Microspheres
The invention refers to malondialdehyde (MDA) - microspheres.
Background
Microparticles are small vesicles that are released from activated cells or cells undergoing apoptosis. They can be found circulating in the blood, for example when they originate form leukocytes, endothelial cells, red blood cells or platelets. In addition, they can also found in other bodily fluids as well as tissues. Microparticles circulating in blood plasma are often procoagulant shed membrane fragments described as "platelet dust". They usually are >100 nm in diameter and derived from the plasma membrane. Smaller vesicles (40 to 100 nm) originating from the
endoplasmic membranes are described as exosomes, and larger particles (>1 ,5 μιτι) containing nuclear material are best known as apoptotic bodies. Microparticles typically are different in lipid and protein composition from the parental cells they derive from, which provides an explanation for their possible pathophsyiological effects. Moreover microparticles released from activated cells may be different from microparticles shed after cell activation. For example, increased numbers of circulating endothelial microparticles have been identified in individuals with certain diseases, including acute coronary syndrome, hypertension, diabetes, prothrombotic states, the antiphospholipid antibody syndrome, multiple sclerosis and cerebral malaria. The endothelial microparticles in some of these disease states have been shown to have arrays of cell surface molecules reflecting a state of endothelial dysfunction. Therefore, endothelial microparticles may be useful as an indicator or index of the functional state of the endothelium in disease, and may potentially play key roles in the pathogenesis of certain diseases. The plasma level of endothelial microparticles is, for instance, a specific marker of endothelial dysfunction in patients with cardiovascular disease. Circulating levels of microparticles are also augmented in patients with autoimmune and thrombotic disease. Both microparticles and apoptotic bodies externalize the anionic phospholipid phosphatitylserine, which plays a significant role in the activation of coagulation and complement cascades. A review of circulating microparticles, their pathophysiology and clinical implications is provided by Piccin et al (2007. Blood Reviews 21 , 157-171 ).
Microorganisms or toxins that successfully enter an organism will encounter the cells and mechanisms of the innate immune system. The innate response is usually triggered when microbes are identified by pattern recognition receptors, which recognize components that are conserved among broad groups of microorganisms, or when damaged, injured or stressed cells send out alarm signals, so called "danger signals", many of which are recognized by the same receptors as those that recognize pathogens. Innate immune defenses have a limited repertoire. This system does not confer long-lasting immunity against a pathogen, as found in immunological memory of adaptive responses. The innate immune system is the dominant system of host defense in most organisms.
Inflammation is one of the first responses of the immune system to infection or any other insult, e.g. mechanical. The symptoms of inflammation are redness and swelling, which are caused by increased blood flow into a tissue. Inflammation is promoted by eicosanoids and cytokines, which are released by injured or infected cells. Eicosanoids include prostaglandins that produce fever and the dilation of blood vessels associated with inflammation, and leukotrienes that attract leukocytes.
Common cytokines include interleukins that are responsible for communication between white blood cells; chemokines that promote chemotaxis; and interferons that have anti-viral effects, such as shutting down protein synthesis in the host cell. Growth factors and cytotoxic factors may also be released. These cytokines and other chemicals recruit immune cells to the site of infection and promote healing of any damaged tissue following the removal of pathogens.
Natural antibodies (NAbs) play an important immunobiological role in the natural defense mechanism. NAbs spontaneously arise without prior infection or immune exposure. In mice, they are predominantly derived from B-1 cells. Natural antibodies exhibit a remarkably conserved repertoire, which has been suggested to represent a primitive layer of the immune system as a product of natural selection. NAbs are typically regarded as "polyreactive" in that they bind to a number of self or foreign antigens. This pattern of broad reactivity of a preformed pool of antibodies is required for the rapid and immediate recognition and protection against invading pathogens. On the other hand, natural antibodies may also play a role in the recognition and removal of senescent cells, cell debris, and other (neo-)self-antigens and thereby possess another so called "house-keeping" function in neutralizing and removing body waste and protecting from autoimmunity. There has been evidence of pathways in which NAbs may contribute in the elimination of self-antigens exposed during stress, tissue damage, or even conventional cell turnover. Atherosclerosis is a disease of the vascular wall that leads to myocardial infarction, heart failure, peripheral vascular disease, and stroke. Although multiple risk factors have been identified that contribute variably to lesion formation, the growth of the atherosclerotic lesion is both initiated and sustained by increased levels of LDL and low and/or dysfunctional HDL. In the past decade, inflammatory processes have been identified as equally important factor contributing to atherosclerotic lesion formation. Atherosclerosis develops over decades and is believed to progress from intimal thickening to ever more complex lesions involving the accumulation of cells derived from the circulation, proliferation of inherent vascular wall cells, and synthesis of extracellular matrix and lipid accumulation, both extracellular bound to matrix and intracellular, within macrophage foam cells. Macrophage cholesteryl ester formation is believed to be attributable in large part to enhanced and unregulated uptake of oxidized, aggregated, and variously otherwise modified LDLs and possibly other lipoproteins and disturbed cellular responses that are unable to mediate the export of the accumulated cholesterol load. As the lesions progress, many of the lipid-filled cells undergo apoptosis but are not sufficiently cleared, leading to an abnormal
accumulation of apoptotic cells in the lesion. Under these conditions, apoptotic cells may undergo secondary necrosis, yielding the acellular gruel characteristic of the advanced atherosclerotic plaques. Smooth muscle cell proliferation and secretion of a thick collagen cap may stabilize the lesion, but eventually vulnerable areas of the plaque erode or rupture, leading to thrombosis, ischemia, and clinical events or even death.
