Lp(a).

[0102] This disclosure also relates to a single-chain variable antibody fragment ("scFv") that selectively binds to lipoprotein (a) in an isoform-independent manner, V H , V L and provides a complementarity-determining region. The scFv of this disclosure is soluble and readily synthesizable. Furthermore, vectors containing sequences encoding the scFv disclosed herein have enabled the creation of transgenic mouse models for further research into various diseases and studies.

[0103] This disclosure provides biologically active specific antibody sequences and antibody sequence fragments, but is not limited to polypeptides having these specific sequences. In some embodiments, variants having percent sequence identity with the specific antibody sequences of this disclosure are also provided. In some embodiments, such variants may be selected for use based on one or more desired enhanced properties. It should be recognized that certain variants may have improved functionality associated with one desired property (e.g., binding affinity, avoidance of undesirable side effects) but less desirable functionality associated with another desired property (e.g., stability, specificity).

[0104] In some embodiments, modifications (may be multiple) to the amino acid sequences of antibodies and fragments described herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or fragment. Amino acid sequence variants of antibodies can be prepared by introducing appropriate changes to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody or antibody fragment, and / or insertions into residues and / or substitutions of residues. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct has the desired properties, including binding to at least Lp(a). Amino acid modifications can be introduced into the amino acid sequence of the antibody of interest at the time the sequence is constructed.

[0105] A useful method for identifying specific residues or regions of an antibody that are favorable sites for mutagenesis is called “alanine scanning mutagenesis,” as described by Cunningham and Wells (1989) Science, 244:1081-1085 (the entire work is incorporated herein by reference). Here, a target residue or group of residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituted with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) that affects the interaction between the amino acid and the antigen. These amino acid positions that demonstrate functional sensitivity to the substitution are then purified by introducing further or other variants at or for the substitution site. Thus, the sites for introducing amino acid sequence mutations are predetermined, but the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, Ala scanning or random mutagenesis is performed at the target codon or region, and the expressed immunoglobulin is screened for the desired activity.

[0106] Amino acid sequence insertions include fusions of amino and / or carboxyl terminals ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionine residue. Other insertion variants of antibody molecules include fusion of the antibody to the N-terminus or C-terminus with enzymes that increase the serum half-life of the antibody (e.g., for ADEPT) or polypeptides. Polyhistidine tags are also useful for purification.

[0107] In certain embodiments, the antibodies or antibody fragments of this disclosure are modified to increase or decrease the degree to which the antibody is glycosylated. Polypeptide glycosylation is typically either N-linked or O-linked. N-linking refers to the binding of an asparagine residue of the carbohydrate moiety to a side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for enzymatic binding of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (most commonly serine or threonine), although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0108] The addition or deletion of a glycosylation site to an antibody is conveniently achieved by modifying the amino acid sequence such that (in the case of an N-linked glycosylation site) one or more of the tripeptide sequences described above are created or removed. The modification can also be carried out by adding, deleting, or substituting one or more serine or threonine residues into the original antibody sequence (in the case of an O-linked glycosylation site).

[0109] If an antibody contains an Fc region, the carbohydrate bound to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides commonly bound by an N-bond to the Asn of the CH2 domain of the Fc region. (See, for example, Wright et al. (1997) TIBTECH 15:26-32, which is incorporated herein by reference in its entirety.) Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may be performed to produce antibody variants having certain improved properties.

