Generation and characterization of LPA-KIV9, a murine monoclonal antibody binding a single site on apolipoprotein (a)

Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV2 repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV2. We generated hybridomas, screened them, and successfully produced a KIV2-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV9 without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence 4076LETPTVV4082 on KIV9. In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements. Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV2 repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV2. We generated hybridomas, screened them, and successfully produced a KIV2-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV9 without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence 4076LETPTVV4082 on KIV9. In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements. Lipoprotein (a) [Lp(a)] is composed of apo(a) covalently bound to apoB-100 (1Kronenberg F. Utermann G. Lipoprotein(a): resurrected by genetics.J. Intern. Med. 2013; 273: 6-30Crossref PubMed Scopus (332) Google Scholar). Apo(a) consists of 10 unique kringle (K)IV repeats that are present in one copy, except for KIV2, which is present in a variable number of identical copies at the protein level (1 to >40). It also contains one copy of KV and an inactive protease-like domain. The apo(a) protein displays broad size heterogeneity due to a variable number of KIV2 repeats among individuals and populations. Plasma Lp(a) levels are genetically determined by the production and secretion rate of apo(a) by hepatocytes, with isoforms containing a small number of KIV2 repeats being secreted more efficiently, leading to an inverse association of KIV2 repeat number and plasma Lp(a) levels (2Schmidt K. Noureen A. Kronenberg F. Utermann G. Structure, function, and genetics of lipoprotein(a).J. Lipid Res. 2016; 57: 1339-1359Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Elevated Lp(a) is highly prevalent, with an estimated 20% of the population having levels >50 mg/dl (>125 nmol/l), the threshold above which risk accrues in statin-treated patients (3Tsimikas S. Fazio S. Ferdinand K.C. Ginsberg H.N. Koschinsky M.L. Marcovina S.M. Moriarty P.M. Rader D.J. Remaley A.T. Reyes-Soffer G. et al.NHLBI Working Group recommendations to reduce lipoprotein(a)-mediated risk of cardiovascular disease and aortic stenosis.J. Am. Coll. Cardiol. 2018; 71: 177-192Crossref PubMed Scopus (231) Google Scholar, 4Willeit P. Ridker P.M. Nestel P.J. Simes J. Tonkin A.M. Pedersen T.R. Schwartz G.G. Olsson A.G. Colhoun H.M. Kronenberg F. et al.Baseline and on-statin treatment lipoprotein(a) levels for prediction of cardiovascular events: individual patient-data meta-analysis of statin outcome trials.Lancet. 2018; 392: 1311-1320Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Lp(a) is also a target of therapy, with recent studies showing that antisense oligonucleotides may lower Lp(a) by over 80% (5Tsimikas S. Karwatowska-Prokopczuk E. Gouni-Berthold I. Tardif J.C. Baum S.J. Steinhagen-Thiessen E. Shapiro M.D. Stroes E.S. Moriarty P.M. Nordestgaard B.G. et al.Lipoprotein(a) reduction in persons with cardiovascular disease.N. Engl. J. Med. 2020; 382: 244-255Crossref PubMed Scopus (330) Google Scholar, 6Tsimikas S. Viney N.J. Hughes S.G. Singleton W. Graham M.J. Baker B.F. Burkey J.L. Yang Q. Marcovina S.M. Geary R.S. et al.Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.Lancet. 2015; 386: 1472-1483Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 7Viney N.J. van Capelleveen J.C. Geary R.S. Xia S. Tami J.A. Yu R.Z. Marcovina S.M. Hughes S.G. Graham M.J. Crooke R.M. et al.Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.Lancet. 