The present invention relates to IgM antibodies against lipid-related antigens such as antibodies against malondialdehyde (anti-MDA) and/or antibodies against phosphorylcholine (anti-PC) for use in combination therapy with an antineoplastic agent such as methotrexate in mammals such as humans in order to prevent the development of or to treat a mammal suffering from rheumatic diseases, such as active rheumatoid arthritis, osteoarthritis, active juvenile idiopathic arthritis (JIA), psoriasis and severe psoriatic arthritis; cancer such as breast, skin, head, neck, lung or bone-marrow cancer; SLE or CVD. The invention also relates to compositions such as a vaccine comprising lipid-related antigens or antibodies against lipid-related antigens such as malondialdehyde and/or phosphorylcholine and one or more antineoplastic agent such as methotrexate and to the use of such compositions. Background

We determined that antibodies against danger associated molecular patterns (DAMP) and pathogen associated molecular patterns (PAMP) are associated with protection in rheumatic disease, cardiovascular disease and atherosclerosis and also several other chronic inflammatory conditions, findings largely confirmed by others. Underlying mechanisms include protection against inflammatory and other effects of oxidized low density lipoprotein and related inflammatory phospholipids including decreased foam cell formation and an anti-inflammatory effect. ^1"3 Another mechanism is clearance of dead and damaged cells. ⁴ DAMPs PAMPs where antibodies also have protective properties also include other compounds and antigens as oxidized cardiolipin and phosphatidylserine, oxLDL, apoptotic, necrotic and/or damaged cells, microvesicles, mitochondria. ^5"7

Methotrexate (lUPAC name (2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino] benzoyl]amino]pentanedioic acid) is an antimetabolite and antifolate agent with antineoplastic and immunosuppressant activities. Methotrexate is known to bind to and inhibit the enzyme dihydrofolate reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis and, subsequently, inhibition of DNA and RNA syntheses. Methotrexate also exhibits potent immunosuppressant activity although the mechanism(s) of actions is unclear. Currently, several pharmacological mechanisms of methotrexate action have been suggested, including the inhibition of purine and pyrimidine synthesis, suppression of transmethylation reactions with accumulation of polyamines, reduction of antigen-dependent T-cell proliferation, and promotion of adenosine release with adenosine-mediated suppression of inflammation. It is possible that a combination of these mechanisms is responsible for the antiinflammatory effects of methotrexate ⁸ . Methotrexate is known as a disease-modifying anti-rheumatic drug (DMARD) being one of the mainstays of treatment for inflammatory forms of arthritis, including rheumatoid arthritis (RA) psoriatic arthritis and juvenile idiopathic arthritis, because it not only reduces pain and swelling, but also slows joint damage and disease progression over time. It can be used either as first-line therapy or as second-line therapy that is given when initial treatment (first-line therapy) does not work, or stops working.

Methotrexate has been part of RA treatment for three decades. Researchers first developed this drug in the 1940s as a cancer treatment. In the 1980s it was found that methotrexate relieved pain, swelling and other symptoms in people with RA and it soon became the treatment of choice for patients with this condition and other forms of inflammatory arthritis.

However, not everyone who takes methotrexate gains adequate relief from joint pain, swelling, morning stiffness and other symptoms. If the therapeutic effect is not sufficiently effective in a patient, methotrexate treatment is most often combined with other DMARDs, such as leflunomide (Arava), cyclosporine (Neoral), sulfasalazine (Azulfadine) and hydroxychloroquine (Plaquenil).

In patients that do not respond well to a DMARD - alone or in combination - methotrexate treatment may be combined with biologic drugs. These drugs inhibit part of the overactive immune system response that contributes to many types of inflammatory arthritis. The most common biologic combination is with tumor necrosis factor (TNF) inhibitors such as etanercept (Enbrel), adalimumab (Humira), infliximab (Remicade) or certolizumab pegol (Cimzia). These drugs block TNF, a protein that promotes inflammation and damages joints. Methotrexate treatment may also sometimes be combined effectively with other biologies, such as abatacept (Orencia), rituximab (Rituxan) and tocilizumab (Actemra) or small molecule antirheumatic drugs. Methotrexate is also sometimes administered in combination with sulfasalazine and chloroquine with or without one or more corticosteroids such as prednisone. Methotrexate is moreover commonly used to treat certain types of cancer of the breast, skin, head, neck and lung because methotrexate interferes with the growth of certain cells of the body, especially cells that reproduce quickly, such as cancer cells, bone marrow cells, and skin cells. Methotrexate is further used to treat patients suffering from Bowel Disease (IBD) such as Crohn's Disease or Ulcerative Colitis.Malondialdehyde is the dialdehyde of malonic acid and a byproduct of lipid metabolism in the body. Malondialdehyde is highly reactive and is one of the many reactive electrophile species that cause toxic stress in cells and form covalent protein adducts, called advanced lipoxidation end products (ALE). This compound also forms mutagenic DNA adducts when it reacts with deoxyadenosine and deoxyguanosine in DNA.