[0110] For example, antibody variants are provided that have a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. Such variants may have improved ADCC function. See, for example, U.S. Patent Application Publication US2003 / 0157108 (Presta, L.) and U.S. Patent Application Publication 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include U.S. Patent Application Publication 2003 / 0157108, International Publication 2000 / 61739, International Publication 2001 / 29246, U.S. Patent Application Publication 2003 / 0115614, U.S. Patent Application Publication 2002 / 0164328, and U.S. Patent Application Publication 200 U.S. Patent Application Publication No. 4 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, International Publication No. 2003 / 085119, International Publication No. 2003 / 084570, International Publication No. 2005 / 035586. This includes International Publication No. 2005 / 035778, International Publication No. 2005 / 053742, International Publication No. 2002 / 031140, Okazaki et al. J.Mol.Biol.336:1239-1249(2004), and Yamane-Ohnuki et al. Biotech.Bioeng.87:614(2004) (each of these publications is incorporated herein by reference in its entirety).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986, U.S. Patent Application Publication No. 2003 / 0157108 A1, Presta, L., and International Publication No. 2004 / 056312 A1, Adams et al., particularly Example 11), as well as knockout cell lines such as those containing the alpha-1,6-fucosyltransferase gene, FUT8, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al.) See also al., Biotechnol. Bioeng., 94(4):680-688 (2006), and International Publication No. 2003 / 085107 (each of these documents is incorporated herein by reference in its entirety).

[0111] Antibody variants are further provided with bifid oligosaccharides, for example, a branched oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation function and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication No. 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.) (each of these documents is incorporated herein by reference in its entirety). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication No. 1997 / 30087 (Patel et al.), International Publication No. 1998 / 58964 (Raju, S.), and International Publication No. 1999 / 22764 (Raju, S.) (each of these publications is incorporated herein by reference in its entirety).

[0112] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that further improve or inhibit / reduce ADCC. Such substitutions may occur in combination with any of the above modifications.

[0113] In certain embodiments, this disclosure intends to describe antibody variants having some, but not all, effector functions, which are desirable candidates for many applications where the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In certain embodiments, the Fc activity of the antibody is measured to ensure that only the desired properties are maintained. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks certain binding but retains other bindings. Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I., et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), and U.S. Patent No. 5,821,337 (e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)) (each of these publications is incorporated herein by reference in its entirety). Alternatively, non-radioactive assay methods may be used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998) (the entire model is incorporated herein by reference). Clq binding assays may also be performed to confirm that the antibody is unable to bind Clq and therefore lacks CDC activity.To evaluate complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004) (each of these publications is incorporated herein by reference in its entirety)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol. 18(12):1759-1769 (2006) (the entirety is incorporated herein by reference)).

[0114] Other antibody variants having one or more amino acid substitutions are also provided. Sites targeted for substitutional mutagenesis include hypervariable regions, but FR modifications are also possible. Conservative substitutions can also be performed. Amino acid substitutions can be introduced into the antibody of interest, and the product is screened for desired activities such as improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0115] This disclosure provides an antibody or antibody fragment that can bind to Lp(a) in an isoform-independent manner, wherein the antibody or antibody fragment has a variable heavy chain (V H ) domain and / or variable light chain (V L ) including domains, (a)V H The domain contains an amino acid sequence that includes one, two, and three complementarity-determining regions (CDRs), and these CDRs include: (i) Sequence ID 9 or its variant, (ii) Sequence ID No. 10 or its variant, and (iii) Includes, but is not limited to, SEQ ID NO: 11 or its variants, (b)V LThe domain contains an amino acid sequence that includes one, two, or three complementarity-determining regions (CDRs), and these CDRs include: (i) Sequence ID 12 or its variant, (ii) Sequence ID No. 13 or its variant, and (iii) Includes, but is not limited to, SEQ ID NO: 14 or its variants.

[0116] In one embodiment, the antibody or antibody fragment comprises an amino acid sequence including a CDR containing SEQ ID NOs. 9, 10, and 11. H V containing an amino acid sequence including a domain and / or a CDR including SEQ ID NOs. 12, 13, and 14 L Includes the domain.

[0117] In one embodiment, the disclosure provides an antibody or scFv selected from the group consisting of antibodies or scFv having heavy chain domains and light chain domains including complementarity-determining regions of SEQ ID NOs. 9, 10, 11, 12, 13, and 14. In one embodiment, the scFv is linked to an Fc region.

[0118] In one embodiment, the disclosure provides an antibody comprising a light chain variable region described in SEQ ID NO: 16, lacking a signal peptide. In another embodiment, the disclosure provides an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 18, lacking a signal peptide.