2016; 388: 2239-2253Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), and a phase 3 clinical trial that is now underway (https://clinicaltrials.gov/ct2/show/NCT04023552). Multiple national and international societies have suggested that Lp(a) be measured in individuals at moderate to high risk for CVD to enhance risk prediction or for considerations as a risk enhancer (8Catapano A.L. Graham I. De Backer G. Wiklund O. Chapman M.J. Drexel H. Hoes A.W. Jennings C.S. Landmesser U. Pedersen T.R. et al.2016 ESC/EAS guidelines for the management of dyslipidaemias.Eur. Heart J. 2016; 37: 2999-3058Crossref PubMed Scopus (2042) Google Scholar, 9Anderson T.J. Gregoire J. Pearson G.J. Barry A.R. Couture P. Dawes M. Francis G.A. Genest Jr., J. Grover S. Gupta M. et al.2016 Canadian Cardiovascular Society guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult.Can. J. Cardiol. 2016; 32: 1263-1282Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 10Stefanutti C. Julius U. Watts G.F. Harada-Shiba M. Cossu M. Schettler V.J. De Silvestro G. Soran H. Van Lennep J.R. Pisciotta L. et al.Toward an international consensus–integrating lipoprotein apheresis and new lipid-lowering drugs.J. Clin. Lipidol. 2017; 11: 858-871.e3Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Grundy S.M. Stone N.J. Bailey A.L. Beam C. Birtcher K.K. Blumenthal R.S. Braun L.T. de Ferranti S. Faiella-Tommasino J. Forman D.E. et al.2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA guideline on the management of blood cholesterol: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines.J. Am. Coll. Cardiol. 2019; 73: 3168-3209Crossref PubMed Scopus (704) Google Scholar, 12Wilson D.P. Jacobson T.A. Jones P.H. Koschinsky M.L. McNeal C.J. Nordestgaard B.G. Orringer C.E. Use of lipoprotein(a) in clinical practice: a biomarker whose time has come. A scientific statement from the National Lipid Association.J. Clin. Lipidol. 2019; 13: 374-392Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 13Cegla J. Neely R.D.G. France M. Ferns G. Byrne C.D. Halcox J. Datta D. Capps N. Shoulders C. Qureshi N. et al.HEART UK consensus statement on Lipoprotein(a): a call to action.Atherosclerosis. 2019; 291: 62-70Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 14Mach F. Baigent C. Catapano A.L. Koskinas K.C. Casula M. Badimon L. Chapman M.J. De Backer G.G. Delgado V. Ference B.A. et al.2019 ESC/EAS guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk.Eur. Heart J. 2020; 41: 111-188Crossref PubMed Scopus (3066) Google Scholar). Many commercially available assays exist to measure plasma Lp(a), but assay reagents and methodologies vary among manufacturers and have not been globally standardized. It is anticipated that the testing of Lp(a) values will expand with recent European guidelines that have recommended that every adult person have Lp(a) measured at least once in their lifetime (14Mach F. Baigent C. Catapano A.L. Koskinas K.C. Casula M. Badimon L. Chapman M.J. De Backer G.G. Delgado V. Ference B.A. et al.2019 ESC/EAS guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk.Eur. Heart J. 2020; 41: 111-188Crossref PubMed Scopus (3066) Google Scholar), as well as by the development of new therapies capable of substantially lowering Lp(a) (5Tsimikas S. Karwatowska-Prokopczuk E. Gouni-Berthold I. Tardif J.C. Baum S.J. Steinhagen-Thiessen E. Shapiro M.D. Stroes E.S. Moriarty P.M. Nordestgaard B.G. et al.Lipoprotein(a) reduction in persons with cardiovascular disease.N. Engl. J. Med. 2020; 382: 244-255Crossref PubMed Scopus (330) Google Scholar). To date, only one isoform-independent antibody binding to KIV9 on apo(a) has been described and well-characterized, but its peptide epitope has not been reported. This antibody has been used to develop an ELISA method, and the ELISA has been used to assign a target value to the WHO/IFCC reference material, and it is also used as a comparison method to validate commercial assays. However, while the method has been used in a variety of clinical studies, it is not at present commercially available (15Marcovina S.M. Albers J.J. Gabel B. Koschinsky M.L. Gaur V.P. Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a).Clin. Chem. 1995; 41: 246-255Crossref PubMed Scopus (272) Google Scholar). In this study, we describe the generation and characterization of a new murine monoclonal antibody that binds to a defined peptide sequence present only once in KIV9 that can be used in both basic and clinical aspects of Lp(a) research and Lp(a) measurements. To generate immunogens, we used two approaches. First, we generated several truncated apo(a) proteins for use as immunogens. One construct spanned the entire KIV10-KV sequence, and its generation and purification are described in the supplemental material. A second large apo(a) construct, designated 8K-IV, contained one copy of KIV1, one copy of KIV2, a fusion of KIV3 and KIV5, and individual kringles, KIV6 to KIV10, KV, and the protease-like domain, as previously described (16Schneider M. Witztum J.L. Young S.G. Ludwig E.H. Miller E.R. Tsimikas S. Curtiss L.K. Marcovina S.M. Taylor J.M. Lawn R.M. et al.High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins.J. Lipid Res. 2005; 46: 769-778Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Second, as a different strategy, we generated several unique short peptides derived from apo(a) but not present on plasminogen, which were conjugated to keyhole limpet hemocyanin for immunization. These included peptide GDGRSYRGISSTTVT present in one copy on KIV9 and peptide MNPRKLFDYC present in one copy on KV. Screening antigens included Lp(a) purified from a single donor as previously described (17van der Valk F.M. Bekkering S. Kroon J. Yeang C. Van den Bossche J. van Buul J.D. Ravandi A. Nederveen A.J. Verberne H.J. Scipione C. et al.Oxidized phospholipids on lipoprotein(a) elicit arterial wall inflammation and an inflammatory monocyte response in humans.Circulation. 2016; 134: 611-624Crossref PubMed Scopus (277) Google Scholar), human plasminogen (R&D, Minneapolis, MN), and a variety of recombinant apo(a) constructs, including two KIV2 constructs containing either three or five copies, various constructs spanning consecutively from KIV6 to the protease domain (KIV6-P, KIV7-P, KIV8-P, KIV9-P, KIV10-P, respectively), a 17 kringle human construct with eight KIV2 repeats, a 17 kringle construct without the protease domain (17KΔP), and a 17 kringle construct without KV (17KΔV) (18Hancock M.A. Boffa M.B. Marcovina S.M. Nesheim M.E. Koschinsky M.L. Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces.J. Biol. Chem. 2003; 278: 23260-23269Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Leibundgut G. Scipione C. Yin H. Schneider M. Boffa M.B. Green S. Yang X. Dennis E. Witztum J.L. Koschinsky M.L. et al.Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a).J. Lipid Res. 2013; 54: 2815-2830Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The disulfide bond between apoB-100 and apo(a) is composed of cysteine 4236 of apoB-100 and cysteine 4057 on apo(a) (20Cheesman E.J. Sharp R.J. Zlot C.H. Liu C.Y. Taylor S. Marcovina S.M. Young S.G. McCormick S.P. An analysis of the interaction between mouse apolipoprotein B100 and apolipoprotein(a).J. Biol. Chem. 2000; 275: 28195-28200Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Murine monoclonal antibody MB47, specific for human apoB-100 (21Young S.G. Smith R.S. Hogle D.M. Curtiss L.K. Witztum J.L. Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B.Clin. Chem. 1986; 32: 1484-1490Crossref PubMed Scopus (76) Google Scholar), whose epitope on apoB is between residues 3350 and 3506 (22Weisgraber K.H. Innerarity T.L. Newhouse Y.M. Young S.G. Arnold K.S. Krauss R.M. Vega G.L. Grundy S.M. Mahley R.W. Familial defective apolipoprotein B-100: enhanced binding of monoclonal antibody MB47 to abnormal low density lipoproteins.Proc. Natl. Acad. Sci. USA. 1988; 85: 9758-9762Crossref PubMed Scopus (52) Google Scholar), was used in an ELISA format to measure Lp(a). Similarly, murine monoclonal antibody LPA4 (23Tsimikas S. Lau H.K. Han K.R. Shortal B. Miller E.R. Segev A. Curtiss L.K. Witztum J.L. Strauss B.H. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): short-term and long-term immunologic responses to oxidized low-density lipoprotein.Circulation. 