Polyclonal antibodies for measuring lipoprotein(a) concentration

Abstract

The present disclosure relates to the field of biology. In particular, the present disclosure relates to polyclonal antibodies specific for lipoprotein (a) and methods of preparation and use related thereto. In some aspects, the anti-Lp(a) polyclonal antibodies are generated using a non-human animal host inoculated with a recombinant antigen comprising a sequence of an endogenous protein or portion thereof, modified to include one or more epitopes representative of individual amino acids or segments of amino acids in the polypeptide sequence of human Lp(a).

Description

POLYCLONAL ANTIBODIES FOR MEASURING LIPOPROTEIN(A) CONCENTRATIONCross-Reference to Related Application

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 562,144, filed March 6, 2024, the content of which is hereby incorporated by reference in its entirety.Reference to Sequence Listing

[0002] The contents of the electronic sequence listing (“039062.00175. xml” (17 KB), which was created on March 6, 2025, is hereby incorporated by reference in its entirety.Technical Field

[0003] The present disclosure relates to the field of biology. In particular, the present disclosure relates to polyclonal antibodies for lipoprotein (a) and methods of preparation and use related thereto.Background

[0004] Lipoprotein(a) (“Lp(a)”) is a liver-derived macromolecular complex found in human plasma that combines structural elements from the lipoprotein and blood clotting systems and that is associated with premature coronary heart disease and stroke. It is assembled from a low-density lipoprotein (“LDL”) particle containing one molecule of apolipoprotein B- 100 covalently linked to apolipoprotein(a) (“apo(a)”), a plasminogen-like glycoprotein characterized by a series of tandem kringle domains. Each kringle domain comprises a tripleloop topology with three conserved disulfide bridges. The kringle domains of apo(a) share a high homology to the kringle type IV and kringle type V domains of plasminogen, and are thus referred to as “type IV” and “type V” kringle domains, respectively.

[0005] In particular, apo(a) is composed of 10 different kringle type IV domains (classified as subtypes 1 to 10) which share high inter-domain homology, covalently linked to a single kringle type V domain, and a serine protease domain. Nine of the ten kringle type IV domains (subtypes IVi, IV3, IV4, IVs, IVe, IV7, IVs, IV9, IV10) are present as a single copy, while the number of kringle type IV, subtype 2 (IV2) domains can vary between 1 to >40 copies. This variability is responsible for the great heterogeneity of isoforms present in the general population and is associated with differences in the risk of cardiovascular disease, as well as other medical conditions. Kronenberg, F., et al. “Lipoprotein (a): resurrected by genetics.” J. Internal Medicine 273.1 (2013): 6-30. For example, low molecular weight isoforms (less than or equal to 22 total kringle type IV repeats) have been shown to be associated with a four to five-fold increase in Lp(a) plasma concentration, as compared to higher molecular weight isoforms. Kronenberg, F., et al. “Human genetics and the causal role of lipoprotein (a) for various diseases.” Cardiovascular Drugs and Therapy 30.1 (2016): 87-100.

[0006] The complete molecular structure of apo(a) has yet to be published. However, the structures of several of its kringle type IV domains, and the sole kringle type V domain, are available. In particular, structures for subtypes IVe, IV7, IVs, IV10, and type V, have been deposited in the Protein Data Bank (PDB) database as PDB identification numbers: 1 JFN, 1171, 2FEB, 1KIV, and 4BVV, respectively. All of these kringle domains show a very high degree of structural homology, with the highest found among the subtypes of kringle type IV domains, which each share a common kringle domain fold consisting of a single polypeptide chain with a length of approximately 80 amino acids, and a disulfide bond pattern comprising six cysteine residues joined in a 1-6, 2-4, and 3-5 scheme. Trexler, M., et al. “Folding autonomy of the kringle 4 fragment of human plasminogen.” Proceedings of the National Academy of Sciences 80.9 (1983): 2457-2461.

[0007] In vitro and animal studies have implicated plasma Lp(a) as a key biomarker for processes related to cardiovascular disease, including atherosclerosis. Boffa, M. et al. “Lipoprotein (a) as a risk factor for atherosclerosis and thrombosis: mechanistic insights from animal models.” Clinical Biochemistry 37.5 (2004): 333-343. In particular, higher concentrations of plasma Lp(a) tend to be associated with increased risk for future cardiovascular disease. Despite notable advances in the prevention and treatment of cardiovascular disease, it remains a leading cause of morbidity and mortality. Accordingly, there exists a need for tools and methods for the detection and measurement of Lp(a), for example, for use in assays to identify subjects that may be at increased risk of cardiovascular disease and other medical conditions associated with Lp(a).Brief Summary of the Disclosure

[0008] Provided herein are recombinant antigens, and methods for making and using the same, including methods for generating polyclonal antibodies specific for selected epitopes. In some aspects, these constructs and methods can be used to generate polyclonal antibodies specific for human Lp(a). For example, a non-human animal host (for example, a rabbit) may be inoculated with a recombinant antigen comprising one or more directly or indirectly-linked human Lp(a) kringle type IV domains, or fragments or variants thereof. In other aspects, the non-human animal host may be inoculated with a chimeric antigen comprising least one non- human plasminogen kringle type IV domain that has been modified to incorporate one or more heterologous amino acids (for example, to mimic epitopes present on a human Lp(a) kringle type IV domain). These and other constructs, as well as related methods of preparation and use, shall be described in further detail herein and illustrated in the accompanying drawings.

[0009] In a first general aspect, the disclosure provides recombinant proteins that may, for example, be used as antigens to generate polyclonal antibodies as described herein.

[0010] In some aspects, the recombinant protein may comprise one or more directly or indirectly linked human kringle type IV domains, or fragments thereof. In some aspects, each fragment may comprise at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 contiguous amino acids of a human kringle type IV domain. In some aspects, each human kringle type IV domain comprises a polypeptide sequence represented by any one of SEQ ID NOs: 1-9, or a polypeptide sequence sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with any one of SEQ ID NOs: 1-9. In some aspects, the recombinant protein comprises 1-10 of the directly or indirectly linked human kringle type IV domains.

[0011] In some aspects, the recombinant protein may comprise a chimeric construct comprising a non-human protein sequence (for example, the sequence of a non-human plasminogen), or a fragment or variant thereof, which has been modified to incorporate one or more epitopes designed to generate polyclonal antibodies specific for human Lp(a).

[0012] In some aspects, the recombinant protein may comprise one or more non-human plasminogen kringle type IV domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 10, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 10. In some aspects, the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NO 10. In some aspects, the one or more amino acid substitutions comprise one or more of the amino acid substitutions shown in FIGS. 6-16.

[0013] In some aspects, the recombinant protein may comprise one or more non-human plasminogen kringle type II domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 11, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 11. In some aspects, the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQID NO 11. In some aspects, the one or more amino acid substitution comprise one or more of the amino acid substitutions shown in FIGS. 6-16.

[0014] In some aspects, the recombinant protein may comprise one or more non-human plasminogen kringle type III domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 12, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 12. In some aspects, the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NO 12. In some aspects, the one or more amino acid substitution comprise one or more of the amino acid substitutions shown in FIGS. 6-16.

[0015] In some aspects, the recombinant protein may comprise one or more non-human plasminogen kringle type V domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 13, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 13. In some aspects, the fragment comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 contiguous amino acids of SEQ ID NO 13. In some aspects, the one or more amino acid substitution comprise one or more of the amino acid substitutions shown in FIGS. 6-16.

[0016] In some aspects, the recombinant protein may comprise one or more non-human plasminogen serine protease domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 14, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 14. In some aspects, the fragment comprises at least , 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 or 230 contiguous amino acids of SEQ ID NO 14. In some aspects, the one or more amino acid substitution comprise one or more of the amino acid substitutions shown in FIGS. 6-16.

[0017] In some aspects, the recombinant protein may comprise any combination of nonhuman plasminogen kringle type II, III, IV, and V domains, or plasminogen serine protease domains, with any of the amino acid substitutions described herein. In some aspects, the plasminogen kringle type II, III, IV, and V domain(s), and / or the plasminogen serine protease domain(s) are connected by one or more linker sequences. Each linker may comprise any of the linker sequences disclosed herein.

[0018] In a second general aspect, the disclosure provides methods for generating antibodies specific for human Lp(a), comprising: a) inoculating a non-human animal host with any of the recombinant antigens described herein; b) obtaining serum from the animal host comprising one or more polyclonal antibodies; c) generating polyclonal antibodies specific for Lp(a) by purifying the serum. In some aspects, the serum is purified by contacting the serum with a screening agent to isolate one or more off-target, non-specific, and / or cross-reactive antibodies. In some aspects, the screening agent comprises: (i) a human plasminogen kringle type IV domain, or a fragment thereof, and / or (ii) a human Lp(a) kringle type IV, subtype 2 domain, or a fragment thereof. In some aspects, the human plasminogen kringle type IV domain, or a fragment thereof, and / or the human Lp(A) kringle type IV subtype 2 domain, or fragment thereof, is affixed to a solid support when contacted with the serum. In some aspects, the human plasminogen kringle type IV domain, or the fragment thereof, and / or the human Lp(a) kringle type IV subtype 2 domain, or the fragment thereof, is affixed to a bead when contacted with the serum. In some aspects, the purifying step comprises a precipitation reaction or adsorption chromatography, using the human plasminogen kringle IV domain, or a fragment thereof, and / or the human Lp(a) kringle IV subtype 2 domain, or a fragment thereof.