[0119] In one embodiment, the disclosure provides an scFv comprising a linker between a light chain variable region and a heavy chain variable region. The linker can be any number of commonly used peptide linkers. In one embodiment, the linker comprises one or more repeating units of GGGS (SEQ ID NO: 19). The repeats of GGGS (SEQ ID NO: 19) may be two, three, four or more times.

[0120] In another embodiment, the disclosure provides an scFv comprising a light chain variable region of SEQ ID NO: 16 linked to the heavy chain variable region of SEQ ID NO: 18 by a peptide linker. In another embodiment, the disclosure provides an scFv comprising a light chain having a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16, and a heavy chain having a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18, comprising CDRs of SEQ ID NOs: 9, 10, 11, 12, 13, and 14, wherein the scFv selectively binds to lipoprotein (a).

[0121] In yet another embodiment, a fusion construct comprising a first domain or a variant or fragment thereof comprising SEQ ID NOs: 16 and / or 18 is operably linked to (i) a detectable label or (ii) a second domain comprising the polypeptide of interest. Those skilled in the art will recognize that such a fusion construct can be generated using chemical or molecular biological techniques that link a coding sequence comprising the sequence or variant thereof of SEQ ID NOs: 15 and / or 17 to, for example, the coding sequence of the polypeptide of interest. The coding sequence and domain may be separated by a linker or directly linked.

[0122] In yet another embodiment, the disclosure includes an scFv comprising a light chain variable region of SEQ ID NO: 16 linked to a heavy chain variable region of SEQ ID NO: 18 by a peptide linker.

[0123] Nucleic acid molecules encoding the amino acid sequences of antibodies, antibody fragments, and antibody variants are prepared by various methods known in the art. Methods for preparing variants include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or preparation of variant or non-variant versions of previously prepared antibodies by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0124] In certain embodiments, the Disclosure provides a polynucleotide encoding the heavy chain of an antibody or antibody fragment of the Disclosure, comprising (i) a sequence encoding the polypeptide of SEQ ID NO: 18, (ii) a sequence comprising SEQ ID NO: 17, or (iii) a sequence that is at least 80% identical and / or hybridizes to the sequence comprising SEQ ID NO: 17.

[0125] In certain embodiments, the Disclosure provides a polynucleotide encoding the light chain of an antibody or antibody fragment of the Disclosure, comprising (i) a sequence encoding the polypeptide of SEQ ID NO: 16, (ii) a sequence comprising SEQ ID NO: 15, or (iii) a sequence that is at least 80% identical and / or hybridizes to the sequence comprising SEQ ID NO: 15.

[0126] This disclosure further provides scFv disclosed herein, further comprising an antibody fragment crystallizable region ("Fc"). In certain embodiments, the Fc region is derived from a human or humanized antibody and may be modified to modulate the biological activity of the domain. The Fc region is the tail region of the antibody that interacts with cell surface receptors called Fc receptors and several proteins of the complement system. This property allows the antibody to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody, while in IgM and IgE, the Fc region has three heavy chain constant domains (C) in each polypeptide chain. HThe Fc region of IgG includes domains 2-4). The Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycan bound to this site is primarily a complex core-fucosylated dianthentory structure. Furthermore, small amounts of these N-glycans also have bifurcation GlcNAc and α-2,6-linked sialic acid residues. The other part of the antibody, called the Fab region, contains a variable region that defines specific targets to which the antibody can bind. The scFv of this disclosure consists of elements from the Fab region. In contrast, the Fc regions of all antibodies in a class are the same for each species, and they are constant rather than variable. Thus, the Fc region is sometimes called the “fragment constant region”. Thus, polynucleotide and polypeptide sequences encoding the Fc region for countless species have already been determined and will be known to those skilled in the art. In certain embodiments, this disclosure provides antibodies and antibody fragment polynucleotides, or variants thereof, encoding SEQ ID NOs. 16 and / or 18, further comprising polynucleotide sequences encoding the Fc region.

[0127] In certain embodiments, the disclosure provides polynucleotide sequences disclosed herein (e.g., SEQ ID NOs: 15 and / or 17) further comprising polynucleotide sequences encoding an Fc region.

