PRECLINICAL STUDY: EDITORIAL COMMENT
Regression of Atherosclerosis With Therapeutic AntibodiesPipe Cleaner or Pipe Dream?*
Geoffrey S. Ginsburg, MD, PhD, FACC*
Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina.
* Reprint requests and correspondence: Dr. Geoffrey S. Ginsburg, Center for Genomic Medicine, Duke Institute for Genome Sciences and Policy, Box 3382, 101 Science Drive, Durham, North Carolina 27708. (Email: geoffrey.ginsburg{at}duke.edu).
Oxidized low-density lipoprotein (oxLDL) is an immunogen that plays an important role in the initiation and progression of atherosclerosis (1). Active immunization with oxLDL in model systems prone to atherosclerosis results in a heightened adaptive immune response and protection against lesion formation (2). Indeed, in vitro oxidation of low-density lipoprotein (LDL) results in multiple oxidation-specific epitopes (3,4) in the peptide sequences of its protein component, apolipoprotein B (apoB), and in its phospholipid moieties. Together, these are highly immunogenic and can be used to generate immunoglobulin (Ig) G antibodies that in turn lead to inhibition of atherosclerosis in the murine LDL receptor knockout and apolipoprotein E knockout (ApoE
–/–) models of atherosclerosis. It has been suggested that anti-oxLDL antibodies might possess pathogenic as well as protective effects (5). The pathogenic effects of anti-oxLDL antibodies is supported by observations of elevated IgG seen in lesions (6) in the sera of patients with coronary disease (7,8), peripheral vascular disease (9), and with syndromes associated with accelerated atherosclerosis such as hypertension, diabetes, and immune-mediated diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis) (10,11). However, others have speculated that the higher levels of anti-oxLDL antibodies, in particular IgM autoantibodies, observed in children compared with levels in adults might serve a protective purpose. Furthermore, other studies have demonstrated an inverse association of intimal media thickness in the carotid arteries with anti-oxLDL antibody levels in serum (12). Several monoclonal antibodies (mAbs) have been developed that bind epitopes on oxLDL and are used to assay oxLDL levels in plasma or in vascular lesions. Assays using these antibodies have been developed as probes to cultivate a greater understanding of the clinical utility of anti-oxLDL as a circulating biomarker for atherosclerosis (13).
Based on observations that a large number of malondialdehyde-modified sequences in apoB are recognized in human plasma and that immunization of ApoE
–/– mice with these peptides resulted in inhibition of atherosclerosis development, Schiopu et al. (14) developed a series of mAbs against apoB peptides 661 to 680. These antibodies, when injected into the ApoE
–/– mice, reduced the extent of atherosclerosis development as well as the plaque content of oxLDL epitopes (14), providing support for the hypothesis that passive immunity with anti-oxLDL might be protective against atherosclerosis. These results were observed despite the fact that these mice developed significant titers of antihuman antibodies. Other groups have confirmed and extended this finding using alternative antibody preparations against different epitopes on oxLDL (15,16). In the current issue of the Journal, this group has extended their work to show that anti-oxLDL antibodies can induce regression of established atherosclerosis in a mouse model (17). The effects on pre-existing vascular atherosclerosis of recombinant IgG antibodies against the oxLDL apoB epitope between amino acids 661 and 680 were assessed in an experimental model of atherosclerosis. The administration of 3 weekly doses of the antibodies led to a rapid and significant regression of existing lesions. This observation, if confirmed, implies that passive immunity could have an important role in ameliorating established atherosclerotic vascular disease.