[0019] In a third general aspect, the disclosure provides polyclonal antibodies specific forLp(a), for example, generated by any of the methods describes herein. In some aspects, the polyclonal antibodies: a) are insensitive as to the size of the apo(a) component of Lp(a); b) arespecific for one or more kringle type IV domains of Lp(a); c) are specific for a kringle type V domain of Lp(a); d) are specific for a linker sequence adjacent to a kringle type IV domain of Lp(a); e) are specific for a serine protease domain of Lp(a); f) do not display cross-reactivity against human plasminogen; and / or g) display any combination of the foregoing properties. In some aspects, the polyclonal antibodies specific for Lp(a) display a titer to a human plasminogen kringle type IV domain and / or a human Lp(a) kringle type IV, subtype 2 domain, that is less than 20% of a total titer to human Lp(a).

[0020] In a fourth general aspect, the disclosure provides methods for measuring an Lp(a) concentration in a biological sample obtained from a subject in need thereof, comprising: a) obtaining the biological sample from the subject, wherein the biological sample comprises whole blood, serum, or plasma; b) optionally, purifying the biological specimen to enrich or remove one or more components; and c) measuring the concentration of Lp(a) in the biological sample by performing an immunoassay using any of the polyclonal antibodies specific for Lp(a) described herein. In some aspects, the immunoassay is an ELISA assay, a Western blot, a particle enhanced turbidimetric (PET) assay, a competition assay, or any other immunoassay known in the art. In some aspects, such methods may further comprise a step of evaluating the subject’s risk of cardiovascular disease based on the measured concentration of Lp(a). In some aspects, the disclosure provides kits for performing any of the methods described herein. For example, a kit may comprise one or more polyclonal antibodies specific for Lp(a), such as those generated using the methods and / or antigens described herein, plus one or more buffers, reference samples (for example, comprising a known amount of Lp(a)), or other reagents.

[0021] To the accomplishment of the foregoing and related ends, the one or more aspects comprise the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative features of the one or more aspects. These features are indicative, however, of but a few of the variousways in which the principles of various aspects may be employed, and this description is intended to include all such aspects and their equivalents.Description of the Figures

[0022] The drawings set forth herein illustrate and describe exemplary aspects of the disclosure and are not meant to limit the scope of the invention as defined by the claims.

[0023] FIG. 1A is a schematic diagram showing the domain architecture of rabbit plasminogen, human plasminogen, and human apo(a).

[0024] FIG. IB is a schematic diagram showing the domain architecture of two antigen and one absorption protein used to elicit and purify polyclonal antibodies for Lp(a).

[0025] FIG. 1C is a schematic diagram showing the domain architecture of five exemplary chimeric antigen constructs that may be used, for example, as an inoculant to generate polyclonal antibodies for Lp(a).

[0026] FIG. 2 illustrates an annotated polypeptide sequence showing an antigen comprising consecutive kringle type IV domains (types 5-7-9, highlighted bold) separated by front (green) and end (blue) linkers from each respective kringle type IV domain.

[0027] FIG. 3 illustrates an annotated polypeptide sequence showing a second antigen comprising consecutive kringle type IV domains (types 4-6-8, highlighted bold) separated by front (green) and end (blue) linkers from each respective kringle type IV domain.

[0028] FIG. 4 illustrates an annotated polypeptide sequence showing an adsorption protein comprising consecutive kringle type IV domains (KIV1-2, followed by human Plasminogen KIV, highlighted bold) separated by front (green) and end (blue) linkers from each respective kringle type IV domain.

[0029] FIG. 5 illustrates an annotated polypeptide sequence showing a rabbit plasminogen kringle type IV domain (bold) with surrounding linker sequences (green).

[0030] FIGs. 6-16 each illustrate an annotated polypeptide sequence of a chimeric antigen according to the disclosure, which may be used to elicit specific reactivity against human Lp(a) while reducing or minimizing cross reactivity against human plasminogen.

[0031] FIG. 17 illustrates a pairwise sequence alignment comparing the polypeptide sequences of rabbit plasminogen and human plasminogen.

[0032] FIG. 18 illustrates a multiple sequence alignment comparing the upstream and downstream linker sequences adjacent to several kringle type IV domains of human Lp(a) and the kringle type IV domain of rabbit plasminogen. FIG. 19 is a percent identity matrix showing the inter-sequence similarity of each of the sequences shown in FIG. 18.

[0033] FIG. 20 illustrates nine pairwise sequence alignments, each comparing the sequence of a single Lp(a) kringle type IV domain (IVi, IV3, IV4, IVs, IVe, IV7, IVs, IV9, and IV10) against the sequence of kringle domain IV2, the varying domain of apo(a).

[0034] FIG. 21 illustrates nine pairwise sequence alignments, each comparing a linker sequence separating a single Lp(a) kringle type IV domain (IVi, IV3, IV4, IVs, IVe, IV7, IVs, IV9, and IV10) against a linker sequence separating kringle domain IV2, the varying domain of human Lp(a).

[0035] FIG. 22 illustrates four pairwise sequence alignments used to identify unique epitopes for the human apo(a) kringle type V domain. In this figure, 11 potential epitopes for constructs according to the disclosure are flagged with an asterisk. Each epitope, or any combination thereof, may be used to elicit polyclonal antibodies insensitive to the size of apo(a) and specific for the kringle type V domain of the apo(a) component of human Lp(a).

[0036] FIG. 23 illustrates three pairwise sequence alignments used to identify unique epitopes for the human apo(a) serine protease domain. In this figure, 15 potential epitopes for constructs according to the disclosure are flagged with an asterisk. Each epitope, or anycombination thereof, may be used to elicit polyclonal antibodies insensitive to the size of apo(a) and specific for human Lp(a).

[0037] FIG. 24 shows a charged surface plot of predicted kringle type V domain models (rows A and B showing rotated views).

[0038] FIG. 25 is a multiple sequence alignment of the rabbit and human plasminogen kringle type IV domain sequences, and several human Lp(a) kringle type IV domain sequences. Amino acid differences are color-coded to identify positive (blue), negative (red) charged, hydrophobic (yellow), polar uncharged (grey) and special cases (green). A set of exemplary preferred epitopes that may be graft onto the rabbit plasminogen kringle backbone of chimeric constructs according to the disclosure are indicated with an asterisk (*).

[0039] FIG. 26 shows a structural comparison of the protease domains from rabbit plasminogen (FIG. 26A, blue) and human Lp(a) (FIG. 26B, gray). Rabbit plasminogen protease carries an additional stretch of nine amino acids colored in wheat (FIG. 26A) and potential surface exposed epitopes are shown in stick mode and colored in red (FIG. 26B). FIG. 26C and FIG. 26D show electrostatics of the two protease domains. The surface areas with + / - the nine amino acid stretch are significantly different in surface charge and should generate rabbit antibodies binding to human Lp(a). Catalytic triangle is shown in stick mode and colored yellow (FIG. 26A and FIG. 26B). FIG. 27 is a multiple sequence alignment comparing the sequences of these protease domains with colored-coded annotation. Amino acid differences are color-coded to identify positive (blue), negative (red) charged, hydrophobic (yellow), polar uncharged (grey) and special cases (green).

[0040] FIGs. 28-29 show chromatograms of size exclusion chromatography (“SEC”) purification of Antigen I (SEQ ID NO: 15) and Antigen II (SEQ ID NO: 16), respectively. For reference, the sequence of Antigens I and II are also shown in FIGs. 2-3, respectively. FIGs.30-31 show SDS-gels generated using Antigen I and II fractions collected from this SEC purification.

[0041] FIG. 32-33 are graphs showing the results of turbidimetric analyses Antigens I and II (FIG. 32), and of anti-sera produced by rabbit hosts following inoculation with Antigen I or Antigen II (FIG. 33). As illustrated by FIG. 32, an antibody induced by immunization with native antigen Lp(a) particles [anti-Lp(a) pAb] agglutinates both Antigen I (blue circles) and Antigen II (orange circles). The same antibody could also react to the Adsorption protein (grey circles).

[0042] FIG. 34 is a graph showing the results of a turbidimetric analysis of antibodies generated by a rabbit inoculated with Antigen I. In particular, anti-serum from a rabbit immunized with Antigen I was analyzed for reactivity toward native Lp(a) particles (blue diamonds, left) and compared to anti-Lp(a) pAb (orange square). FIG. 35 is a graph showing the results of a dynamic light scattering study of the Lp(a) particles used in this turbidimetric analysis.

[0043] FIG. 36 shows a pair of graphs of ELISA data for antibodies produced according to the disclosure. In this case, ELISA plates were coated with human plasminogen KIV (blue lines) or human Apo(a) KIV2 (orange lines). The dark blue and - orange lines were obtained using raw IgG fractions whereas the respective light-colored lines show the results after purification of the IgG fraction on a column loaded with the adsorption protein.

[0044] FIG. 37 is a chart showing the level of serum immunoglobulin (Ig) in rabbits immunized with an exemplary construct according to the disclosure.

[0045] FIGs. 38-40 are graphs showing the results of ELISA assays to assess reactivity against KIVs (FIG. 38), KIV7 (FIG. 39), and KIV9 (FIG. 40), before and after adsorption purification of antigen I derived IgG fraction was tested. Dark blue, green, and peach coloreddata lines were obtained using raw IgG fractions. Light blue, orange and grey data lines were obtained using adsorption-purified IgG fractions.

[0046] FIGs. 41-42 are graphs showing the results of a tubidimetric analysis of polyclonal antibodies produced using Antigen I. As illustrated by this figure, polyclonal antibody produced using Antigen I recognized both Antigen I (blue circles) and the Adsorption (orange circles) protein (FIG. 41). After using the adsorption protein as a bait on an adsorption column, the polyclonal antibody selectively recognizes Antigen I and not the adsorption protein (FIG. 42). Blue dots: antigen I and orange dots: adsorption protein.