Oxidation-specific epitopes are a class of pathogen-associated molecular patterns (PAMPs) that are recognized by natural antibodies and other innate and adaptive immune receptors. Physiological and pathological stress can lead to the generation of oxidation-specific epitopes, which are considered altered self or neoself- antigens on membranes of lipoproteins as well as (apoptotic or necrotic) cells and cellular debris, which are subsequently recognized by natural antibodies, scavenger receptors, and other innate effector proteins via these motifs. In many, if not all, cases, molecular mimicry exists between oxidation-specific epitopes of self-antigens and epitopes of microbes, which represent the "conserved" patterns on various pathogens to which NAbs bind, termed "pathogen-associated molecular patterns" (PAMPs) (Shaw P. X. et al. 2000. J. Clin. Invest.105: 1731-1740). Thus, as a result of oxidative stress, oxidation-specific epitopes constitute one category of "altered self," which represents "danger signals" (e.g. PAMPs) that are recognized and defended against by multiple arcs of innate immunity.
Typically, peroxidation of the abundant phospholipid phosphatidylcholine is initiated at the oxidationprone sn-2 polyunsaturated fatty acid. Decomposition of the oxidized fatty acid generates a wide spectrum of reactive molecular species, such as malondialdehyde (MDA) with its many condensation products and 4-hydroxynonenal (HNN), as well as the "core aldehyde" of the residual oxidized phospholipid (OxPL) backbone, yielding 1 -palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine
(POVPC), which contains the phosphorylcholine (PC) head group. These reactive aldehydes can modify autologous molecules, including both the protein moiety of LDL, apolipoprotein B (apoB), and other lipid molecules, such as amine-containing phospholipids (e.g., phosphatidylserine). Thus, altered lipids as well as oxidized lipid- protein adducts are formed, yielding, for example, MDA-modified lysines on proteins as well as aminophospholipids, as well as OxPLs and OxPLprotein/lipid adducts.
MDA is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct, immunogenic protein adduct, termed the Malonacetaldehyde (MAA) adduct. Proteins modified in the presence of high concentrations of MDA can also produce MAA-modified proteins.
Chang M.K. et al. (1999 Proc. Natl. Acad. Sci. USA. 96: 6353-6358.) have shown that the membranes of apoptotic cells are enriched in oxidized PC
phospholipids, suggesting that apoptotic cells bearing oxidation specific epitopes should be immunogenic. It was demonstrated that a number of the different oxidation- specific IgM monoclonal antibodies that bound OxLDL, such as EO6, and EO14, which binds to MDA-LDL, bound to cell surface determinants on apoptotic cells but not viable cells. Furthermore, each of these antibodies could inhibit the uptake of apoptotic cells by macrophages in an additive manner. These oxidation-specific neoepitopes are evidently PAMPs representing "eat me" signals to innate immunity, as manifested by macrophage scavenger receptors and natural antibodies. It is likely that there are many other such neoepitopes generated as a result of stress-induced alterations in native structure. These epitopes can be generated by adduct formation between reactive lipid moieties and proteins or other lipids, generating entirely novel structures, such as MDA-lysine adducts on LDL (Binder C.J. et al. 2005 J. Lipid Res. 46: 1353- 1363).
Importantly, it was found that >20% of all circulating micropartides isolated from the plasma of healthy volunteers have IgM antibodies bound to their surface.
Characterization of the binding specificties of these IgM antibodies after elution from isolated micropartides revealed that within the microparticle-bound IgM preparation there is an enrichment for IgM with specificity for oxidation-specific epitopes, most prominently malondialdehyde (MDA), when compared to plasma IgM. In addition, the presence of oxidation-epitopes on the surface of these same circulating micropartides was assessed by flow cytometry using IgM monoclonal antibodies with specificity for defined oxidation-epitopes. Consistent with the characterization of the eluted IgM, mAbs against MDA-epitopes stained up to 40% of all circulating micropartides, and mAbs against oxidized PtC (Phosphatidylcholine) as well as oxidized cardiolipin stained between 10-15%. Thus, a significant portion of circulating micropartides in healthy volunteers carries oxidation-epitopes, partially bound by endogenous IgM Abs. It was further found that these characteristics were to a large extent associated with red blood cell derived micropartides, and to a lesser degree with leukocyte or platelet derived micropartides. These data indicate that circulating micropartides are physiological carriers of oxidation-specific epitopes, most prominently MDA epitopes, and as such represent major prototypic targets of natural IgM antibodies. The presence of these oxidation-epitopes on the surface of circulating micropartides provides a mechanism for their pro-inflammatory effects. Indeed, circulating
micropartides induce the production of I L-1 β by macrophages in vitro, and this activity can be neutralized using a natural IgM mAb directed against MDA-epitopes. Thus, natural IgM Abs with specificity for oxidation epitopes provide "house keeping" functions by clearing and neutralizing the activity of micropartides, which is particularly important in atherosclerosis, where increased accumulation of micropartides has been reported. Moreover, micropartides seem to be a most relevant physiological carrier for oxidation-specific epitopes.
Binder et al (The Journal of Clinical Investigation 1 14(3) 427-437 (2004)) discloses oxidation specific neoepitopes, such as MDA-modified LDL. Immunization of mice with MDA-LDL as active ingredient results in the activation of Th2 cells with specificity for the MDA modification. Anti-inflamnnatory N-fatty acid-amino acid conjugates and J2 prostanoid-amino acid conjugates is described by US2006/0014820A1 . The active ingredient may be incorporated into microspheres, e.g. albumin crosslinked by glutaraldehyde or starch microspheres. The microspheres can be hardened by cross-linking procedures, such as heat treatment or by using chemical cross-linking agents, inter alia by
malondialdehyde.
Summary
There is a need to better understand the natural defense mechanism in detail and to provide treatment of diseases resulting from an imbalance of the innate immune system. It is, thus, an object of the invention to provide tools specifically bearing oxidation-specific epitopes, for use in identifying or providing diagnosis or treatment of disease conditions associated with oxidative stress.