[0128] In a further embodiment, the Disclosure provides a vector comprising a polynucleotide sequence encoding an antibody or antibody fragment, wherein the polynucleotide comprises a sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% sequence identity to SEQ ID NO: 15 and / or 17, and encoding a polypeptide that specifically binds to lipoprotein (a). In a further embodiment, the Disclosure provides a vector comprising a polynucleotide sequence encoding an antibody or antibody fragment, wherein the polynucleotide comprises a sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% sequence identity to SEQ ID NO: 15 and / or 17, and comprising the CDRs of SEQ ID NOs: 9, 10, 11, 12, 13, and 14.

[0129] The polynucleotide sequences encoding the polypeptide components of the antibodies or antibody fragments of this disclosure can be obtained using standard recombination techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the polypeptide-encoding sequences are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of this disclosure. The selection of a suitable vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed using the vector. Each vector contains a variety of components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, origins of replication, selection marker genes, promoters, ribosome-binding sites (RBS), signal sequences, heterologous nucleic acid inserts, and transcription termination sequences.

[0130] Generally, plasmid vectors containing replicons (or multiple replicons) and regulatory sequences derived from a species compatible with the host cell are used in association with these hosts. The vectors typically carry replication sites, as well as marking sequences that allow for phenotypic selection in transformed cells. For example, Escherichia coli is typically transformed using pBR322, a plasmid derived from the Escherichia coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing a convenient means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages also contain, or can be modified to contain, promoters that microorganisms can use for the expression of endogenous proteins. Examples of pBR322 derivatives used for the expression of specific antibodies are described in detail in U.S. Patent No. 5,648,237 by Carter et al.

[0131] Furthermore, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, bacteriophage vectors can be used to create recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0132] The expression vectors disclosed herein may comprise two or more promoter-cistron pairs encoding each of the polypeptide components. The promoter is a non-translational regulatory sequence located upstream (5' end) of the cistron and regulates its expression. Prokaryotic promoters are generally classified into two classes: inductive and constitutive. Inductive promoters are those that initiate an increase in the level of transcription of the cistron under their control in response to changes in culture conditions (e.g., presence or absence of nutrients or temperature changes).

[0133] Numerous promoters recognized by various potential host cells are well known. A selected promoter can be operably ligated to cistron DNA encoding a light or heavy chain by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both natural promoter sequences and many heterologous promoters can be used to induce amplification and / or expression of a target gene. In some embodiments, heterologous promoters are utilized because they generally allow for higher transcription and higher yields of the expressed target gene compared to natural target polypeptide promoters.

[0134] Promoter suitable for use with prokaryotic hosts include the PhoA promoter, the 3-galactamase and lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria (such as other known bacterial promoters or phage promoters) are equally suitable. Since their nucleotide sequences are publicly available, skilled technicians can ligate them into activatable cistrons encoding the target light and heavy chains using linkers or adapters to supply any required restriction enzyme sites (Siebenlist et al. (1980) Cell 20:269).