[0047] FIG. 43 shows an SDS-gel (A) and Western blot (B), generate using a Roche Tina2 calibrator. The Western blot was first probed with a rabbit anti-human Lp(a) antibody that had been adsorption purified to remove binding to kringle type IV2. Secondly, a goat anti-rabbit IgG antibody labelled with HRP was used to visualize the Apo(a) containing bands. Lane 1 : size marker. Lanes 2-6: Tina2 calibrator (1 pL) levels 5-1. Lane 7: empty. Lanes 8-10 Tina2 calibrator (3 pL) levels 5, 3, and 1. Level 5 is the highest concentration and 1 the lowest.

[0048] FIG. 44 is a schematic showing an exemplary workflow for adsorption purification of antibodies generated according to the disclosure.

[0049] FIGs. 45-52 show graphs illustrating the results of ELISA assays using plates coated with various proteins, for example, native human plasminogen (FIG. 45), “hplg”), an adsorption domain (FIG. 46), recombinant Lp(a) kringle type IV2 domain (FIG. 47), or native Lp(a) (FIG. 48).

[0050] FIG. 53 depicts photographs of Western blots produced using antibodies generated by the present methods. The results demonstrate that cross-reactivity against plasminogen is removed (arrows), and that the kringle type IV2 domain sensitive part is removed (circles) when comparing the relative intensities. M: marker; 1-3: Donor 1,2,3; 4: native human plasminogen; 5: native LDL; 6: native Lp(a).

[0051] FIG. 54 depicts a photograph of a Western blot produced using antibodies generated by the present methods. Rabbit anti-plasminogen antibody were used as the primary pAb, and swine anti-rabbit coupled with HRP was used as the secondary pAb. DAB+ ( Agilent®) was used to develop the membrane and shows that detection limit is below 16 ng.

[0052] FIG. 55 depicts a photograph of an SDS-PAGE gel showing the expression of exemplary chimeric constructs according to the disclosure. M: marker; 1 : cDI.l; 2: cDI.2; 3: cDII.l; 4: cDII.2; 5: cDIII. l; 6: human plasminogen; 7: rabbit plasminogen (kringle II-III-IV); 8: rabbit plasminogen kringle IV; 9: kringle IVi- IV2- IV2- IV2.

[0053] FIG. 56 depicts a multiple sequence alignment (MSA) showing the alignment of several non-human plasminogen protein sequences.

[0054] FIG. 57 depicts the results of ELISA experiments when coating the plate with recombinantly expressed human apo(a) serine protease and various polyclonal antibodies. The results show that inoculation with Lp(a) particles also give rise to reactivity towards the protease. In contrast, all earlier recombinant antigens did not contain the protease.

[0055] FIG. 58 depicts chimeric constructs based on rabbit plasminogen serine protease carrying selected human epitopes from apo(a) serine protease. Expression of chimeric protein was confirmed by SDS-PAGE.

[0056] FIG. 59 depicts chimeric constructs based on rabbit plasminogen kringle 4 carrying human apo(a) kringle 4 or 9 epitopes. These chimeric proteins are predicted to direct specific response towards apo(a) kringle 4 or 9, respectively.

[0057] FIG. 60 depicts apo(a) on LDL. Optimal specificity to achieve efficient agglutination is indicated by arrows pointing to kringle 4, 9 and protease domains. Specific targeting of these epitopes is based on their uniqueness and positions on the lipoprotein particle.Detailed Description

[0058] The detailed description set forth below in connection with the appended drawings is intended as a description of various configurations and is not intended to represent the only configurations in which the concepts described herein may be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of various concepts. However, it will be apparent to those skilled in the art that these concepts may be practiced without these specific details.

[0059] Lp(a) is a lipoprotein particle containing one molecule of apoB-100 wrapped around a particle that has primarily a core of cholesteryl ester and triglyceride with phospholipids and unesterified cholesterol at its surface. A hydrophilic, highly-glycosylated protein known as apo(a) is covalently attached to apoB-100 by a single disulfide bridge, differentiates Lp(a) from LDL. Brunner, C., et al. “Cys4057 of apolipoprotein (a) is essential for lipoprotein (a) assembly.” PNAS 90.24 (1993): 11643-11647. Apo(a) is part of the plasminogen gene superfamily, and its presence imparts synthetic and catabolic properties to Lp(a) along with size heterogeneity. Hoek, Y, et al. “The apolipoprotein (a) kringle IV repeats which differ from the major repeat kringle are present in variably sized isoforms.” Human Molecular Genetics 2.4 (1993): 361-366. As shown by the domain architecture schematics provided in FIG. 1A, apo(a) shares a high degree of sequence homology with several regions of the serine protease zymogen plasminogen, including the protease domain, and its kringle domains, which as explained above are tri-loop polypeptides stabilized by three internal disulfide bridges. Apo(a)’s inactive carboxy -terminal protease-like domain and kringle type V domain both exhibit ~85% homology with human plasminogen, and apo(a) includes at least 10 copies of the plasminogen-like kringle type IV domain, in tandem. This high sequence homology between apo(a) and plasminogen has presented a significant challenge to the development of suitable immunoassays for the accurate measurement of Lp(a).

[0060] Moreover, as shown by FIG. 1A, apo(a) isoforms may include multiple identical copies of the kringle type IV, subtype 2 domain (“IV2”) (for example, 1 to >40 copies). The existence of different, genetically determined apo(a) isoform sizes further complicates the development of immunoassays for Lp(a) due to size heterogeneity issues. In particular, the number of antigenic determinants per particle available to interact with anti-Lp(a) antibodies will vary in different samples and potentially among calibrators, resulting in apo(a) sizedependent inaccuracies, particularly for immunoassays based on polyclonal antibodies. In view of the high degree of size variation of apo(a), it is practically impossible to select an assay calibrator with the same apo(a) size present in individual samples to be analyzed, and a size difference between Lp(a) molecules in the samples versus those in the calibrator will result in over or underestimation of the subject’s Lp(a) concentration. Marcovina, S., et al. “Lipoprotein (a) measurements for clinical application.” J. Lipid Res. 2016 Apr;57(4):526-37.

[0061] Efforts have made to mitigate this issue using carefully designed calibrators where the Lp(a) used at each calibration point has a selected number of kringle IV2 repeats. There is a tendency for people with a low number of IV2 repeats to have a higher concentration of Lp(a) particles. Thus, a calibrator with a low concentration contains particles with a high number of IV2 repeats and high-concentration calibrators can be made using Lp(a) particles with a lower number of IV2 repeats, to better fit the calibrator to patients in either cohort. However, these specialized calibrators do not provide a complete solution, as they must be pre-configured for specific groups (for example, low or high-concentration patients), and are not necessarily accurate due to intra-subject Lp(a) size heterogeneity. An individual may carry any combination of high or low repeat alleles and while the number of repeats is genetically determined, the exact concentration is variable. Accordingly, while some degree of calibration is available, it is practically impossible to develop a patient-specific calibrator, resulting inongoing accuracy issues that limit the effective use of Lp(a) as a biomarker for cardiovascular disease and other conditions.

[0062] The present disclosure addresses these and other shortcomings of current methods, in addition to providing various other improvements and advantages, by providing immunoassays capable of accurately measuring Lp(a) concentration in the blood, serum, or plasma of human subjects, without the need for special calibrators. Such assays are made possible by the use of polyclonal antibodies generated using the recombinant antigens and methods described herein, which in some aspects may be used to generate polyclonal antibodies specific for Lp(a) that are insensitive as to the size of apo(a) and / or which display minimal cross-reactivity against human plasminogen, among other properties. Accordingly, the present disclosure provides immunoassays that can be used to measure Lp(a) in a biological sample using a standard calibrator (for example, based on serial dilution), allowing for both increased accuracy and better use of Lp(a) as a clinical biomarker.

[0063] Recombinant Antigen Constructs / Chimeric Constructs

[0064] Polyclonal antibodies display a broad degree of reactivity towards multiple epitopes ensuring a robust reaction even in the face of diversity of the target or environmental changes. To obtain polyclonal antibodies, a host animal (for example, a rabbit) is typically immunized with a heterologous or recombinant protein, or a fragment thereof, after which the humoral immune system selects antibody-producing B-cell clones for expansion and maturation. At later stages these B-cells will further diversify through mutagenesis and selection of high affinity immunoglobulin genes. The adaptive immune system of vertebrates is thus capable of generating antibodies against almost any foreign protein.

[0065] The adaptive immune system is also capable of distinguishing “self’ from “nonself’ (for example, foreign or abnormal) antigens. This failure to respond to “self’ antigens, called “immunological tolerance” is understood to be the consequence of deletion (negativeselection) of lymphocytes that recognized a “self’ antigen before reaching maturity, in addition to other less well understood mechanisms. Kisielow, P. “How does the immune system learn to distinguish between good and evil? The first definitive studies of T cell central tolerance and positive selection.” Immunogenetics 71, 513-518 (2019). As explained in further detail herein, in some aspects the present methods leverage this mechanism of immunological tolerance to generate polyclonal antibodies specific for portions of Lp(a). In particular, a recombinant antigen may be designed based on an endogenous protein sequence expressed by an animal host, which has been modified to incorporate one or more single-nucleotide polymorphisms (“SNPs”) and / or mutated segments (consisting of a plurality of amino acid insertions, deletions, and / or substitutions). Such constructs are designed to elicit the generation of polyclonal antibodies directed to the mutated epitope(s), in view of immunological tolerance to the unmodified “self’ portion of the recombinant antigen.