The object is solved by the subject matter of the claims. Thus, the invention refers to a microsphere based on MDA coupled to a synthetic backbone, also called synthetic microsphere, with a size ranging between 500 nm and 500 μιτι, preferably
900 nm - 300 μιτι, more preferred at least 1 μιτι.
The preferred microsphere according to the invention is based on a solid material selected from the group consisting of a polymeric thermoplast, a hydrogel, silica particle, latex and magnetic beads. This includes the preferred materials of polymers, like polystyrene and polylysine, alginates and agarose, such as Sepharose or similar materials used as carriers for separation or chromatographic purposes.
According to the invention the microsphere preferably additionally comprises epitopes of at least one of MAA, 4-hydroxynonenal or oxidized phospholipids.
The preferred microsphere according to the invention comprises MDA to bind to at least 100 ng serum antibody per gram, e.g. based on the weight of Sepharose beads, in a saturation assay. The appropriate saturation binding assay typically would be a potency assay.
Specifically the microsphere according to the invention would have a capacity to deplete serum IgM, in particular MDA-specific antibodies.
Preferably the microsphere according to the invention would be coated by MDA, for example at least 10% of available ε-amino groups are coated by MDA. This is particularly preferred when polylysine or lysine containing bridging molecules is employed as a backbone. The preferred binding of the MDA epitope and eventual other oxidation specific epitopes to the backbone is a covalent binding, either directly or via a bridging structure, like polylysine or any other epsilon-aminogroup carrier. Alternatively, the epitopes are bound via affinity binding partners or ligands, employing binding partners with a high binding affinity as a mediator of binding or an affinity binding bridge.
According to a preferred aspect of the present invention the microsphere is labelled, including dye labels.
The microsphere according to the invention preferably forms an immune complex with an MDA specific antibody, or a fragment or derivative thereof, such antibody being of any isotype. Such immune complex comprises the MDA epitope bound to a respective immune binding partner.
According to a specific aspect of the invention the microsphere, including the immune complex preparation of the microsphere, is preferably provided for analytic, diagnostic or pharmaceutic purposes.
Specifically such a microsphere is used in the manufacture of a pharmaceutical preparation for the prevention or treatment of oxidative stress disease in a patient. Thus, the microsphere according to the invention is specifically used for the prevention or treatment of oxidative stress disease in a patient. Therefore the microsphere may be used directly to formulate the pharmaceutical preparation of microspheres, or may be employed as a tool to select and provide specific binders or reactands for use in the development of pharmaceuticals.
According to a specific embodiment of the invention the microsphere is provided in a diagnostic preparation for use in an assay for determining oxidative stress disease or the risk of developing such disease in a patient by qualitative and/or quantitative measurement. Specifically, an assay for the physiological MDA binding potential in a patient's sample, such as blood, plasma or serum, gives an indication of the risk of developing a respective disease, thus is considered a prognostic factor. In a
competitive assay system, the endogenous MDA is determined to indicate the risk and stage of the disease progression. Thus, according to a specific aspect the present invention relates to a method of prognosis of oxidative stress disease or the risk of developing such disease in a patient, comprising
- providing microspheres according to the invention,
- providing a sample of said patient, - contacting said sample with said microspheres to effect binding of said microspheres to oxidation epitopes of said sample, and
- determining said binding as a prognostic factor of oxidative stress disease. The relevant disease is typically related to oxidative stress associated with cardiovascular disease, the metabolic syndrome and obesity, autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, cancer and conditions caused by cancer treatment, age related macular degeneration, Alzheimer's disease, brain senescence, alcoholic liver disease, ischemic reperfusion injury, diabetic nephropathy, nephritis, acute lung injury, and infectious diseases, or any inflammatory conditions associated or caused therewith.
According to a further embodiment of the invention there is provided a method of selecting an antagonist of oxidative stress disease from a repertoire of agents possibly binding to oxidation epitopes, e.g. of oxidized lipoproteins, circulating microparticles, apoptotic or necrotic cells, or cellular debris, comprising:
- providing a microsphere according to the invention,
providing the repertoire of agents from synthetic or natural sources, selecting an agent from the repertoire that specifically binds to the MDA epitope of said microsphere, and
determining the potency of said agent to neutralize said microsphere. Preferably said agent is selected from the group consisting of polypeptides, such as antibodies and antibody fragments or derivatives, reactive proteins, peptides, enzymes and small molecules.
Preferably said potency is determined by a specific binding assay, such as an immunoassay. The potency may also be determined by a functional assay to neutralize MDA and/ or MAA-mediated effects.
In a preferred method according to the invention a repertoire of agents is selected, each having said potency. This repertoire may be further characterized to provide a lead compound for developing a drug.
Other features and advantages of the present invention will become apparent from the detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Detailed Description
The term "backbone" refers to the carrier or framework of a microsphere, which may be loaded with ligands, such as antigens or binding epitopes, within the
framework and/ or on the surface of a backbone particle.
The term "microsphere" refers to small synthetic, spherical particles
manufactured from various natural and synthetic materials, including glass
microspheres, polymer microspheres and ceramic microspheres, e.g. in the solid or semi-solid state, including hollow microspheres, with varying density, sphericity, uniformity, particle size and particle size distribution.
The term "oxidative stress" refers to an imbalance of the immune and/ or complement system caused by environmental or pathological factors, which triggers the development of oxidation specific epitopes as a measure of oxidative stress disease.
The term "oxidation specific epitope" refers to neo-epitopes on structures, like lipids, lipoproteins, proteins or dead cells, including drusen and debris, which develop due to reactions caused by oxidative stress, in particular with oxidized lipoproteins. Apoptotic and necrotic cells are known to carry oxidation specific epitopes. Among those epitopes is MDA/MAA or epitopes on MDA/MAA-adducted proteins, alone or associated with a carrier, e.g. a lipid or lipoprotein, such as LDL or other epitopes formed by oxidation of endogenous structures.