While oxidation of polyunsaturated fatty acids is the major source of MDA in vivo, other minor sources exists such as byproducts of free radical generation by ionizing radiation and of the biosynthesis of prostaglandins. Consistent evidence is available for the reaction between MDA and cellular macromolecules such as proteins, RNA and DNA. DA reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine which may be mutagenic and these can be quantified in several human tissues. Oxidative stress is an imbalance between oxidants and antioxidants on a cellular or individual level. Oxidative damage is one result of such an imbalance and includes oxidative modification of cellular macromolecules, induction of cell death by apoptosis or necrosis, as well as structural tissue damage.

Phosphorylcholine is the hydrophilic polar head group of some phospholipids, which is composed of a negatively charged phosphate bonded to a small, positively charged choline group. Phosphorylcholine is part of platelet-activating factor; the phospholipid phosphatidylcholine as well as sphingomyelin. Treatment of cell membranes by certain enzymes renders the phosphorylcholine moiety exposed to the external aqueous phase, and thus accessible for recognition by the immune system. Antibodies against phosphorylcholine are naturally occurring autoantibodies that are created by CD5+/B-1 B cells and are referred to as non-pathogenic autoantibodies.

Anti-PC are natural antibodies, which were described in early studies as being of major importance in the early response to lethal infections with PC-exposing Meningococcae in mice ¹⁰ . PC may play different roles in immune reactions, which may be both protective and deleterious ¹¹ . It has been suggested that anti-PC are atheroprotective in humans, since anti-PC are negatively associated with atherosclerosis development ¹⁷ . Further, low anti-PC levels are independently associated with increased risk of CVD ¹⁴ . Also, it has previously been reported that individuals from Kitava, New Guinea, with a traditional life-style, have high anti-PC levels. Interestingly, these individuals do not seem to suffer from CVD and only to a very limited degree from rheumatic diseases ¹⁵ . In line with clinical studies, mice experiments indicate that both active and passive immunization with anti-PC lead to decreased development of atherosclerosis ^{16, 1?} .

Anti-PC would appear to have several effects in relation to preventing diseases. Firstly, anti-PC have anti-inflammatory and inhibiting effects on inflammatory phospholipids, such as platelet activating factor (PAF) ³ . Another mechanism seems to be inhibition of macrophage uptake of oxLDL by anti-PC ¹⁴ . Increased levels of anti-PC could in principle ameliorate disease manifestations through inhibition of PAF, with implications for chronic inflammatory diseases in general ¹⁸ . A third mechanism would appear to be prevention of plaque rupture by the inhibition of the cytotoxic effects of lysophosphatidylcholine, with implications for acute cardiovascular diseases in particular (WO201 1/107291).

During LDL-oxidation, an array of compounds are generated, both protein (apoB)- and lipid derived. Two lipid epitopes, MDA and phosphorylcholine (PC) appear to be of special interest. An antibody response is abundant in humans, and may have atheroprotective properties.1

We recently reported that antibodies against both MDA, when conjugated with albumin, forming a stable product, are T cell dependent in humans and together, strongly and negatively associated with atherosclerosis and vulnerable plaques and also with risk of CVD. ^{9 10}

The antibodies could either be endogenously produced as a result of active immunization (vaccination), or exogenously administered (passive immunization). Antibodies against MDA and/or PC have previously been described in various connections with antineoplastic agents such as methotrexate, but the correlation between the levels of anti-MDA and/or anti-PC and an antineoplastic agent such as methotrexate has not previously been disclosed:

EP2742948 relates to the use of cardiotophin-1 for the prevention and/or treatment of acute renal injury in mice. The kidneys of mice subjected to Ischemia-reperfusion induced acute kidney injury demonstrated an increase in MDA levels, suggesting an increased lipid peroxidation in renal tissues. Tissue levels of MDA observed in anti-CT- -treated mice were significantly higher than those observed in the saline control. The Ischemia-reperfusion induced acute kidney injury can be induced by various antineoplastic agents including methotrexate.