Immune-mediated therapies have existed for more than 100 years with passive antibody therapy, or "serum therapy," being a mainstay of treatment for pneumonia, meningitis, and a variety of other infectious diseases (18). These crude heterologous extracts from sera were fraught with variability in efficacy and risk of allergic reactions and serum sickness. Today antibody purification, hybridoma technology, and the development of mouse–human chimeric and humanized mAbs have markedly improved the specificity and reduced the potential for toxicity from mAb therapies. The latter have provided for reduced immunogenicity and longer half-lives, issues that have plagued many biologic candidates for human use. More recently phage display, the ability to manipulate various antibody regions and enhance functionality, and the ability to express human antibodies in different species (even in plants) have led to a reduction in both the toxicity and variability seen in antibody preparations of the past. Over the past 2 decades, more than 20 antibodies have been approved by the Food and Drug Administration, all of which were monoclonal. The vast majority of these were developed with a therapeutic goal of inhibition of host cell function targeting cellular antigens to interfere with cell function and achieve a therapeutic effect. Abciximab, a Fab fragment preparation of the mouse–human chimeric antibody c7E3 with specificity for glycoprotein IIb/IIIa in human platelets, is the only antibody therapeutic approved for human use clinically in cardiovascular disease in the setting of acute myocardial infarction and high-risk percutaneous coronary interventions. Despite these successes and the compelling data from animal models described in this issue and elsewhere, the path to therapeutic antibody therapy for regression of established atherosclerosis will be arduous. There are several major issues that must be addressed for these achievements to translate into clinical utility.
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Should an Anti-oxLDL Be Polyclonal or Monoclonal?
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The major question here is whether the therapeutic target of oxidation of LDL is a single epitope or multiple epitopes. Several oxidation-specific epitopes on LDL exist, including those on apoB and its lipids; however, it is uncertain if a single therapeutic antibody will be sufficient to result in clinically significant regression of disease. It may be that in humans a combination of mAbs will be required to provide a diversity of biologic function. Thus, for any individual patient, the activity of a polyclonal preparation will reside in a subfraction of the total Ig preparation, therefore requiring large amounts of protein to achieve the desired effect and exposing patients to higher probabilities of toxicity (discussed later) and higher costs associated with preparation of Ig.
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What About Antibody Specificity?
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A major advantage of antibody therapeutics is the potential to design therapies with great specificity for a particular cell type or functional receptor. Whether this advantage can be achieved with anti-oxLDL antibodies is difficult to ascertain at this time. For example, how antigenically diverse is oxLDL in humans? Are all epitopes present in susceptible individuals or do individuals exhibit temporal changes in the oxLDL epitopes that are expressed? Are these epitopes present in normal tissues, and if so, what are the consequences of exposure to them in this respect? Clearly, additional experiments are required to address these issues.
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Should the Therapeutic Antibody Be Heterologous or Homologous?
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Schiopu et al. (14,17) used a recombinant human IgG that, in mice, results in significant titers of antihuman antibodies. In humans, a human antibody preparation would be a very reasonable approach to minimize the potential of immune-mediated sensitivity reactions (e.g., human antimouse antibodies) seen in heterologous antibody products. However, even humanized therapeutic antibodies have resulted in human antichimeric and human antihuman neutralizing antibodies that have limited therapeutic effectiveness or induced adverse reactions in humans. Although the use of homologous antibodies is expected to come with a reduced risk of toxicologic side effects, these reactions would need to be extensively studied in the first human studies using this approach. In addition, the reduction in the likelihood of hypersensitivity afforded by a human antibody may carry an additional financial burden. Given the fact that most human preparations of antibody may be 100-fold greater in cost than heterologous preparations plus the likelihood that anti-oxLDL therapy would be a lifelong treatment, cost is a significant factor.
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Should an Intact Antibody or a Fab Fragment Be Used and What Dose Would Be Optimal?
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Fab fragments have the advantage of lower immunogenicity and shorter half-lives. The half-life, dose, and dose interval will need to be established in humans to carry out a successful clinical trial. The antibody used by Schiopu et al. (14,17) is an intact human IgG with a short half-life, evidenced by the fact that it is absent from the circulation of mice within 2 weeks. The utility of the murine data for dosing in humans is likely to be minimal; therefore, human studies and empiric dosing studies will need to be carried out to ascertain the appropriate dosing schedules in humans. A major issue that will need to be resolved in future studies of this approach is the extent to which the disease progresses following cessation of any antibody therapy and thus how long after therapy would it need to be re-initiated, assuming the disease returns. In the current study (17), animals were sacrificed just 2 weeks following the last of 3 doses, which is not a long enough duration to assist us in answering this important question.