The term "oxidative stress disease" refers to human or veterinary diseases, either chronic or acute, caused or associated with oxidative stress, including MDA- or MAA-induced inflammation and MDA- or MAA-induced complement activation, thus e.g. triggering local or systemic inflammatory reactions. Among the oxidative stress diseases there is cardiovascular disease, the metabolic syndrome and obesity, autoimmune diseases, including rheumatoid arthritis, cancer and conditions caused by cancer treatment, age related macular degeneration, Alzheimer's disease, brain senescence, alcoholic liver disease, ischemic reperfusion injury, diabetic nephropathy, nephritis, acute lung injury, and infectious diseases, or any inflammatory conditions associated or caused therewith. "Oxidative stress disease conditions" are generally understood as those pathological conditions associated with oxidative stress.
Specifically the invention refers to the condition or disease that benefits from the inhibition of MDA- and/or MAA-mediated effects. The term "repertoire" refers to a population of diverse variants, for example nucleic acid variants which differ in nucleotide sequence, peptide or polypeptide variants which differ in amino acid sequence, or small molecules. A library according to the invention will encompass such a repertoire. According to the present invention, a repertoire of agents is designed to possess a binding site for an oxidized epitope present on a microsphere.
The term "treatment" refers to both prophylactic and therapeutic treatment of patients. The term also includes treatment for in vivo or ex vivo diagnostic purposes. Treatment may be either human or veterinary, including treatment of mammalians in general. As used herein, and as well understood in the art, treatment is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Palliating a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder. The term prevention or prophylaxis, or synonym thereto, as used herein refers to a reduction in the risk or probability of a patient becoming afflicted with the disease or manifesting a symptom associated with the disease.
Thus, the invention refers to a microsphere of a specific size having MDA as a specific binding partner to agents that would possibly agonize or antagonize the function of circulating microparticles in vivo. Contrary to circulating microparticles the microspheres according to the invention are standardized, e.g. to comprise an MDA coating, e.g. wherein at least 10%, preferably at least 20, more preferably at least 30, 40 or 50% of available or accessible ε-amino groups are coated by MDA, which can be determined by a standard TNBS assay for free amino groups, e.g. as described by Habeeb AF. Anal Biochem. 1966 Mar;14(3):328-36.
The standardized preparations are specifically used for medical or scientific purposes. The further feature of binding capacity is mainly dependent on the number of accessible MDA epitopes on the surface of the microsphere. Specifically the capacity to bind serum IgM is determined as a quality control, e.g. by a respective antibody depletion assay. In an exemplary assay a 10% normal human plasma (NHS) solution is incubated with a specific amount of MDA-modified microspheres, which is the same amount as the maximum amount of control microspheres that would not significantly deplete IgM and/or less than 15% of MDA specific IgM antibodies in this assay. Upon reacting and centrifugation, the supernatant is analyzed for the presence of total IgM levels and IgM titers to MDA-modified BSA. The depletion is positive if >5%, specifically >10% of total IgM are depleted in the supernatant and/or >50%, specifically >70% of MDA-specific IgM antibodies are depleted.
The standardized preparations are specifically provided as liquid suspensions or lyophilized, specifically as storage stable preparations. Exemplary preparations are comprised in test-kits for scientific or medical use, e.g. as research tools or diagnostic tools.
Besides the MDA epitope there are preferably further oxidized epitopes comprised in the microsphere according to the invention, which were found to play a significant role in oxidative stress disease conditions. It surprisingly turned out that the MDA-microsphere according to the invention was advantageous over plain MDA- lipoproteins, which are available in the non-particulate form. Through the association with the microsphere backbone and the respective coating, the MDA epitopes are apparently more accessible to agents involved in the innate immune system, complement activation and inflammatory processes in general. Thus, the
microparticles according to the invention constitute an efficient and reliable binding partner to the relevant native structures and drugs, as they mimic naturally occurring targets carrying these modifications.
The preferred backbone of the microsphere according to the present invention is a synthetic backbone, e.g. not biogenic and formed by polymers, e.g. thermosplastic polymers. Preferred backbones comprise e.g. polyethylene, polystyrene, cellulose acetate, fluoroplastics, polyacrylate, polyamide, polycarbonate, polyester,
polyethylene, polylactide, polypropylene and polyvinyl acetate, or an amino acid copolymer, or a polylysine. Polystyrene microspheres are typically used in biomedical applications due to their ability to facilitate procedures such as cell sorting and immunoprecipitation. Proteins and ligands absorb onto polystyrene readily and permanently, which makes polystyrene microspheres suitable for medical research and biological laboratory experiments. Polyethylene microspheres are commonly used for analytical purposes, e.g. for flow visualization and fluid flow analysis.
Preferred amino acid copolymers include Gly and Ser residues, and Lys, Cys, or other appropriate residues, for providing conjugation sites. Polylysine is particularly preferred in the manufacture of a microsphere according to the invention.
Silica or glass microspheres are commonly used in medical technology.
A hydrogel backbone as used according to the invention is a network of polymer chains that are water-insoluble, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymers. Common ingredients are e.g. polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with an abundance of hydrophilic groups. Preferred hydrogels e.g. are composed of polyvinyl alcohol, polyacrylic acid, Poly(2-hydroxyethyl methacrylate) (pHEMA) and/ or polyethylene glycol (PEG). Preferred natural hydrogel materials include dextran, agarose, crosslinked, beaded-form of agarose (Sepharose), methylcellulose, hyaluronan, and other naturally derived polymers. Preferably cellulose or agarose is treated with specific enzymes, e.g., cellulase and agarase, to yield different lengths.