[0066] For example, in some aspects, a recombinant antigen may comprise one or more nonhuman (for example, rabbit) plasminogen kringle type IV domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 10, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 10. These amino acid substitutions may include SNPs and / or segments comprising a plurality of contiguous amino acid substitutions, which correspond to amino acids for in a kringle type IV domain of human Lp(a). In some aspects, the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NO: 10. In some aspects, the one or more amino acid substitutions comprise one or more of the amino acid substitutions shown in any one of FIGs. 6-16.

[0067] This technique can also be applied to non-human (for example, rabbit) plasminogen kringle type II, II and V domains. For example, a recombinant antigen may comprise one or more non-human (for example, rabbit) plasminogen kringle type II, III, or V domains, eachsharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 11, 12, or 13, respectively, and comprising one or more amino acid substitutions as compared to SEQ ID NOs: 11, 12, or 13, respectively. In some aspects, the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NOs: 11, 12, or 13. In some aspects, the one or more amino acid substitution comprise one or more of the amino acid substitutions shown in any one of FIGs. 6-16.

[0068] In some aspects, a recombinant antigen may comprise one or more non-human plasminogen kringle type II, III, IV, or V domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with one or more of kringle type II, III, IV, or V domain(s) present in a rabbit (Oryctolagus ciiniciilus) plasminogen, such as the sequence represented by NCBI Accession No. XP_051711119.1, a mouse plasminogen (for example, NCBI Accession No. NP_032903.3), a rat plasminogen (for example, NCBI Accession No. NP_445943.1), a bovine plasminogen (for example, NCBI Accession No. NP_776376.1), a hamster plasminogen (for example, NCBI Accession No. XP_040614106.1), or an equine plasminogen (for example, NCBI Accession Nos. XP_014593633.1 or XP_001500552.3), or a fragment or variant of any of the foregoing plasminogen sequences. In each case, the recombinant antigen may comprise one or more amino acid insertions, deletions, or substitutions in each of the non-human plasminogen kringle type II, III, IV, or V domains, as compared to the native sequence, which humanize the domain (e.g., changes which incorporate the amino acid(s) present at the corresponding position in a human kringle type IV domain when aligned pairwise with the native sequence). FIG. 56 depicts an MSA showing several non-human plasminogen protein sequences aligned using the Clustal Omega alignment program. See Wilm, F. et al., “Fast, scalable generation of high- quality protein multiple sequence alignments using Clustal Omega,” Molecular Systems Biology 7 Article No. 539.

[0069] This general technique can also be extended to the protease domain of a non-human (for example, rabbit) plasminogen. In some aspects, the recombinant protein may comprise one or more non-human plasminogen serine protease domains, each sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 14, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 14. In some aspects, the fragment comprises at least , 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 or 230 contiguous amino acids of SEQ ID NO: 14. In some aspects, the one or more amino acid substitution comprise one or more of the amino acid substitutions shown in any one of FIGs. 6-16.

[0070] FIG. IB illustrates exemplary antigen constructs according to this aspect of the disclosure. In brief, chimeric immunogens may be designed that have a backbone derived from rabbit plasminogen. Unique epitopes from human Lp(a) kringle type IVi, and / or type IV3-IV10 domains, or from the Lp(a) protease domain, may then transferred to the rabbit backbone by substituting specific amino acids. Chimeric designs I-III each illustrate a construct comprising three rabbit plasminogen kringle type IV domains that have been modified to incorporate amino acid substitutions that introduce epitopes found on the human Lp(a) type IV kringle domain. Furthermore, in Chimeric design I the native rabbit kringle type IV linkers surrounding the domain were used, whereas in Chimeric design II, the native linkers were replaced with the linkers separating the kringle domains in Lp(a), for example, IVs, IV7, and IV9 or IV4, IVe, and IVs, respectively. Chimeric design III illustrates an alternative design comprising rabbit plasminogen kringle type II, III, and IV domains that have similarly been modified to incorporate amino acid substitutions that introduce epitopes found on the human Lp(a) type IV kringle domain. Chimeric designs IV and V illustrate alternative designs comprising one or more linked rabbit plasminogen protease domains alone (IV) or further linked to one or more kringle domains (V), wherein one or more of the protease and / or kringle domains have beenmodified to incorporate amino acid substitutions that introduce epitopes found on the human Lp(a) type IV kringle domain. Constructs produced according to chimeric designs I-V may be used to immunize a host animal (for example, a rabbit), eliciting antibodies highly specific for epitopes that are not present in the human kringle type IV2 repeats or the kringle IV domain in human plasminogen.

[0071] In alternative aspects, antibodies against Lp(a) may be generated using a different strategy. In this case, a host animal (for example, a rabbit) may be immunized with constructs comprising three native human kringle domains and the resulting antibodies may be screened with a bait to remove undesired antibodies (for example, which react to undesired epitopes in human plasminogen). FIG. IB illustrates exemplary antigens (Antigen I and II) and an exemplary adsorption protein that can be used as a bait in methods according to this alternative strategy. As illustrated by this example, an antigen comprising three or more kringle domains (for example, “Antigen I”, IV5-IV7-IV9; or “Antigen II”, IV4-IV6-IV8) may be used as an immunogen. The kringle domains may be connected via their individual native linkers (blue, yellow, grey and purple, turquoise and black in FIG. IB) or via any other linker known in the art or described herein. A bait comprising one or more domains for which cross-reactivity is undesirable may be used to screen the population of antibodies generated by the animal host post-inoculation. For example, the “adsorption protein” shown in FIG. IB illustrates the use of a bait comprising human Lp(a) kringle type IVi and IV2 domains, as well as a human plasminogen kringle type IV domain separated by native human Lp(a) kringle type IVi and IV2 linkers.

[0072] Chimeric antigen constructs according to the disclosure may be used to generate polyclonal antibodies against Lp(a) while reducing or minimizing cross-reactivity against human plasminogen and sensitivity to the number of repeats of kringle IV2 found in different apo(a) isoforms. By grafting unique epitopes from human apo(a) kringle IVi, and / or IV3-IV10onto non-human (for example, rabbit) plasminogen kringle domains, an immune response is trigger which elicits specific reactivity against Lp(a) epitopes without causing reactivity against human plasminogen. Similarly, (for example, using a bait described herein) antibodies that display undesirable cross-reactivity (for example, against human plasminogen) may be removed from the population of antibodies generated by the animal host post-inoculation. FIGs. 2-4 show annotated polypeptide sequences of exemplary antigen and bait proteins that may be used in the present methods.

[0073] FIGs. 6-16 illustrate additional exemplary chimeric constructs according to the disclosure based on this general design, which may be used to elicit specific reactivity against human Lp(a) while avoiding cross reactivity against human plasminogen. For example, FIG. 6 illustrates a construct wherein the sequence of a rabbit plasminogen kringle type IV domain (cyan) has been modified to incorporate epitopes based on human Lp(a) kringle IV subtypes IVs, IV7, and IV9, respectively (purple). The chimeric kringle domains are joined by linkers (green) which each comprise a portion of the endogenous linker sequence found adjacent to the rabbit plasminogen kringle type IV domain. An additional linker (green) is present between the first chimeric kringle domain and the N-terminus. Optionally, the N-terminus of the chimeric antigen may further comprise a signal-peptide for example, for HEK-cell expression), a purification or selection tag (for example, a His-tag used for IMAC purification) and / or a cleavage tag (for example, a TEV cleavage site used for cleavage of the purification or selection tag).

[0074] As illustrated by this example, one or more rabbit plasminogen kringle type IV domains and / or protease domains, may be used as a backbone that can be modified to include amino acid substitutions. The amino acid substitutions may comprise one or more of the exemplary substitutions shown in FIGs. 6-16. In other words, a chimeric construct according to any of the aspects described herein may include one or more rabbit plasminogen domains(for example, as a backbone) wherein the sequence of the domain has been modified to incorporate one or more insertions, deletions, and / or substitutions described herein. Such substitutions may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8 ,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions (or a number of substitutions within a range defined by any pair of the foregoing numbers) selected from the set of purple-highlighted substitutions shown in FIGs. 6-16. For example, as shown by FIG. 6 a chimeric construct may comprise one or more rabbit plasminogen kringle type IV domains, optionally joined by one or more linkers, wherein each rabbit plasminogen kringle type IV domain includes one or more independently selected substitutions. In the example shown in this figure, the first rabbit plasminogen kringle type IV domain comprises mutations at positions 3-5, 32, 34, 47, 55, 56, 58 and 64 (annotated in purple), wherein each mutation is a substitution that introduces an amino acid found at the corresponding position when the rabbit plasminogen kringle type IV domain is aligned against a human kringle type IV domain. The human kringle type IV domain may comprise, for example, a human Lp(a) kringle type IV4, IV5, IVe, IV7, IVs, or IV9 domain. It is understood that in some aspects, the one or more rabbit plasminogen domains may similarly comprise one or more insertions or deletions as compared to a native rabbit plasminogen domain (or any plasminogen domain described herein). Each insertion and each deletion, if any are present, may comprise one or more amino acids that are added or deleted as compared to a native rabbit plasminogen domain (or any plasminogen domain described herein). Any such insertions or deletions may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that are newly added, or omitted, as compared to the baseline native rabbit plasminogen domain (or other plasminogen domain described herein), as the case may be.

[0075] In cases where a chimeric construct includes another type of domain (for example, the protease domain of rabbit plasminogen) as a backbone, the domain may similarly bemodified to incorporate any number (for example, 1-50, inclusive of all integer values therein) mutations, wherein each mutation is a substitution that introduces an amino acid found at the corresponding position when the domain in question is aligned against a human counterpart domain, for example, the human Lp(a) protease domain, in the case of rabbit plasminogen’s protease domain being selected as a backbone.