[0135] In one embodiment, each cistron in a recombinant vector contains a secretory signal sequence component that directs the transposition of the expressed polypeptide across the membrane. Generally, the signal sequence may be a component of the vector or a part of the target polypeptide DNA inserted into the vector (see Tables A and B and SEQ ID NOs. 16 and 18). The signal sequence selected for the purposes of this disclosure should be recognized and processed by the host cell (e.g., cleaved by a signal peptidase). In the case of prokaryotic host cells that do not recognize and process signal sequences specific to the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or the thermostable enterotoxin II (STII) reader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0136] In another embodiment, immunoglobulin production according to this disclosure can occur within the cytoplasm of the host cell and therefore does not require the presence of a secretory signaling sequence within each cistron. In this respect, the immunoglobulin light and heavy chains are expressed, folded, and assembled within the cytoplasm to form a functional immunoglobulin. Certain host strains (e.g., *Escherichia coli* trxB strain) provide cytoplasmic conditions favorable for disulfide bond formation, thereby enabling proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0137] Suitable prokaryotic host cells for expressing the antibodies of this disclosure include archaea and bacteria, such as Gram-negative or Gram-positive bacteria. Examples of useful bacteria include Escherichia species (e.g., Escherichia coli), bacilli (e.g., Bacillus subtilis), Enterobacteriaceae, Pseudomonas species (e.g., Pseudomonas aeruginosa), Salmonella tiphimurium, Serratia marcescens, Klebsiella species, Proteus, Shigella, Rhizobia, Vitreosilla, or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, Escherichia coli cells are used as the host of this disclosure. Examples of Escherichia coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219, ATCC deposit number 27,325) and its derivatives, and strain 33D3 (US 5,639,635). Other strains and their derivatives, such as Escherichia coli 294 (ATCC 31,446), Escherichia coli B, Escherichia coli X 1776 (ATCC 31,537), and Escherichia coli RV308, are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above bacteria having defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally important to select appropriate bacteria considering the replication potential of the replicons in bacterial cells. For example, when supplying replicons using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410, Escherichia coli, Serratia, or Salmonella species can be suitable hosts. Typically, the host cells should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0138] Host cells are transformed with the expression vector described above and cultured in a conventional nutrient medium appropriately modified for promoter induction, transformant selection, or amplification of the gene encoding the desired sequence.

[0139] Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate, either as an extrachromosomal element or by chromosomal integrants. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is typically used for bacterial cells that contain a substantial cell wall barrier. Another method of transformation uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0140] The prokaryotic cells used to produce the polypeptides of this disclosure are known in the art and are grown in a medium suitable for culturing selected host cells. Examples of suitable media include Luria broth (LB) and necessary nutritional supplements. In some embodiments, the medium also includes a selective agent selected based on the construction of the expression vector to selectively enable the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0141] Any other necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included in appropriate concentrations, either alone or as a mixture with other supplements or media, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolates, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at an appropriate temperature.

[0142] In one embodiment, the expressed polypeptide is secreted into the periplasm of host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganism by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell fragments or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred to a culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated to further purify the produced protein. The expressed polypeptide can be further isolated and identified using known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting assays.

[0143] Large-scale or small-scale fermentation can be used and optimized using skills well known in this art.

[0144] Standard protein purification methods known in the art can be used. The following procedure is an example of a suitable purification procedure, namely fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or a cation exchange resin such as DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration.

[0145] The Disclosure further provides an expression vector replicable in a prokaryotic or eukaryotic system, comprising at least one polynucleotide encoding a light chain and / or heavy chain as disclosed herein. In one embodiment, the Disclosure provides an expression vector comprising a polynucleotide sequence encoding a first sequence having at least 100%, at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% identity to SEQ ID NO: 16. In another embodiment, a second vector or identical vector comprises a second polynucleotide encoding a second sequence having at least 100%, at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% identity to SEQ ID NO: 18. In a further embodiment, the vector, when expressed, yields a soluble form of scFv or antibody and has the ability to bind to Lp(a) in an isoform-independent manner.

[0146] This disclosure further provides expression vectors encoding scFv or antibodies disclosed herein, which are transferred into a suitable host organism. Suitable host organisms are microorganisms, yeasts, or mammalian cell lines. Typically, mammalian cell lines are monocyte-derived (e.g., macrophages, monocytes, and neutrophils), lymphocyte-derived (e.g., myeloma, hybridoma, and normal immortalized B cells), parenchymal cells (e.g., hepatocytes), and non-parenchymal cells (e.g., astrocytes).

[0147] The scFv disclosed herein, including the Fc region, can be used for a number of possible therapeutic applications. For example, they can be used as anti-atherosclerotic agents.

[0148] In one embodiment of this disclosure, a method is provided for determining the Lp(a) level and / or predisposition to coronary artery disease in a subject. This method comprises determining the plasma Lp(a) level of the subject and correlating the Lp(a) level to a control or normal level, where an increased level of Lp(a) indicates predisposition to coronary artery disease. The amount of Lp(a) can be detected using an antibody, antibody fragment, or binding domain containing a CDR for light and heavy chains described herein that specifically binds to Lp(a) in an isoform-independent manner. This disclosure further utilizes a secondary antibody that binds to OxPL such as EO6, MB47, LPA4, or a secondary antibody that binds to another epitope of Lp(a) (e.g., KIV9). See International Publication 2021 / 222181 (the disclosure of which is incorporated herein in its entirety by reference).