Suitable alginate and alginate-hydroxypropylmethylcellulose (HPMC)
microspheres may be prepared by an emulsification method.
Another preferred material for use in the microsphere according to the invention is latex, either from natural or synthetic sources.
Magnetic beads, e.g. made of nanometric-sized iron oxide particles
encapsulated or glued together with polymers, may be particularly preferred in specific assays where binders to the microsphere would be separated from the medium through employing a magnetic field.
The MDA epitope as used according to the invention is preferably provided in the enol form, predominantely as the transisomer. It is typically generated in situ by hydrolysis of 1 ,1 ,3,3-tetramethoxypropane. The MDA adduct formation typically occurs at a pH of 7.4 or - in a modified protocol - in the presence of 200mM Acetaldehyde at a pH of 4.8. The modified molecule is then typically coupled via amide linkage to the bead backbone. Uncoupled protein is removed by extensive washing. However, it is also possible to first couple the carrier molecule to the microsphere and modify it thereafter with MDA/MAA or to directly modify the beads without using a carrier. Additional epitopes as preferably provided with the microsphere according to the invention are other oxidation-epitopes, such as MDA condensation products, e.g. MAA, 4-hydroxynonenal or oxidized phospholipids, such as oxidized phosphatidylcholine and cardiolipin. According to a particularly preferred embodiment the microsphere according to the invention is devoid of tissue factor. Contrary to some native circulating microparticles, the preferred microspheres according to the invention, thus, would not provoke undesired procoagulant side effects caused by tissue factor. In this regard it is also preferred that the microspheres according to the invention for pharmaceutic use do have a limited procoagulant activity or do not carry procoagulant phospholipids.
Typically the microsphere according to the invention is prepared as follows: The carrier molecule is either (1 ) modified first with the epitopes and then covalently coupled to the spherical backbone, or (2) first coupled to the beads and then modified with the epitopes, or (3) the spherical backbone is directly modified with the epitopes without the use of a carrier molecule. It is possible to couple different epitopes following the same protocol.
MDA and eventually further antigens can be coupled directly to the derivatized, activated backbone. Bifunctional cross-linkers suitable for conjugating the activated backbone and the antigens can be selected based on the properties desired and the specific substances to be cross-linked. These heterobifunctional reagents are available from several commercial sources, e.g. GE healthcare, Polysciences Inc., Sigma.
The preferred microspheres according to the present invention comprise free amino groups, which are modified to the extent of at least 10%, preferably at least 25%, more preferred at least 50%, 60%, 70%, 80%, 90% up to complete modification, to obtain a high density of MDA and optionally further epitopes. Thereby the preferred microsphere according to the invention is capable of binding at least 100 ng serum antibody per gram, preferably at least 1 g per gram, as measured in a standardized saturation assay. According to a preferred assay both MDA-modified and unmodified microspheres were incubated with mouse and human serum samples in order to investigate IgM antibody binding to MDA epitopes. By performing an anti-lgM FACS analysis it was found that only MDA-modified microspheres, but not unmodified microspheres, were able to bind endogenous IgM antibodies.
Immune complexes of the microsphere according to the invention may be particularly preferred to provide an immunogen to provoke an immune response, or to manufacture specific binders to such immune complexes. The preferred immune complex comprises either natural antibodies, serum immunoglobulins, preferably IgG, IgA and/or IgM, fragments or derivatives thereof, or recombinant immunoglobulins or respective binders based on immunoglobulin domains.
Preferably the microsphere according to the invention is used for analytical, diagnostic or pharmaceutic purposes.
Methods for specifically detecting and quantifying a binder include
immunoassays. Binders are preferably selected from native binders of structures, like oxidized lipoproteins and/or circulating microparticles, apoptotic cells, cellular debris and necrotic cells, and those agents that interfere with these native binders, These assays are e.g. (a) classical direct immunoassays, such as immunodiffusion,
Immunoelectrophoresis, agglutination and immunoprecipitation assays, and (b) methods such as immunofluorescence, radioimmunoassay (RIA), enzyme- immunoassay (EIA) and Western blot assays. These approaches exploit the specificity of the microsphere-binder interactions.
In specific cases it is preferred to employ a label to provide a detectable signal, preferably a fluorophore, chemical dye, radioactive binding agent, chemiluminescent binding agent, electrochemiluminescent agent, magnetic binding agent, paramagnetic binding agent, promagnetic binding agent, enzyme that yield a colored product, enzyme that yield a chemiluminescent product, or an enzyme that yields a magnetic product. In some aspects, the detectable label is generated indirectly from the activation of a target molecule.
The preferred diagnostic preparation comprising a microsphere according to the invention is used in an assay, typically comprising the steps of
- providing a sample of body tissue or body fluid, such as blood plasma or serum,
- providing a microsphere according to the invention, which is optionally bound to a solid surface,
- incubating said microsphere with said sample, and
- determining the reaction products of said sample with said microsphere. The level of reaction product in said sample is indicative of the protection against oxidative stress disease conditions, e.g. enabling a respective prognosis.
Likewise the microsphere according to the invention may be used in a competition assay for diagnostic purposes, e.g. to determine the total fraction of binders or the endogenous level of specific oxidized epitopes, indicative for the state of the oxidative stress disease in a patient. For example, a Latex enhanced immunoassay or Latex enhanced agglutination assay is preferably employed to test 1 ) MDA-binding IgM, 2) MDA-binding complement Factor H, 3) MDA-binding protein X and 4) global MDA-binding activity (i.e. all MDA-binding activity or proteins that have a potency to complex with endogenous MDA). If the level or quality of oxidative stress is high, most engogenous MDA-binding proteins could be complexed already, thus global binding activity with free MDA-binding capacity could be low. The diagnostic assay according to the invention is particularly useful to determine the oxidative stress factor in a sample, supporting a prognosis or risk determination of cardiovascular and other diseases with oxidative stress, to monitor therapy of oxidative stress disease and/or to diagnose oxidative stress disease conditions.