2004; 109: PubMed Scopus Google that binds to the epitope present on KIV5, and of apo(a) was used in as previously described S. Fazio S. Viney N.J. Xia S. Witztum J.L. Marcovina S.M. of lipoprotein(a) and to lipoprotein(a) and apolipoprotein(a) isoform Clin. Lipidol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). The sequence of by LPA4 is also present on KIV2 and is as LPA4 also binds to KIV2, as previously described S. Fazio S. Viney N.J. Xia S. Witztum J.L. Marcovina S.M. of lipoprotein(a) and to lipoprotein(a) and apolipoprotein(a) isoform Clin. Lipidol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). were to by the and Use of the of BALB/C were immunized with keyhole limpet peptides GDGRSYRGISSTTVT and as well as apo(a) construct but produced specific antibodies or the antibodies bound to KIV2 plasminogen, was with and the results are not A BALB/C mouse was immunized with a as previously described A. P. C. D. Taylor A.M. Tsimikas S. Witztum J.L. Miller A monoclonal antibody to oxidized with and apoB-100 in human Lipid Res. 2019; Full Text Full Text PDF PubMed Scopus Google Scholar), with a of of the apo(a) construct, at with three with A of of in was the the mouse was were and with a and the were in were to and To antibodies specific to Lp(a) without binding to plasminogen or KIV2, from the were screened by ELISA as described To that bound Lp(a) but not KIV2 or plasminogen were by in without and by The and of two were and were found to be identical in The were by in in a with with 3 and antibody purified with over of antibody was the mouse monoclonal antibody This monoclonal antibody was designated as of antibodies present in immunized mouse of and testing the purified monoclonal antibody were by ELISA as previously described A. P. C. D. Taylor A.M. Tsimikas S. Witztum J.L. Miller A monoclonal antibody to oxidized with and apoB-100 in human Lipid Res. 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). In were with Lp(a), apo(a) KIV2 construct, or plasminogen at in at sites were with in and for at of mouse plasma to in or were and for at antibodies were an and on a binding was as over An of apo(a) constructs (18Hancock M.A. Boffa M.B. Marcovina S.M. Nesheim M.E. Koschinsky M.L. Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces.J. Biol. Chem. 2003; 278: 23260-23269Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Leibundgut G. Scipione C. Yin H. Schneider M. Boffa M.B. Green S. Yang X. Dennis E. Witztum J.L. Koschinsky M.L. et al.Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a).J. Lipid Res. 2013; 54: 2815-2830Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Marcovina S.M. Koschinsky M.L. of the of apo(a) kringle in Biol. PubMed Scopus Google was to and analysis with LPA-KIV9. were in and on a were to a and the were with in and for 1 with antibodies (1 in at by with antibodies conjugated with for 1 at and by with A was also with antigens in on the and were as described above and by a and an reagent was from the the of was either antisense or the of of and were to the of of of antibody were a was to for with of The consensus sequence was A peptide spanning of peptides by only one was on LPA-KIV9 binding was antibody conjugated with and analysis was used to the epitope on a of M. S. A. Arnold K. G. F. M. L. et protein and Res. PubMed Scopus Google Scholar). The was plasminogen, with sequence The sequence of apo(a) was from to analysis and analysis were with version the the apo(a) construct, mouse plasma was screened for the generation of monoclonal antibodies binding Lp(a). Plasma contained binding to the apo(a) construct and Lp(a), with only levels of binding to the construct were and were and screened by ELISA for production of antibodies Lp(a), in that were and Two were for their binding to Lp(a) but not were to two of to were for binding to Lp(a) but not to or plasminogen the two were found to have identical in their and and were derived from the The are in One was in and the antibody LPA-KIV9 was purified and to be an by