[0149] This method can be performed on a biological sample obtained from a subject. The biological sample can be, for example, blood, serum, or plasma.

[0150] In some embodiments, antibodies, antibody fragments, or binding domains are immobilized on a substrate to form an array. In one embodiment, the substrate is a microtiter plate, glass slide, nylon slide, nitrocellulose membrane, or PVDF membrane. In another embodiment, the substrate is latex beads, glass beads, or microspheres.

[0151] Exemplary biochemical tests for identifying Lp(a) use standardized test formats such as enzyme-linked immunosorbent assays or ELISA tests, but the information provided herein may be applied to the development of other biochemical or diagnostic tests, and is not limited to the development of ELISA tests. A variety of commercially available ELISA kits are available. Other assay formats, such as assay formats containing latex beads, can be adapted to use the antibodies or antibody fragments of this disclosure.

[0152] In one embodiment, the immunoassay can be performed by first capturing Lp(a) on a microtiter well with the antibody of the present disclosure, and then detecting Lp(a) with a labeled antibody. In another embodiment, a bead-based assay can be used with fluorescent barcoded beads containing antibodies or antibody fragments linked to beads, where the assay is examined using flow cytometry. Bead-based immunoassays enable several times higher multiple detection of targets than is possible with conventional ELISA formats, saving time and samples.

[0153] This disclosure also provides that the scFv or antibody of this disclosure can be used as an anti-atherosclerotic agent for the treatment of cardiovascular disease and CAVS. The scFv or antibody of this disclosure can be used to treat patients at high risk of coronary artery disease ("CAD") by inhibiting clot formation induced by the prothrombotic activity of Lp(a).

[0154] The scFVs and antibodies disclosed herein bind to Lp(a) in an isoform-independent manner and can block plaque, clot formation, and / or atheroma formation. In vivo use of the scFVs, antibodies, or antibody fragments disclosed herein is expected to be useful for blocking Lp(a) biological effects in many different situations.

[0155] As used herein, “treatment” refers to a clinical intervention in an attempt to alter the natural course of the individual or cell being treated, which may be carried out either for prevention or during the course of clinicopathology. Desired effects of treatment include prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the scFV, antibody, or antibody fragment of this disclosure is used to delay the onset of disease or disability.

[0156] An “individual,” “subject,” or “patient” is a vertebrate. In certain embodiments, a vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cattle, horses, and sus), sport animals, pet / companion animals (such as cats and dogs), primates, mice, and rats. In certain embodiments, a mammal is a human.

[0157] "Effective dose" refers to the effective amount in terms of dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0158] The “therapeutic effective dose” of a substance / molecule, agonist, or antagonist in this disclosure may vary depending on factors such as the individual’s disease state, age, sex, and weight, as well as the substance / molecule, agonist, or antagonist’s ability to induce a desired response in the individual. The therapeutic effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the substance / molecule, agonist, or antagonist. The “preventive effective dose” refers to the effective dose and duration required to achieve the desired preventive outcome. Typically, but not always, the preventive effective dose is less than the therapeutic effective dose because the preventive dose is used in the subject before or earlier in the disease.

[0159] Therapeutic formulations comprising the antibody or fragment thereof of this disclosure are prepared for storage by mixing the antibody or fragment having the desired purity with any physiologically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacy 20th edition (2000)) in the form of an aqueous solution, lyophilized formulation, or other dry formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, histidine and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), and low molecular weight (about 10 residues). The material comprises polypeptides (less than 100%), proteins such as serum albumin, gelatin, or immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants or polyethylene glycol (PEG).

[0160] The formulations described herein may also contain one or more active compounds necessary for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0161] These active ingredients can be encapsulated, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington: The Science and Practice of Pharmacy 20th edition (2000).