The preferred pharmaceutic purpose is for preparing pharmaceutical
compounds, such as pro-inflammatory agents, immunogens, vaccines, agonists or antagonists of circulating microparticles, or else apheresis to separates out a
respective binder by extracorporeal therapy.
According to the invention a microsphere may be used for screening respective binders, functional agonists or antagonists from a repertoire of binding agents.
The preferably used method for screening such agents is either an
immunoassay or a functional assay to determine the effect of binding, such as effects on macrophages or complement factors. Functional agents directed to such
microspheres and therapeutics derived from such antibodies are also provided. High throughput and parallel screening for potentially therapeutic agents are feasible to a skilled person employing the microsphere according to the invention as ligand to the binding agents.
The repertoire is typically provided as a library of agents, e.g. obtained by site directed mutagenesis or recombinant library methods. A variety of display approaches are employed for the engineering of variants of a scaffold. Phage, yeast, transgenic mouse or in vitro display, such as ribosomal display of libraries is e.g. used for finding binders against targets. Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. Other libraries comprise native antibodies expressed from human B cells.
The preferred agents are provided in a repertoire of synthetic agents, including recombinant agents, e.g. by combinatorial libraries, or natural sources. Specific agents are selected from polypeptides, such as antibodies, in particular natural antibodies, fragments or derivatives thereof, including Fv, Fab, F(ab')2 fragments or combinations of respective antibody domains, reactive proteins or (pro)enzymes or cofactors, such as innate effector proteins, peptides, such as antimicrobial peptides, small molecules, i. e. molecules having molecular weight of up to about 1000 daltons, which are suitable for pharmaceutical use, especially those which are small molecule inhibitors.
Benzodiazepines belong to a preferred chemical class, which has already yielded useful pharmacological agents. Preferred binding agents may be selected from native human antibodies, immortalized B cells expressing such antibodies and non- immortalized B cell libraries comprising B cells expressing such antibodies. Besides the screening for respective binders of the MDA microsphere, further agents with pharmacologic activity may be screened, which interfere with such binding in an agonistic or antagonistic way.
Respective repertoires of agents may be provided by the skilled person employing well-known techniques. In combinatorial chemistry a large number of novel compounds is generated, which can be screened to identify lead compounds for pharmaceutical research and drug development. Theoretically, the total number of compounds which may be produced for a given library is limited only by the number of reagents available to form substituents on the variable positions on the library's molecular scaffold. The combinatorial process lends itself to automation, both in the generation of compounds and in their biological screening, thereby enhancing greatly the opportunity and efficiency of drug discovery. Specifically preferred combinatorial libraries are composed of peptides, in which all or selected amino acid positions were randomized. Nucleic acids are also used in combinatorial libraries. Their great advantage is the ease with which a nucleic acid with appropriate binding activity can be amplified. As a result, combinatorial libraries composed of nucleic acids can be of low redundancy and hence, of high diversity.
The preferred potency assay to determine the binding activity of the agents binding to the microsphere according to the invention is performed as follows: In a specially designed competition assay, the antigen of interest is coated at a limiting concentration on a plate or immobilized by other means. The purified agent identified as binding to the microsphere is added. Addition of increasing concentrations of antigen or microspheres should decrease the binding. Alternatively, a fixed amount of the agent can be depleted from asolution with increasing amounts of microspheres, and the remaining supernatant can be tested for specific binding to the microspheres. Methods for screening agents potentially binding to said microspheres from a repertoire of agents for the type of activities mentioned above are known to those skilled in the art, and e.g. reviewed by Haab BB (Molecular & Cellular Proteomics 4:377-383 (2005)).
Like circulating microparticles the microspheres according to the invention would be recognized by natural IgM antibodies, which would have direct implications for "house keeping" functions and atherosclerosis. Circulating microparticles are membrane derived vesicles from activated or apoptotic cells, and their plasma levels have been shown to be increased in cardiovascular diseases, where they exert various pathological activities, including pro-inflammatory and pro-thrombotic properties. It has been found that about 20% of all circulating microparticles isolated from healthy volunteers have IgM antibodies bound to their surface. Moreover, characterization of IgM antibodies eluted from isolated microparticles revealed that within this preparation there is enrichment for IgM with specificity for oxidation epitopes, most prominently MDA, when compared to plasma IgM. In addition, the presence of oxidation-epitopes was characterized by flow cytometry using specific IgM monoclonal antibodies.
Consistent with the characterization of the eluted IgM, monoclonal antibodies against MDA-epitopes stained up to 40% of all circulating microparticles, and monoclonal antibodies against the phosphorcholine headgroup of oxidized phosphatidylcholine as well as oxidized cardiolipin stained between 10-15%. Thus, a significant portion of circulating microparticles in healthy volunteers carries oxidation-epitopes, partially bound by IgM antibodies. It was further found that these characteristics were mostly associated with red blood cell derived microparticles, and to a lesser degree with leukocyte or platelet derived microparticles. Moreover, circulating microparticles induced the production of IL-1 β by macrophages in vitro, and this activity could be neutralized using an IgM monoclonal antibody directed against MDA-epitopes. Thus, natural IgM antibodies with specificity for oxidation epitopes may provide "house keeping" functions by clearing and neutralizing the activity of microparticles, which is particularly important in atherosclerosis, where increased accumulation of
microparticles has been reported. The relevant models may be employed and the respective natural antibodies or other binders may be selected using the microsphere according to the invention.