ELISA of the purified LPA-KIV9 a specific with Lp(a), with with or plasminogen of LPA-KIV9. LPA-KIV9 and and sites are To the site on apo(a) to which LPA-KIV9 we its binding to an of constructs by and LPA-KIV9 with constructs containing various of kringle including KIV7-P, KIV8-P, and containing from and KIV9 to of protease domain, It also the 17 kringle human construct, the construct KV, and the construct the protease-like domain However, LPA-KIV9 not with and the only constructs not containing with the epitope being present on KIV9. This specific binding was with showing that the of KIV9 was for LPA-KIV9 A peptide spanning KIV9 of in was LPA-KIV9 bound to peptide on KIV9 which the epitope at This epitope is of cysteine 4057 on apo(a) in in that the disulfide bond with cysteine 4236 of apoB-100 (20Cheesman E.J. Sharp R.J. Zlot C.H. Liu C.Y. Taylor S. Marcovina S.M. Young S.G. McCormick S.P. An analysis of the interaction between mouse apolipoprotein B100 and apolipoprotein(a).J. Biol. Chem. 2000; 275: 28195-28200Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The of the epitope on apo(a) was by a showing that it is a epitope the of KIV9 in in and three from This the generation and characterization of a murine monoclonal antibody, that a unique epitope on KIV9 of apo(a) present only in one The generation of this new monoclonal antibody will for development of an isoform-independent assay for the of plasma Lp(a) levels. of binding only one site on it may address of the of clinical Lp(a) assays that polyclonal antibodies binding multiple sites on apo(a) that can lead to either or of Lp(a) levels. In an National and Working Group recommended the development of a globally of Lp(a) to commercial (3Tsimikas S. Fazio S. Ferdinand K.C. Ginsberg H.N. Koschinsky M.L. Marcovina S.M. Moriarty P.M. Rader D.J. Remaley A.T. Reyes-Soffer G. et al.NHLBI Working Group recommendations to reduce lipoprotein(a)-mediated risk of cardiovascular disease and aortic stenosis.J. Am. Coll. Cardiol. 2018; 71: 177-192Crossref PubMed Scopus (231) Google and that total Lp(a) in be and values in to a reference of results by the different be an is A recent Lp(a) levels by commercially available that commercial assays are and their the in a not on apo(a) H. B. H. G. M. W. of lipoprotein (a) measured by commercially available 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). commercially available assays use polyclonal which are a of antibodies that may the apo(a) including the potential for binding multiple within the KIV2 assay are derived from plasma of multiple but generally apo(a) isoforms present in the being to the polyclonal antibodies more to apo(a) which are found in with low plasma Lp(a) and to In antibodies to small which are found in Lp(a) in with high plasma Lp(a) and to values S. Fazio S. Viney N.J. Xia S. Witztum J.L. Marcovina S.M. of lipoprotein(a) and to lipoprotein(a) and apolipoprotein(a) isoform Clin. Lipidol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Albers J.J. Lipoprotein (a) measurements for clinical Lipid Res. 2016; 57: Full Text Full Text PDF PubMed Scopus Google Scholar). The in monoclonal antibodies unique to Lp(a) the KIV2 region that also not with plasminogen, which has to apo(a) A.M. Lawn R.M. sequence of human apolipoprotein(a) is to PubMed Scopus Google Scholar), that only a number of sites are available to generate with in the study, an and to develop antibodies with multiple immunogens, only two were as being isoform and only binding one site, and both were identical in mapping that the epitope of LPA-KIV9 from the and may be more clinical will be on assay to measure Lp(a). of isoform independence in with isoform size will be once a assay method is In conclusion, the murine monoclonal antibody, may be useful in to measure Lp(a) in both research and clinical that the within this and of this are available from the The for and for in the and of LPA-KIV9. with kringle kringle lipoprotein (a)

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