[0076] FIG. 7 illustrates a similar construct, wherein the sequence of a rabbit plasminogen kringle type IV domain has instead been modified to incorporate epitopes based on human Lp(a) kringle IV subtypes IV4, IVe, and IVs, respectively. The chimeric kringle domains are joined by linkers (green) which each comprise a portion of the endogenous linker sequence found adjacent to the rabbit plasminogen kringle type IV domain.

[0077] FIG. 8 illustrates a construct wherein the sequence of a rabbit plasminogen kringle type IV domain (cyan) has been modified to incorporate epitopes based on human Lp(a) kringle IV subtypes IV5, IV7, and IV9, respectively (purple). The chimeric kringle domains are joined by linkers (blue and green) which each comprise portions of the endogenous linker sequences found adjacent to one or more human apo(a) kringle domains. An additional linker (green) is present between the first chimeric kringle domain and the N-terminus. Optionally, the N- terminus of the chimeric antigen may further comprise a signal-peptide (for example, for HEK- cell expression), a purification or selection tag (for example, a His-tag used for IMAC purification) and / or a cleavage tag (for example, a TEV cleavage site used for cleavage of the purification or selection tag).

[0078] FIG. 9 illustrates a similar construct, wherein the sequence of a rabbit plasminogen kringle type IV domain (cyan) has instead been modified to incorporate epitopes based on human Lp (a) kringle IV subtypes IV4, IVe, and IVs, respectively. The chimeric kringle domains are joined by linkers (blue and green) which each comprise portions of the endogenous linker sequences found adjacent to human apo(a).

[0079] FIG. 10 illustrates a construct wherein the sequence of a rabbit plasminogen kringle type II (top, grey), type III (middle, grey), and type IV domain (bottom, cyan) has been modified to incorporate epitopes based on human Lp (a) kringle IV subtypes IVs, IV7, and IV9, respectively (purple). The chimeric kringle domains are joined by linkers (green) which each comprise portions of the endogenous linker sequences found adjacent to each of the associated kringle domains (i.e., kringle types II, III, and IV, respectively) in rabbit plasminogen. An additional linker (green) is present between the first chimeric kringle domain and the N- terminus. The N-terminus of the chimeric antigen may further comprise, for example, a signal- peptide used for HEK-cell expression, a His-tag used for IMAC purification and / or a TEV cleavage site used for cleavage of the His-tag, respectively.

[0080] FIG. 11 illustrates a similar construct, wherein the sequence of a rabbit plasminogen kringle type II (top, grey), type III (middle, grey), and type IV domain (bottom, cyan) has been modified to incorporate epitopes based on human Lp(a) kringle IV subtypes IV4, IVe, and IVs, respectively (purple). The chimeric kringle domains are joined by linkers (green) which each comprise portions of the endogenous linker sequences found adjacent to each of the associated kringle domains (i.e., kringle types II, III, and IV, respectively) in rabbit plasminogen.

[0081] FIGs. 12-16 depict other alternative designs for a chimeric construct according to the disclosure (for example, using various combinations of the kringle and other domains, and linker sequences, described herein.

[0082] Potential epitopes for use in the recombinant antigenic constructs described herein were initially identified by aligning the polypeptide sequence of rabbit plasminogen against human plasminogen and identifying sequence differences. FIG. 17 illustrates a pairwise sequence alignment comparing the polypeptide sequences of rabbit plasminogen and human plasminogen. As shown by this figure, plasminogen consists of a PAN domain (red), fivekringle domains (shaded cyan and green to highlight the rabbit and human domains, respectively) and a serine protease domain (shaded yellow and pink to highlight the rabbit and human domains, respectively), wherein each domain is separated by a linker of varying length.

[0083] FIG. 18 illustrates a multiple sequence alignment comparing the upstream and downstream linker sequences adjacent to several kringle type IV domains of human Lp (a) and the kringle type IV domain of human plasminogen. FIG. 19 is a percent identity matrix showing the inter-sequence similarity of each of the sequences shown in FIG. 18.

[0084] After this initial selection process, candidate epitopes were screened by alignment against the sequence of Lp (a) kringle IV2 to determine whether the candidate epitopes were also present in Lp(a) kringle IV2. As explained above, the apo(a) component of Lp(a) may comprise a variable number of repeating kringle IV2 domains. As such, size-insensitive polyclonal antibodies for Lp(a) are typically desirable, and candidate epitopes shared with Lp(a) kringle IV2 may be screened out. FIG. 20 illustrates nine pairwise sequence alignments, each comparing the sequence of a single Lp(a) kringle type IV domain (IVi, IV3, IV4, IV5, IVe, IV7, IVs, IV9, and IV10) against the sequence of kringle domain IV2, the varying domain of apo(a).

[0085] In some aspects, chimeric antigens according to the disclosure may include linker sequences, for example, between adjacent kringle domains and / or between an initial kringle domain and an N-terminal signal peptide (and optional purification or other tag sequences), as shown by the exemplary constructs depicted in FIGs. 6-16. In some aspects, a linker may comprise a sequence present in the polypeptide sequence of Lp(a), for example, a linker sequence separating two adjacent kringle domains of Lp(a), or a fragment thereof. In other aspects, a linker sequence may comprise a sequence present in the polypeptide sequence of a non-human plasminogen (for example, a linker sequence separating two adjacent kringle domains), or a fragment thereof. In some aspects, the linker sequence may contain one or moremutations as compared to an endogenous linker sequence of a non-human (for example, rabbit) host used to generate polyclonal antibodies against Lp(a). Each mutation may comprise a SNP or segment consisting of a plurality of adjacent amino acid substitutions and, in some aspects, the mutations may comprise amino acid substitutions that introduce epitopes present in human Lp(a).

[0086] FIG. 21 illustrates nine pairwise sequence alignments, each comparing a linker sequence separating a single Lp(a) kringle type IV domain (IVi, IV3, IV4, IVs, IVe, IV7, IVs, IV9, and IV10) against a linker sequence separating kringle domain IV2, the varying domain of human Lp(a). To identify potential epitopes, each linker sequence separating a single kringle type IV domain was compared against linker sequences separating kringle IV2 from kringle IVi and kringle IV3. The linkers were divided into two regions, a front and end of the domain. Potential epitopes showing positional changes as compared to kringle IV2 are highlighted in bold in this figure. These candidate epitopes were then aligned against rabbit plasminogen (in view of the selection of this non-human animal as the host for antibody generation). All sequential differences may potentially be incorporated into the endogenous linker sequences adjacent to rabbit plasminogen (for example, as SNPs or mutated segments), applying the same principle used to generate polyclonal antibodies against the chimeric kringle type IV domains discussed above.

[0087] FIGs. 22-23 illustrates pairwise alignments used to select potential Lp(a) epitopes for incorporation into a rabbit plasminogen kringle type V domain (FIG. 22) or into the protease domain of rabbit plasminogen (FIG. 23), following the same principles used to generate the chimeric kringle type IV domains discussed above. As illustrated by these figures, 11 potential epitopes were identified that can be used to elicit the generation of size-insensitive polyclonal antibodies specific for the kringle type V domain of apo(a), by aligning the apo(a) kringle type V domain sequence against the sequences of apo(a) kringle type IV2 and thekringle type V domains from rabbit and human plasminogen. An analogous strategy was used to identify 15 potential epitopes for integration into the active protease domain of rabbit plasminogen that would elicit polyclonal antibodies insensitive to size and specific for Lp(a) without cross-reactivity against human plasminogen.

[0088] The preceding passages focused on chimeric antigen constructs that include one or more heterologous epitopes (for example, SNPs or segments of human Lp(a)). However, in other aspects, a recombinant antigen used to generate anti-Lp(a) polyclonal antibodies may comprise one or more domains of Lp(a), or fragments thereof, or a polypeptide sequence that shares at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the polypeptide sequence of an Lp(a) domain or fragment thereof. In some aspects, the fragment comprises at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 contiguous amino acids of a human kringle type IV domain. In some aspects, the recombinant protein comprises 1-10 of the directly or indirectly linked human kringle type IV domains (for example, any combination of Lp(a) kringle type IVi, IV3, IV4, IVs, IVe, IV7, IVs, IV9, and IV10 domains, optionally connected by one or more linker sequences. Each linker sequence may comprise an endogenous linker present in Lp(a), a fragment thereof, or a sequence sharing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the polypeptide sequence of a linker sequence separating two adjacent Lp(a) kringle domains.

[0089] FIG. 24 shows a charged surface plot of predicted kringle type V domain models (rows A and B showing rotated views). FIG. 25 is a multiple sequence alignment of the rabbit and human plasminogen kringle type IV domain sequences, and several human Lp(a) kringle type IV domain sequences. Amino acid differences are color-coded to identify positive (blue), negative (red) charged, hydrophobic (yellow), polar uncharged (grey) and special cases(green). A set of exemplary epitopes that may be graft onto the rabbit plasminogen kringle backbone of chimeric constructs according to the disclosure are indicated with an asterisk (*).

[0090] FIG. 26 shows a structural comparison of the protease domains from rabbit plasminogen and human Lp(a). FIG. 27 is a multiple sequence alignment comparing the sequences of these protease domains with colored-coded annotation. Amino acid differences are color-coded to identify positive (blue), negative (red) charged, hydrophobic (yellow), polar uncharged (grey) and special cases (green). A set of exemplary epitopes that may be graft onto the rabbit plasminogen protease domain backbone of chimeric constructs according to the disclosure are indicated with an asterisk (*).