In respective experiments MDA-microspheres were used to pull down potential candidates from mouse serum and bound proteins were analyzed by tandem mass spectrometry. It tuned out that the plasma glycoprotein Factor H bound exclusively to MAA-modified beads, but not to unmodified beads. Further experiments have proven that complement Factor H was a suitable agent to inhibit the effects of MDA and circulating microparticles carrying MDA-epitopes.
In a further experiment RAW 264.7 cells were incubated with both MDA modified and umodified synthetic microspheres in order to evaluate the MDA mediated cytokine response. Increased Macrophage inflammatory protein 2 (MIP-2) levels were detected by enzyme linked immunosorbent assay (ELISA) in the supernatants of cells incubated with MDA modified microspheres.
The present invention is further illustrated by the following examples without being limited thereto.
Example 1 : Synthetic MDA-microbeads preparation (sepharose)
MAA-modified proteins were prepared by incubating them at a concentration of 2mg/ml for 3 hours at 37°C with 100mM Malondialdehyde (MDA) and 200mM
Acetaldehyde in PBS pH 4.8. MDA (0.5M) was freshly generated from 1 ,1 ,3,3
Tetramethoxypropane by acid hydrolysis: 1 ,1 ,3,3 Tetramethoxypropane was incubated with 12μΙ 4N HCI and 400μΙ H2O at 37°C for 10min. The reaction was stopped by adjusting the pH to 4.8 by addition of 1 N NaOH, and the volume was brought to 1 ml with H2O. After conjugation, MAA-polylysine was extensively dialyzed against PBS to remove any unreacted MDA. Unmodified or MAA-modified polylysine (generated from polylysine 1 .4kD SigmaAldrich) was coupled to NHS-activated sepharose (GE
Healthcare) according to manufacturer's instructions.
Example 2: Synthetic MDA-microbeads preparation (polystyrene)
1 ml of 2.5 % suspension of amino blue dyed microspheres of 1 μΜ size
(Polysciences Inc.) were pelleted (8000rpm; 3min) and resuspended in 1 ml of 8% glutaraldehyde in PBS with gentle end to end mixing overnight at RT. Beads were washed three times with PBS and resuspended in 500μg polylysine (Sigma Aldrich, MW: 500-2000) in PBS and were mixed gently for 5h at RT. Beads were washed three times with PBS and resuspended in 0.5M ethanoalamine for 30min. Finally, beads were resuspended in 1 ml PBS. MDA modification: we prepared 2ml of 0.5 M MDA
(solution A) by mixing 352μΙ MDA (Sigma; 108383), 48 μΙ 4N HCI and 1600 μΙ H2O and we incubated the previously mentioned solution at 37°C for 10 minutes. We adjusted the pH=7. We added 580 ml of solution A and 420μΙ H2O on the beads followed by an incubation of 5 hours at 37°C Example 3: Induction of cytokine secretion in monocytes with MAA-BSA
Human THP-1 monocytic cells were cultured in RPMI-1640 supplemented with 10% FCS. Before stimulation, cells were washed with serum-free RPMI-1640 and incubated with the stimulation medium containing either BSA or MAA-BSA at 50 g/ml in RPMI-1640 at a density of 5x105 cells/ml for 14h. Cells were removed by
centrifugation (500g, 10min) and supernatants stored at -80°C. The supernatants were assayed for the presence of IL8 with a commercially available ELISA-Kit (OptEia Human IL8 ELISA Set, BecktonDickinson) according to manufacturer's instructions. Treatment with MAA-BSA raised the levels of IL8 from about 150pg/ml to about 1650pg/ml.
Example 4: Cytokine secretion of RAW 264.7 cells upon stimulation with MDA modified microspheres.
4x104 RAW 264.7 cells were plated in a 96 well cell culture plate in full cell maintance medium (DMEM, 10%FBS,1 % PS) and incubated at 37°C and 5% CO2 overnight. The second day we washed the cells three times with PBS and added DMEM medium containing 20ul of 2.5 % suspension of either unmodified or MDA modified amino blue dyed microspheres of 1 μΜ size (Polysciences Inc.). The cells were incubated with the microspheres for 8h at 37°C and 5% CO2 and then the supernatant was collected followed by MIP-2 Enzyme linked immunosorbent assay according to the manufacturer's instructions. Results: We found that RAW 264.7 cells incubated with MDA modified microspheres secreted 1700pg of MIP-2 while the cells incubated with unmodified microspheres secreted 700pg. RAW264.7 cells incubated with medium only secreted 500 pg of MIP-2.
Example 5: Circulating microparticles recognized by natural IgM
Circulating microparticles were isolated from plasma of 10 healthy volunteers by sequential centrifugation according to standard isolation procedures and stained with the biotinylated mouse monoclonal IgM-antibodies EO6 (specific for PC of oxidized phospholipids), LRO4 (specific for MDA/MAA modifications), LRO1 (specific for oxidized Cardiolipin), and an irrelevant IgM control Ab at a concentration of 2 g/ml for 30min at 4°C. Bound biotin was detected with Phycoerithrin-conjugated Streptavidin (BD) on FACSCalibur. On average, 10% (EO6), 10% (LRO1 ), 40% (LRO4) of circulating microparticles bound the respective antibodies.
Circulating microparticles were also assayed for the presence of endogenous IgM bound using a Phycoerythrin-conjugated anti-human IgM Ab. On average, 20% of circulating microparticles carried endogenous IgM antibodies. The percentage of IgM- carrying microparticles did not correlate with the total IgM levels in the plasma from which micropartciles were isolated, indicating that the binding of IgM to circulating microparticles occurred in a antigen-specific manner. Microparticle bound IgM Abs were eluted by hypotonic rupture of microparticles, and the specificity of the eluted IgM was determined by chemiluminescence ELISA using MDA-LDL and copper-oxidized LDL as antigens and an alkaline phosphatise -conjugated anti-human IgM Ab. In comparison to an equivalent plasma dilution based on total IgM levels, microparticle associated IgM displayed a 5-fold enriched specificity for MDA-LDL.