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How Would Antibody Therapy Be Combined With Current Drugs or Diet Therapies for Coronary Artery Disease?
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For an antibody therapy approach to succeed clinically, it must have superior efficacy and/or lower toxicity than existing therapies when used alone or in combination with existing therapies. Schiopu et al. (17) switched from an atherogenic to a normal chow diet in their murine studies. The next series of studies on regression should consider the use of these antibodies adjunctively with the standard therapies for atherosclerosis. Moreover, we need to understand the utility of this approach in the setting of a proatherogenic environment, which unfortunately is the world in which we live.
Thus, although the murine studies presented in this issue of the Journal provide an important "proof of concept" that regression of atherosclerosis with an antibody therapeutic is feasible in mice, the hurdles to developing a human therapeutic for regression of disease are significant. Clearly, more studies are needed to identify the dominant antigens mediating atherosclerosis. Further, human validation and long-term administration may be limited by immune reactions to the active agent. Thus, although the concept of antibody-mediated regression of atherosclerosis is attractive, the formidable challenges that must be overcome are significant for this to remain more than just a pipe dream.
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Footnotes
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* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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1. Binder CJ, Chang MK, Shaw PX, et al. Innate and acquired immunity in atherogenesis Nat Med 2002;11:1218-1226.2. Nicoletti A, Kaveri S, Caligiuri G, et al. Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice J Clin Invest 1998;102:910-918.[Web of Science][Medline] 3. Palinski W, Ylä-Herttuala S, Rosenfeld ME, et al. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein Arteriosclerosis 1990;10:325-335.[Abstract/Free Full Text] 4. Palinski W, Witztum JL. Immune responses to oxidative neoepitopes on LDL and phospholipids modulate the development of atherosclerosis J Intern Med 2000;247:371-380.[CrossRef][Web of Science][Medline] 5. Shoenfled Y, Wu R, Dearing LD, Matsuura E. Are anti-oxidized low-density lipoprotein antibodies pathogenic or protective? Circulation 2004;110:2552-2558.[Free Full Text] 6. Ylä-Herttuala S, Palinski W, Butler SW, et al. Rabbit and human atherosclerotic lesions contain IgG that recognized epitopes of oxidized LDL Atertioscler Thromb 1994;14:32-40. 7. Wu R, Nityanand S, Berglund L, et al. Antibodies against cardiolipin and oxidatively modified LDL in 50-year-old men predict myocardial infarction Arterioscler Thromb Vasc Biol 1997;17:3159-3163.[Abstract/Free Full Text] 8. Puurunen M, Manttari M, Manninen V, et al. Antibody against oxidized low-density lipoprotein predicting myocardial infarction Arch Intern Med 1994;154:2605-2609.[Abstract/Free Full Text] 9. Monaco C, Crea F, Niccoli G, et al. Autoantibodies against oxidized low density lipoproteins in patients with stable angina, unstable angina or peripheral vascular disease: pathophysiological implications Eur Heart J 2001;22:1572-1577.[Abstract/Free Full Text] 10. Cvetkovic JT, Wallberg-Jonsson S, Ahmed E, et al. Increased levels of autoantibodies against copper-oxidized low density lipoprotein, malondialdehyde-modified low density lipoprotein and cardiolipin in patients with rheumatoid arthritis Rheumatology (Oxford) 2002;41:988-995.[CrossRef][Medline] 11. Wu R, Svenungsson E, Gunnarsson I, et al. Antibodies to adult human endothelial cells cross-react with oxidized low-density lipoprotein and β2-glycoprotein I (β2-GPI) in systemic lupus erythematosus Clin Exp Immunol 1999;115:561-566.[CrossRef][Web of Science][Medline] 12. Hulthe J, Wiklund O, Hurt-Camejo E, Bondjers G. Antibodies to oxidized LDL in relation to carotid atherosclerosis, cell adhesion molecules, and phospholipase A2
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