[0091] Methods for Generating Lp(a) Polyclonal Antibodies

[0092] In some aspects, the disclosure provides methods for generating antibodies specific for human Lp(a), comprising: a) inoculating a non-human animal host with any of the recombinant antigens (for example, chimeric constructs) described herein; b) obtaining serum from the animal host comprising one or more polyclonal antibodies; c) generating polyclonal antibodies specific for Lp(a) by enriching or purifying the serum. In some aspects, the serum is enriched or purified by contacting the serum with a screening agent to isolate one or more off-target, non-specific, and / or cross-reactive antibodies. In some aspects, the screening agent comprises: (i) a human plasminogen kringle type IV domain, or a fragment thereof, and / or (ii) a human apo(a) kringle type IV2 domain, or a fragment thereof. In some aspects, the human plasminogen kringle type IV domain, or a fragment thereof, and / or the human apo(a) kringle type IV2 domain, or fragment thereof, is affixed to a solid support when contacted with the serum. In some aspects, the human plasminogen kringle type IV domain, or the fragment thereof, and / or the human apo(a) kringle type IV2 domain, or the fragment thereof, is affixed to a bead when contacted with the serum. In some aspects, the purifying step comprises a precipitation reaction or adsorption chromatography, using the human plasminogen kringle IVdomain, or a fragment thereof, and / or the human apo(a) kringle IV2 domain, or a fragment thereof.

[0093] In some aspects, polyclonal antibodies generated using the present methods and antigenic constructs display one or more advantages, including but not limited to: a) being insensitive as to a size of an apo(a) component of Lp(a); b) being specific for one or more kringle type IV domains of Lp(a); c) being specific for a kringle type V domain of apo(a); d) being specific for a linker sequence adjacent to a kringle type IV domain of apo(a); e) being specific for a serine protease domain of apo(a); f) lacking cross-reactivity against human plasminogen; and / or g) displaying any combination of the foregoing properties. In some aspects, the polyclonal antibodies specific for Lp(a) display a titer to a human plasminogen kringle type IV domain and / or a human apo(a) kringle type IV, subtype 2 domain, that is less than 20% of a total titer to human Lp(a).

[0094] In some aspects, the disclosure provides alternative methods for generating antibodies specific for human Lp(a), comprising: a) inoculating a non-human animal host with any of the recombinant antigens described herein (for example, a construct comprising one or more native human kringle domains and / or native protease domains of human apo(a)); b) obtaining serum from the animal host comprising one or more polyclonal antibodies; c) generating polyclonal antibodies specific for Lp(a) by enriching or purifying the serum using a bait protein. In some aspects, the recombinant antigen comprises Antigen I and / or II described herein. The recombinant antigen may comprise, for example, any combination of the native kringle type IV4, IV5, IVe , IV7, IVs, and / or IV9 domain sequences present in human Lp(a), optionally joined by one or more linker or other intervening sequences. In some aspects, the screening step is performed using a bait comprising one or more domains for which crossreactivity is undesirable. Placing the animal host serum (optionally diluted) in combination with the bait will cause antibodies in the serum which cross-react with the target to bind to thebait. The bait may be affixed to a solid surface (for example, beads) in order to pull down or otherwise any such antibodies, leaving antibodies that fail to bind to the bait in solution. In some aspects, the animal serum may be advantageously screened with bait comprising human apo(a) kringle type IVi and / or IV2 domain, and / or a human plasminogen kringle type IV domain, in order to reduce or eliminate the subpopulation of antibodies in the serum which cross-react with these domains. In some aspects, the disclosure provides nucleic acids encoding any of the chimeric constructs or antigens described herein (for example, any polypeptide sequence expressly or implicitly disclosed herein). In some aspects, the nucleic acid is a vector, genomic DNA, or mRNA. In some aspects, the disclosure further provides cells engineered to express any such nucleic acids.

[0095] In some aspects, the animal host is immunized using a nucleic acid (for example, an mRNA) encoding any ay of the chimeric constructs or antigens described herein (for example, any polypeptide sequence expressly or implicitly disclosed herein).

[0096] Assays for Measuring Lp(a)

[0097] In some aspects, the disclosure provides methods for measuring the concentration of Lp(a) in a biological sample (for example, whole blood, serum, plasma) obtained from a subject in need thereof, comprising: a) obtaining the biological sample from the subject; b) optionally, enriching or purifying the biological specimen to enrich or remove one or more components; and c) measuring the concentration of Lp(a) in the biological sample by performing an immunoassay using any of the polyclonal antibodies specific for Lp(a) described herein. In some aspects, the immunoassay is an ELISA assay, a Western blot, a PET assay, a competition assay, or any other immunoassay known in the art. In some aspects, such methods may further comprise a step of evaluating the subject’s risk of cardiovascular disease based on the measured concentration of Lp(a).

[0098] In some aspects, the disclosure provides kits for performing any of the methods described herein. For example, a kit may comprise one or more polyclonal antibodies specific for Lp(a), such as those generated using the methods and / or antigens described herein, plus one or more buffers, reference samples (for example, comprising a known amount of Lp(a)), or other reagents.EXAMPLES

[0099] Several exemplary chimeric constructs according to the disclosure were generated and characterized in order to evaluate the constructs and methods described herein.

[0100] As explained above, methods for generating antibodies specific for human Lp(a) may comprise inoculation with an antigen comprising one or more native domains of human apo(a), followed by a screening step to remove cross-reactive antibodies in the serum produced by the inoculated animal host. In order to evaluate such methods, samples of Antigen I (SEQ ID NO: 15) and Antigen II (SEQ ID NO: 16) were prepared. FIGs. 28-29 show chromatograms of size exclusion chromatography (“SEC”) purification of Antigen I (SEQ ID NO 15) and Antigen II (SEQ ID NO: 16), respectively. For reference, the sequence of Antigens I and II are also shown in FIGs. 2-3, respectively. FIGs. 30-31 show SDS-gels generated using Antigen I and II fractions collected from this SEC purification. Antigen I fractions 4-8 were pooled. Antigen II. Fractions 6-9 were pooled. The pooled antigens were used for immunization. The protein fractions show up as smeared bands as expected for glycosylated proteins such as Lp(a). The band pattern also shows that Antigen I appears to be more glycosylated than Antigen II, which is consistent with the prediction of higher levels of O-glycosylation in Antigen I.

[0101] FIG. 32-33 are graphs showing the results of turbidimetric analyses Antigens I andII (FIG. 32), and of anti-sera produced by rabbit hosts following inoculation with Antigen I or Antigen II (FIG. 33). As illustrated by FIG. 32, an antibody induced by immunization with native antigen Lp(a) particles [anti-Lp(a) pAb] agglutinates both Antigen I (blue circles) andAntigen II (orange circles). The same antibody could also react to the Adsorption protein (grey circles).

[0102] The purified Antigen I and Antigen II samples were used to inoculate separate rabbit hosts. FIG. 33 illustrates the results of a comparison of anti-sera from rabbits immunized with either (A) Antigen I, (B) Antigen II, (C) Antigen I + Antigen II or (D) Lp(a) particles. Blue dots are Antigen I, orange dots are Antigen II and grey dots are adsorption protein. Antiserum raised against Lp(a) particles agglutinate the adsorption protein the best. Indicating the new polyclonal antibody has minimal reactivity against KIV1-2. The anti-serum in A reacts better with Antigen I and that of B reacts better with Antigen II. When the antigens are coimmunized, the result is strong reactivity against both Antigen I and II, with similar excess zone and almost equal intensity. Taken together, these results show that Antigen I and Antigen II contains unique epitopes, which differ from those present in the Adsorption Protein

[0103] As illustrated by FIG. 34, anti-serum from rabbit immunized with Antigen I was analyzed for reactivity toward native Lp(a) particles (blue diamonds, left) and compared to anti-Lp(a) pAb (orange square). The two polyclonal antibodies agglutinated Lp(a) particles similarly. The applied Lp(a) particles had a mean radius of 11 nm (FIG. 35), as measured by Dynamic Light Scattering, consistent with the published Lp(a) radius.

[0104] Subsequent ELISA studies confirmed that an adsorption antigen (i.e., the bait sequence shown in FIG. 4) removes reactivity to both human plasminogen kringle type IV and Lp(a) kringle type IV2. The ELISA data in FIG. 36 shows that no or highly limited reactivity against human plasminogen kringle type IV or Lp(a) kringle type IV2 remains after adsorption. ELISA plates were coated with human plasminogen kringle type IV (blue lines) or human Lp(a) kringle type IV2 (orange lines). Dark blue and - orange lines were obtained using raw IgG fraction whereas light colored lines were derived after purification of the IgG fraction on a column loaded with the adsorption protein.

[0105] Further studies confirmed that triple kringle domain constructs induce extraordinarily high levels of serum immunoglobulin (Ig), as summarized by the table provided in FIG. 37. The total IgG content in the serum of non-immunized rabbits is approximately 10 g / L. A 250 pL sample was processed from rabbits immunized with antigen I.

[0106] To investigate whether any specific reactivity towards kringle type IVs, IV7, or IV9 domains is lost during adsorption, ELISA reactivity against kringle IVs, kringle IV7 and kringle IV9 domains, was tested before and after adsorption purification of an Antigen I derived IgG fraction. See FIGs. 38-40, respectively. Dark blue, green, and peach colored data lines were obtained using raw IgG fraction. Light blue, orange and grey data lines were obtained using adsorption purified IgG fraction.

[0107] As illustrated by FIGs. 41-42, polyclonal antibodies produced using Antigen I were found to recognize both Antigen I (blue circles) and the adsorption protein (orange circles) protein (FIG. 41). After using the adsorption protein as a bait on an adsorption column, the polyclonal antibody selectively recognizes Antigen I and not the adsorption protein (FIG. 42). In this figure, blue dots represent Antigen I and orange dots represent the adsorption protein.