Example 6: Natural antibodies binding to MDA-microsphere (FACS)
Mouse IgM Abs:
30ul of 2.5 % suspension of both MDA modified and unmodified amino blue dyed microspheres of 1 μΜ size (Polysciences Inc.) were pelleted (8000rpm; 3min) and resuspended in 10% mouse serum diluted in PBS-BSA (2%) in a final volume of 10Oul and were incubated at 4°C with gentle end to end mixing for 1 hour. Then, the microspheres were washed three times in PBS-BSA and resuspended in anti-IgM FITC labeled detection antibody (Becton Dickinson) in a final dilution of 1 :600 followed by three washing steps with PBS-BSA (2%).
Results: We found that mouse IgM Abs are recognized only by MDA modified microspheres
Human IgM Abs:
30μΙ of 2.5 % suspension of both MDA modified and unmodified amino blue dyed microspheres of 1 μΜ size (Polysciences Inc.) were pelleted (8000rpm; 3min) and resuspended in 1 % human serum diluted in PBS-BSA (2%) in a final volume of 10Oul and were incubated at 4°C with gentle end to end mixing for 1 hour. Then, the microspheres were washed three times in PBS-BSAand resuspended in anti-IgM biotin antibody (Becton Dickinson) in a final dilution of 1 :600 followed by three washing steps with PBS-BSA (2%). Finally, the beads were resuspended in FITC labeled avidin in a final diluton of 1 :800.
Results: We found that human IgM Abs are recognized only by MDA modified microspheres
Example 7: MDA micropheres to test total MDA reactivity in human serum/ plasma Human serum or plasma samples are diluted in an appropriate dilution buffer. MDA-microspheres based on Latex beads (at approximately 5mg/ml, which is a concentration at which 50% of available MDA-epitopes will be covered by MDA- reactive proteins in NHS) are added and incubated for 30 min at room temperature. Afterwards, an MDA-specific antibody (e.g. murine monoclonal anti-MDA IgG MDA2, J.L. Witztum, UC San Diego; or goat-anti-MDA IgG, Academy Bio-medical Co, USA) conjugated to Biotin is added and incubated at room temperature for 30 min, which allows it to react with the unbound MDA-epitopes on the Latex beads. Agglutination is induced by cross-linking with Avidin-Biotin complexes. The time of agglutination will provide a measure of total MDA-reactivity, which may serve as an indicator of the inflammatory status or inflammatory potential.
Example 8: Intranasal injection of MAA-BSA
Mice were intranasally injected with 50 g of MAA-BSA. After 6h,
bronchoalveolar lavage was performed using BD Venflon (12GA, 1 .77IN, 1 .4x45mm) for 3 times (600μΙ, 500μΙ, 500μΙ). Wash fractions were pooled and total cell number was determined on Casy-Cell-Counter. For differential cell count, cytospins were prepared by centrifugation at 800rpm 15min, dried for 24h at RT and stained with Giemsa and cell populations were counted under the microscope. The concentration of the chemokines CxcM and MIP-2 in the BAL were determined after spinning down cells at 500g for 10min by quantitative ELISA. MAA-BSA injection leads to a 2.4 fold increase in neutrophil number per lung (from 250 in BSA-injected to 600 in MAA- injected). CxcM -Concentration was increased from 40pg/ml to 160pg/ml, whereas MIP- 2 increased from 600pg/ml to 4500pg/ml.
Example 9: Complement activation in human sera with MAA-BSA
Human serum was collected from healthy volunteers in serum tubes (Vacuette 8ml Z Serum Sep Clot Activator) using a 21 G needle (Vacuette blood collection set + luer adapter). After coagulation, the thrombus was removed by centrifugation (15min 2000g 4°C), serum was aliquotized and stored at -80°C. For complement activation, one volume of serum was mixed with one volume of a solution of BSA MAA-BSA in
Veronal Buffered Saline (I mg/ml or less), followed by an incubation for 20min at 37°C. As a positive control, serum was mixed with Cobra Venom Factor in VBS (20U/ml). The complement reaction was stopped by adding 3 volumes of cold sample buffer (Quidel) and C3a generation was determined with Microvue quantitative C3a ELISA (Quidel). Addition of MAA-BSA at a final concentration of 500 g/ml resulted in a 2-fold increased C3a-generation compared to incubation with buffer or native BSA.
Example 10: Identification of Factor H using MDA-Sepharose- microspheres:
Plasma obtained from LDLR-/-RAG-/- mice or human donors were diluted to a concentration of 1 mg/ml total protein. To minimize the amount of proteins binding to unmodified polylysine, plasma dilutions were incubated with PL-coupled beads for 2h at 4°C. After the incubation, supernatant was incubated with either polylysine- or MAA- polylysine-beads for another 2h at 4°C. Beads were washed 3 times with TBS (pH 7.4, 500mM NaCI, 0.5% NP-40, 2mM CaCI2, 1 mM MgCI2). After the last wash, bound proteins were dissociated by adding LDS-sample buffer (Invitrogen) and heating at 95°C for 5min. The supernatants were separated by SDS-PAGE and analyzed by LC- MS-MS on a quadrupole time-of-flight (QTOF) mass spectrometer (Waters) coupled to a nano HPLC system (Agilent). Obtained data was searched against IPI MOUSE database v.3.32 appended with known contaminants (e.g. human keratin proteins). Peptides attributed to factor H were exclusively detected in the MAA-pulldown, but not in the pulldown with unmodified polylysine. The result was verified analyzing the samples by Western-blotting with a CFH-specific antibody.