[0162] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0163] Sustained-release preparations can be prepared. Preferred examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing the immunoglobulins of this disclosure, the matrix being in the form of a molded article, e.g., a film, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT (an injectable microsphere composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over more than 100 days, while certain hydrogels release proteins over shorter periods. If encapsulated immunoglobulins remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, potentially leading to loss of biological activity and altered immunogenicity. Depending on the mechanism involved, reasonable strategies for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thio-disulfide exchange, stabilization can be achieved through modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content, use of appropriate additives, and development of specific polymer matrix compositions.

[0164] The anti-Lp(a) antibodies of this disclosure can be produced transgenically through the generation of mammals or plants that are transgenic to the target immunoglobulin heavy and light chain sequences, and the production of antibodies therefrom in a recoverable form. In connection with transgenic production in mammals, anti-Lp(a) antibodies can be produced in the milk of goats, cattle, or other mammals and recovered therefrom. See, for example, U.S. Patents 5,827,690, 5,756,687, 5,750,172, and 5,741,957 (incorporated herein by reference).

[0165] In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding the anti-Lp(a) antibody or a fragment thereof (e.g., SEQ ID NO: 15 and / or 17) into an animal or plant using standard transgenic techniques. See Hogan and U.S. Patent No. 6,417,429. Transgenic cells used to produce transgenic animals may be embryonic stem cells, somatic cells, or fertilized eggs. Transgenic non-human organisms may be chimeric, non-chimeric heterozygotes, and non-chimeric homozygotes. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1999), Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000), and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999) (all incorporated herein by reference). In some embodiments, the transgenic non-human animal has its target disrupted and replaced by a target construct encoding the heavy and / or light chain of interest. In another embodiment, the transgenic animal comprises and expresses nucleic acid molecules encoding heavy and light chains that specifically bind to an epitope on Lp(a). In some embodiments, the transgenic animal comprises nucleic acid molecules encoding a modified antibody, such as a single-chain antibody, a chimeric antibody, or a humanized antibody. Anti-Lp(a) antibodies can be produced in any transgenic animal. In another embodiment, the non-human animal is a mouse, rat, sheep, pig, goat, cattle, or horse. The non-human transgenic animal expresses the encoded polypeptide in blood, milk, urine, saliva, tears, mucus, and other bodily fluids.

[0166] The following embodiments are intended to illustrate the disclosure and not to limit it. They are typical of what may be used, but alternatively, other procedures known to those skilled in the art may be used. [Examples]

[0167] The following peptides were produced.

number

[0168] The peptide was resuspended in sterile PBS (1 mg / ml). The resuspended peptide was used as an antigen for immunization.

[0169] BALB / C female mice (n=2) were injected with SQ along with 250 ug of antigen (50 ug of five pooled peptides) in 250 ul of incomplete Freund's adjuvant on days 0, 14, and 28. IgG titers for K8 were tested in immunized mouse plasma on day 38 (Figure 2), followed by a fourth SQ boost (250 ug / 250 ul in IcFA) on day 42.

[0170] Three days after IV boost (125 ug of peptide mixture in PBS), splenocytes from mouse #2 were fused with myeloma cells (p3X63Ag8.653) using the ClonaCell®-HY hybridoma kit (StemCell Technologies (Cambridge MA)), and the fused cells were resuspended in semi-solid HAT hybridoma selective medium.

[0171] After 10 days, approximately 300 colonies were transferred to ClonaCell®-HY growth medium (DMEM, pre-selected serum, HT, gentamicin, and supplements).

[0172] To select colonies expressing apo(a)-specific antibodies without binding to plasminogen or KIV2, conditioned media from hybridoma cells were screened against an antigen array by ELISA (Figure 3).

[0173]

number

[0174]

number

[0175] Many embodiments are described herein. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. An antibody or antibody fragment that recognizes and binds to lipoprotein (a), wherein the antibody or antibody fragment has a variable heavy chain (V Ｈ ) domain and / or variable light chain (V Ｌ ) including domains, (a) The above V Ｈ The domain includes an amino acid sequence comprising a complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 9 or its variant, SEQ ID NO: 10 or its variant, and SEQ ID NO: 11 or its variant. (b) The above V Ｌ An antibody or antibody fragment comprising an amino acid sequence in which the domain includes a complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 12 or its variant, SEQ ID NO: 13 or its variant, and SEQ ID NO: 14 or its variant.