[0108] Subsequent analysis demonstrated that Roche® Tina2 calibrator levels 1-5 contains Apo(a) with increasing numbers of kringle type IV2 repeats (FIG. 43). The samples were reduced to break disulfide bridges and free Apo(a) from Apo(B100) and size separated on an SDS-PAGE gel (A). Size separated protein bands were transferred to a nylon membrane by electroporation (B). The membrane was first probed with a rabbit anti-human Apo(a) antibody that had been adsorption purified to remove binding to kringle type IV2. Secondly, a goat antirabbit IgG antibody labelled with HRP was used to visualize the Apo(a) containing bands. Lane 1 : size marker. Lanes 2-6: Tina2 calibrator (1 pL) levels 5-1. Lane 7: empty. Lanes 8-10 Tina2 calibrator (3 pL) levels 5, 3, and 1. Level 5 is the highest concentration and 1 the lowest. Open bars to the right illustrates that the two selected lower band sizes are parallel whereas the upperband is at an angle showing that the Apo(a) repeat band is growing in size as the Apo(a) kringle type IV2 repeat number increases. Middle bar is the expected size of Apo(a) without kringle type IV2 repeats.

[0109] FIG. 44 is a schematic showing an exemplary workflow for adsorption purification of antibodies generated according to the disclosure. Cross-reactivity to human plasminogen is adsorbed by loading the Ig fraction from rabbits immunized with Antigen I onto columns covalently coupled with native human plasminogen (“hplg,” black square). The flow-through contains anti-Lp(a) reactivity and is denoted “IgG Apo(a) KIV2-sensitive.” Further adsorption purification was done by running this fraction onto a column with immobilized adsorption protein (“Ad,” red square). This purification step resulted in anti-Apo(a) reactivity insensitive to kringle type IV2 repeats and the fraction is denoted “IgG Apo(a) KIV2-insensitive.”

[0110] As illustrated by FIGs. 45-48, adsorption purification of an immunoglobulin fraction from a rabbit immunized with Antigen I can be made insensitive to human plasminogen and human kringle IV2 domains. ELISA plates were coated with native human plasminogen (hplg) (FIG. 45), adsorption protein (FIG. 46), a recombinant Lp(a) kringle type IV2 domain (FIG. 47) or Apo(a) covalently bound to LDL, Lp(a) (FIG. 48). As shown by FIG. 45, antibodies produced against Antigen I (“IgGAl”) bind to hplg but this reactivity can be removed by adsorption against human plasminogen. Anti-human plasminogen and anti-human Lp(a) polyclonal antibodies served as positive and negative controls, respectively. As shown by FIG. 46, the adsorption protein was bound at the same level by IgGAl, IgGAlKIV2- sensitive and anti-Lp(a) as expected. IgAGlKIV2-insensitive in contrast did not bind to the adsorption protein. As shown by FIG. 47, IgGAl, IgGAl (KIV2-sensitive) and anti-Lp(a) polyclonal antibodies all bound to Apo(a) kringle domain IV2 as expected whereas as IgGAl (KIV2-insensitive did not. As shown by FIG. 48, all of the four polyclonal antibodies bound Lp(a) particles showing they all can bind to the native lipoprotein particle. In conclusion,immunization with Antigen I and subsequent adsorption purification of the Ig fraction using human plasminogen and the adsorption protein makes it possible to produce a polyclonal antibody specific for Lp(a) but lacking interference from kringle type IV2.

[0111] As shown by FIG. 49, all three kringle domains in Antigen I are immunogenic and similar to the native structures. ELISA experiments were conducted with wells coated with one of recombinant apo(a) KIVs, - KIV7 and - KIV9. IgGAl, IgGAl(KIV2-sensitive) and IgGAl(KIV2-insensitive) all bound to these antigens with same reactivity. The correct structure of the recombinant proteins was confirmed by their reactivity to the anti-Lp(a) antibody. To verify the lack of cross reactivity against LDL, ELISA experiments were similarly conducted with LDL coats and no reactivity was seen confirming the absence of anti- Apo(BlOO) reactivity The relatively lower level of anti-Lp(a) reactivity to the kringle domains can be explained by the greater complexity of the anti-Lp(a) antibody. Reactivity to one of many epitopes should be expected to mean that each epitope specific reactivity constitutes a low absolute amount of antibody.

[0112] Western blot analysis supports that the chosen adsorption purification can be used to derive an anti-human Lp(a) polyclonal antibody that is insensitive to repeats of kringle domain type IV2 in Lp(a). See FIG. 50, which contains a Western blot showing that crossreactivity against plasminogen is removed (arrows), and also indicates that the kringle domain type IV2 sensitive part is removed (circles) when comparing the relative intensities. The ratio between the upper bands in lanes 2 and 3 depends on the ability of the applied antibody to recognize Lp(a). When a repeat insensitive antibody is used then a weak band is found in Lane 3 as compared to the upper band in lane 2. This is reversed when a kringle domain type IV2 sensitive antibody is used. The simplest explanation is that donor 3 has a high number of repeats and donor 2 a lower number. The upper band in lane 1 is relatively unaffected by the nature of the applied antibody suggesting that donor 1 has a low kringle domain type IV2 copy numberbut a high level of Lp(a) particles. In this figure: M: marker; 1-3: Donor 1,2,3; 4: native human plasminogen; 5: native LDL; and 6: native Lp(a).

[0113] Additional experiments were conducted using chimeric kringle IV and protease constructs. As shown by FIG. 57, human apo(a) protease can be expressed recombinantly in HEK cells, and anti-Lp(a) antibodies, produced according to the present disclosure, contain reactivity against human apo(a) protease. Lipoprotein(a) consists of low-density lipoprotein (LDL) covalently bound with apo(a). To investigate if anti-Lp(a) antibodies display reactivity against apo(a)’s protease, the protease was recombinantly expressed in HEK cells and used to coat a 96 wells plate at 1 pg / mL (approximately 35 nM). The coat was tested with various primary pAb. Two were insensitive to size variation of apo(a) and produced from recombinant antigen that did not contain the protease. Anti-Lp(a) antibodies were produced by immunizing with native Lp(a) particle and purifying the anti-serum against relevant proteins. Plasminogen (pig) contains a protease with high sequence homology to apo(a) protease, anti-plasminogen, anti-plg, (brown dots, black line) was tested to investigate cross reactivity. The main protein in LDL, apo(b), was used as negative control, as no reactivity would be expected based on a sequence comparison. One lane tested background, “bg,” from secondary goat HRP-anti-rabbit pAb.

[0114] FIG. 58 provides a visualization of protease epitopes. A cassette based on rabbit plasminogen protease was used to carry human Lp(a) epitope. Three cassettes each carrying a single define human epitope are predicted to elicit an immune respond resulting in pAb. Residues in proximity from a structural point of view were grouped and defined as epitopes. Successful expression of chimeric protease was confirmed by the correct band size (SDS- PAGE) of the Epl construct. FIG. 59 shows chimeric constructs with and human epitopes from apo(a) kringle 4 or 9 grafted onto rabbit plasminogen kringle IV, and the respective sequences annotated to highlight the grafted regions. Finally, FIG. 60 depicts an Lp(a) particle andregions of targeted reactivity for optimal agglutination. KIV2 (denoted “2” in this figure) may be expanded and are avoided to obtain a polyclonal response that is size independent. FIG. 60 illustrates how antibodies targeting these epitopes will cover the Lp(a) particles and, without being bound to a theory, it is predicted that the addition of reactivity to the protease domain will complement reactivity to apo(a) kringle 4 and 9. Thus such a polyclonal antibody is expected to give a strong signal in a turbidimetric assay while being insensitive to the number of repeats of apo(a) kringle 2.

[0115] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular compound, composition, article, apparatus, methodology, protocol, and / or reagent, etc., described herein, unless expressly stated as such. In addition, those of ordinary skill in the art will recognize that certain changes, modifications, permutations, alterations, additions, subtractions and sub-combinations thereof can be made in accordance with the teachings herein without departing from the spirit of the present specification.

[0116] Use of the terms “may” or “can” in reference to an embodiment or aspect of an embodiment also carries with it the alternative meaning of “may not” or “cannot.” As such, if the present specification discloses that an embodiment or an aspect of an embodiment may be or can be included as part of the inventive subject matter, then the negative limitation or exclusionary proviso is also explicitly meant, meaning that an embodiment or an aspect of an embodiment may not be or cannot be included as part of the inventive subject matter. In a similar manner, use of the term “optionally” in reference to an embodiment or aspect of anembodiment means that such embodiment or aspect of the embodiment may be included as part of the inventive subject matter or may not be included as part of the inventive subject matter. Whether such a negative limitation or exclusionary proviso applies will be based on whether the negative limitation or exclusionary proviso is recited in the claimed subject matter.

[0117] Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0118] The terms “a,” “an,” “the” and similar references used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Further, ordinal indicators — such as “first,” “second,” “third,” etc. — for identified elements are used to distinguish between the elements, and do not indicate or imply a required or limited number of such elements, and do not indicate a particular position or order of such elements unless otherwise specifically stated. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0119] When used in the claims, whether as filed or added per amendment, the open-ended transitional term “comprising” (and equivalent open-ended transitional phrases thereof like including, containing and having) encompasses all the expressly recited elements, limitations, steps and / or features alone or in combination with unrecited subject matter; the named elements, limitations and / or features are essential, but other unnamed elements, limitations and / or features may be added and still form a construct within the scope of the claim. Specific embodiments disclosed herein may be further limited in the claims using the closed-ended transitional phrases “consisting of’ or “consisting essentially of’ in lieu of or as an amended for “comprising.” When used in the claims, whether as filed or added per amendment, the closed-ended transitional phrase “consisting of’ excludes any element, limitation, step, or feature not expressly recited in the claims. The closed-ended transitional phrase “consisting essentially of’ limits the scope of a claim to the expressly recited elements, limitations, steps and / or features and any other elements, limitations, steps and / or features that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. Thus, the meaning of the open-ended transitional phrase “comprising” is being defined as encompassing all the specifically recited elements, limitations, steps and / or features as well as any optional, additional unspecified ones. The meaning of the closed-ended transitional phrase “consisting of’ is being defined as only including those elements, limitations, steps and / or features specifically recited in the claim whereas the meaning of the closed-ended transitional phrase “consisting essentially of’ is being defined as only including those elements, limitations, steps and / or features specifically recited in the claim and those elements, limitations, steps and / or features that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. Therefore, the open-ended transitional phrase “comprising” (and equivalent open-ended transitional phrases thereof) includes within its meaning, as a limiting case, claimed subject matter specified by the closed-ended transitional phrases “consisting of’ or “consisting essentially of.” As such embodiments described herein or so claimed with the phrase “comprising” are expressly or inherently unambiguously described, enabled and supported herein for the phrases “consisting essentially of’ and “consisting of.”

[0120] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0121] Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

CLAIMSWe claim1. A recombinant protein, comprising one or more directly or indirectly linked human kringle type IV domains.

2. The recombinant protein of claim 1, wherein each human kringle type IV domain comprises a polypeptide sequence represented by any one of SEQ ID NOs: 1-9, or a polypeptide sequence sharing at least 90% sequence identity with any one of SEQ ID NOs: 1-9.

3. The recombinant protein of claim 1, wherein the recombinant protein comprises 1-10 of the directly or indirectly linked human kringle type IV domains.

4. A recombinant protein, comprising: one or more non-human plasminogen kringle type IV domains, each sharing at least 70, 78, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 10 or a fragment thereof, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 10.

5. The recombinant protein of claim 4, wherein the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NO: 10.

6. The recombinant protein of claim 4, wherein the one or more amino acid substitutions comprise one or more of the substitutions shown in FIGs. 6-16.

7. A recombinant protein, comprising: one or more non-human plasminogen kringle type II domains, each sharing at least 90% sequence identity with SEQ ID NO: 11 or a fragment thereof, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 11.

8. The recombinant protein of claim 7, wherein the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NO: 11.

9. The recombinant protein of claim 7, wherein the one or more amino acid substitutions comprise one or more of the amino acid substitutions shown in any one of FIGs. 6-16, or in FIG. 59.

10. A recombinant protein, comprising: one or more non-human plasminogen kringle type III domains, each sharing at least 90% sequence identity with SEQ ID NO: 12 or a fragment thereof, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 12.

11. The recombinant protein of claim 10, wherein the fragment comprises at least 10, 20, 30, 40, or 50 contiguous amino acids of SEQ ID NO: 12.

12. The recombinant protein of claim 10, wherein the one or more amino acid substitutions comprise one or more of the amino acid substitutions shown in any one of FIGs. 6-16.

13. A recombinant protein, comprising: one or more non-human plasminogen kringle type V domains, each sharing at least 90% sequence identity with SEQ ID NO: 13 or a fragment thereof, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 13.

14. The recombinant protein of claim 13, wherein the fragment comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 contiguous amino acids of SEQ ID NO: 13.

15. The recombinant protein of claim 13, wherein the one or more amino acid substitutions comprise one or more of the amino acid substitutions shown in any one of FIGs. 6-16.

16. A recombinant protein, comprising: one or more non-human plasminogen serine protease domains, each sharing at least 90% sequence identity with SEQ ID NO: 14 or a fragment thereof, and comprising one or more amino acid substitutions as compared to SEQ ID NO: 14.

17. The recombinant protein of claim 16, wherein the fragment comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 or 230 contiguous amino acids of SEQ ID NO: 14.

18. The recombinant protein of claim 16, wherein the one or more amino acid substitutions comprise one or more of the substitutions shown in any one of FIGs. 6-16, or in FIG. 58.

19. A method for generating antibodies specific for human lipoprotein a (“Lp(a)”), comprising: a) inoculating a non-human animal host with a recombinant antigen comprising the recombinant protein of any one of claims 1-18; b) obtaining serum from the animal host comprising one or more polyclonal antibodies; c) generating polyclonal antibodies specific for Lp(a) by enriching or purifying the serum.

20. The method of claim 19, wherein the serum is enriched or purified by contacting the serum with a screening agent to isolate one or more off-target, non-specific, and / or cross-reactive antibodies.

21. The method of claim 20, wherein the screening agent comprises: (i) a human plasminogen kringle type IV domain, or a fragment thereof, and / or (ii) a human Lp(a) kringle type IV, subtype 2 domain, or a fragment thereof.

22. The method of claim 21, wherein the human plasminogen kringle type IV domain, or a fragment thereof, and / or the human Lp(a) kringle type IV subtype 2 domain, or fragment thereof, is affixed to a solid support when contacted with the serum.

23. The method of claim 21, wherein the human plasminogen kringle type IV domain, or the fragment thereof, and / or the human Lp(a) kringle type IV subtype 2 domain, or the fragment thereof, is affixed to a bead when contacted with the serum.

24. The method of claim 19, wherein the enriching or purifying step comprises a precipitation reaction or adsorption chromatography, using the human plasminogen kringle IV domain, or a fragment thereof, and / or the human Lp(a) kringle IV type 2 domain, or a fragment thereof.

25. A composition comprising the polyclonal antibodies specific for Lp(a) generated by the method of any one of claims 19-24.

26. The composition of claim 25, wherein the polyclonal antibodies specific for Lp(a): a) are insensitive as to a size of an apo(a) component of Lp(a); b) are specific for one or more kringle type IV domains of Lp(a); c) are specific for a kringle type V domain of Lp(a); d) are specific for a linker sequence adjacent to a kringle type IV domain of Lp(a); e) are specific for a serine protease domain of Lp(a); f) do not display cross-reactivity against human plasminogen; and / or g) any combination of the above.

27. The composition of claim 25, wherein the polyclonal antibodies specific for Lp(a) display a titer to a human plasminogen kringle type IV domain and / or a human Lp(a) kringle type IV, subtype 2 domain, that is less than 20% of a total titer to human Lp(a).

28. A composition comprising polyclonal antibodies specific for Lp(a), wherein the polyclonal antibodies display a titer to a human plasminogen kringle type IV domain and / or a human Lp(a) kringle type IV, subtype 2 domain that is less than 20% of a total titer to human Lp(a).

29. A method for measuring a concentration of Lp(a) in a biological sample obtained from a subject in need thereof, comprising: a) obtaining the biological sample from the subject, wherein the biological sample comprises whole blood, serum, or plasma; b) optionally, enriching or purifying the biological specimen to enrich or remove one or more components; and c) measuring the concentration of Lp(a) in the biological sample by performing an immunoassay using the polyclonal antibodies specific for Lp(a) generated by the method of any one of claims 19-24, or the composition of any one of claims 25-28.

30. The method of claim 29, wherein the immunoassay is an ELISA assay, a Western blot, a particle enhanced turbidimetric (PET) assay, or a competition assay.

31. The method of claims 29 or 30, further comprising:d) evaluating the subject’s risk of cardiovascular disease based on the measured concentration of Lp(a).

32. A kit for measuring a concentration of Lp(a) in a biological sample, comprising: one or more polyclonal antibodies specific for Lp(a) generated by the method of any one of claims 19-24; and at least one buffer and / or reference sample comprising a known amount of Lp(a).

33. A nucleic acid, encoding the recombinant protein of any one of claims 1-18.

34. The nucleic acid of claim 33, wherein the nucleic acid is a genomic DNA, a vector, or an mRNA.

35. A cell engineered to express the nucleic acid of claims 33 or 34.

36. A method for generating antibodies specific for human Lp(a), comprising: a) inoculating a non-human animal host with a recombinant antigen comprising a polypeptide sequence of at least one kringle type IV domain and / or a protease domain, of human Lp(a); b) obtaining serum from the animal host comprising one or more polyclonal antibodies; c) screening the serum using a bait protein configured to bind to one or more polyclonal antibodies in the serum, wherein the bait protein is attached to a solid support; and d) generating polyclonal antibodies specific for Lp(a) by removing the bait protein.

37. The method of claim 36, wherein the recombinant antigen comprises a polypeptide sequence of two or more kringle type IV domains of human Lp(a).

38. The method of claim 36, wherein the recombinant antigen comprises a polypeptide sequence of kringle IVs, IV7, and IV9 domains of human Lp(a), optionally connected by two linker sequences.

39. The method of claim 36, wherein the recombinant antigen comprises a polypeptide sequence of kringle IV4, IVe, and IVs domains of human Lp(a), optionally connected by two linker sequences.

40. The method of any one of claims 36-39, wherein the bait protein comprises a polypeptide sequence of a human Lp(a) kringle type IVi and / or type IV2 domain.

41. The method of any one of claims 36-39, wherein the bait protein comprises a polypeptide sequence of a human plasminogen kringle type IV domain.

42. The method of claims 40 or 41, wherein the bait protein comprises a polypeptide sequence of a human Lp(a) kringle type IVi and type IV2 domains; and a polypeptide sequence of a human plasminogen kringle type IV domain; wherein human Lp(a) kringle type IVi and type IV2 domains, and the human plasminogen kringle type IV domain, are connected by native human Lp(a) kringle type IVi and IV2 linkers.

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