2. The aforementioned V Ｈ The domain contains the amino acid sequence of SEQ ID NO: 18, and / or the V Ｌ The antibody or antibody fragment according to claim 1, wherein the domain comprises the amino acid sequence of SEQ ID NO:

16.

3. The antibody or antibody fragment according to claim 1 or 2, wherein the antibody or antibody fragment is selected from the group consisting of antibodies or scFv having a heavy chain domain and a light chain domain including the complementarity determining region of SEQ ID NOs. 9, 10, 11, 12, 13, and 14.

4. The antibody or antibody fragment according to claim 1, 2, or 3, wherein the heavy chain domain and the light chain domain are linked to the Fc region.

5. The antibody fragment according to claim 1, comprising a single-chain variable fragment ("scFv") that recognizes the KIV4 epitope of lipoprotein (a).

6. The scFv according to claim 5, wherein the scFv is soluble under physiological conditions.

7. The scFv according to claim 5 or 6, wherein the scFv includes a light chain variable region having a sequence that is at least 95% identical to the sequence shown in sequence number 16.

8. The scFv according to claim 7, wherein the scFv includes a heavy chain variable region having a sequence that is at least 95% identical to the sequence shown in Sequence ID No.

18.

9. An antibody comprising the variable light chain and variable heavy chain described in claim 1.

10. The antibody according to claim 9, wherein the antibody is a chimeric antibody.

11. A pharmaceutical composition comprising the antibody according to claim 9 or 10, and a carrier, an excipient, or a stabilizer.

12. An antibody or antibody fragment according to claim 1, bound to a solid substrate.

13. The antibody or antibody fragment according to claim 1, operably linked to a detectable label.

14. A polynucleotide encoding an antibody, antibody fragment, variable light chain, variable heavy chain, or scFv according to claim 1 or 5.

15. The polynucleotide according to claim 14, wherein the polynucleotide comprises (i) a sequence encoding an antibody that hybridizes to a nucleic acid consisting of sequence number 1 and / or 9 and binds to KIV4 of Lp(a), and (ii) a sequence encoding a polypeptide having the sequence of sequence number 16 and / or 18.

16. A vector comprising the polynucleotide described in claim 15.

17. A host cell transformed with the polynucleotide described in claim 15.

18. A host cell transformed with the vector described in claim 16.

19. A transgenic animal comprising the polynucleotide described in claim 15.

20. The transgenic animal according to claim 19, wherein the animal is a mouse.

21. The transgenic animal according to claim 19 or 20, wherein the scFv or antibody is expressed from hepatocytes and / or macrophages.

22. The transgenic animal according to claim 19 or 20, wherein the transgenic animal is used to model coronary artery disease or impairment and / or the effects of atherosclerosis.

23. A method for treating or preventing coronary artery disease or calcified aortic stenosis, comprising administering an antibody or antibody fragment or scFv described in claim 1 or 5 to a subject having coronary artery disease or at risk of having coronary artery disease.

24. A chimeric antigen receptor comprising a binding domain having a VH domain and / or a VL domain as described in claim 1.

25. CAR-T cells comprising the chimeric antigen receptor described in claim 24.

26. An isolated peptide sequence consisting of SEQ ID NO: 1, with 1 to 10 amino acids linked to the N-terminus or C-terminus.

27. An immunological composition comprising the peptide and adjuvant of SEQ ID NO:

1.

28. A pharmaceutical composition comprising the isolated peptide described in claim 26.

29. An antigen epitope specifically recognized by a monoclonal antibody, wherein the monoclonal antibody includes the complementarity-determining regions of SEQ ID NOs: 9, 10, 11, 12, 13, and 14.

30. An antigen epitope containing the sequence of Sequence ID No. 1, or a sequence that is at least 95% to 99% identical thereto.

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