EP2221059 discloses a composition for preventing lifestyle-related diseases by reducing the levels of glucose, malondialdehyde-modified LDL, homocysteine and C- reactive protein in the blood. The composition comprises at least one component selected from vitamin B12, vitamin B6 and folic acid and at least one component selected from zinc, selenium and an antioxidant vitamin.

WO2006086288 relates to a method for inhibiting a disease response wherein dead or dying cells exposing an antigen such as a PC determinant and/or a MDA determinant are put into contact with an antibody that recognizes and binds said determinants thereby inhibiting the pathologic response. The document also describes a kit useful for treating an autoimmune disease or inflammatory disease state comprising methotrexate as a possible second agent.

AU201 1223222 discloses the use of a combination of antibodies against phosphorylcholine or its conjugate, and biologic agents and/or stem cells for treating or preventing autoimmune diseases, chronic inflammatory diseases and cancerous diseases. Some of the patients investigated had been treated with DMARDs such as methotrexate prior to the study. Summary of the invention

The present invention relates to lipid-related antigens such as MDA and PC and to antibodies against lipid-related antigens such as antibodies against malondialdehyde (anti-MDA) and/or antibodies against phosphorylcholine (anti-PC) for use in combination therapy with an antineoplastic agent such as methotrexate in mammals such as humans in order to prevent the development of, or to treat a mammal suffering from rheumatic diseases, such as active rheumatoid arthritis, active juvenile idiopathic arthritis (JIA), psoriasis and severe psoriatic arthritis; cancer such as breast, skin, head, neck, lung or bone-marrow cancer; SLE or CVD. The invention also relates to compositions such as a vaccine comprising lipid-related antigens or antibodies against malondialdehyde and/or antibodies against phosphorylcholine and one or more antineoplastic agent such as methotrexate and to the use of such compositions. It has been found that the folic acid analogue methotrexate reduces the levels of IgM antibodies to malondialdehyde (anti-MDA) and antibodies to phosphorylcholine (anti- PC) in patients during treatment with methotrexate in patients suffering from rheumatic diseases, such as active rheumatoid arthritis, active juvenile idiopathic arthritis (JIA), psoriasis and severe psoriatic arthritis.

It has also been found here that non-responders to the methotrexate treatment in patients suffering from rheumatic diseases, such as active rheumatoid arthritis, active juvenile idiopathic arthritis (JIA), psoriasis and severe psoriatic arthritis can be characterized in that they have a low level of IgM anti-MDA and anti-PC from the outset to treatment with methotrexate.

Methotrexate is commonly used to treat patients suffering from various cancer diseases and rheumatic diseases. With the present invention it is believed that patients receiving treatment with an antineoplastic agent such as methotrexate treatment or patients that are to become treated with methotrexate will benefit more from such treatment if the levels of antibodies to malondialdehyde and/or antibodies to phosphorylcholine or other lipid-related antibodies are increased prior to methotrexate treatment or if administered at the same time- period as the methotrexate treatment or even simultaneously with the methotrexate treatment. Thus, administration of lipid-related antibodies such as anti- MDA and/or anti-PC to the relevant patients is believed to prevent the development of certain disease and to improve the therapeutic effect of the antineoplastic agent such as methotrexate. Since non-responders to methotrexate treatment can be characterized by low levels of anti-MDA and anti-PC compared to the average patient population it is moreover believed that the number of patients responding to methotrexate treatment can be increased by increasing the level of lipid-related antibodies such as anti-MDA and/or anti-PC prior to, during or simultaneously with treatment with an antineoplastic agent such as methotrexate.

The patients that will benefit from a combination therapy with lipid-related antibodies such as anti-MDA and anti-PC and an antineoplastic agent such as methotrexate may suffer from rheumatic diseases, such as active rheumatoid arthritis, active juvenile idiopathic arthritis (JIA), psoriasis and severe psoriatic arthritis or may suffer from cancer, but the beneficial therapeutic effect of administering lipid-related antibodies such as anti-MDA and/or anti-PC in combination with therapy with an antineoplastic agent such as methotrexate may not be limited to these diseases since patients suffering from other anti-inflammatory diseases may also benefit from an increase in these antibodies prior to treatment with an antineoplastic agent. It is further believed that patients in treatment or to become treated with methotrexate in combination with other drugs, such as patients receiving combination therapy with methotrexate, sulfasalazine and chloroquine will benefit from the present invention, including patients receiving combination treatment with methotrexate, sulfasalazine and chloroquine and one or more corticosteroids such as prednisone. The mammal that will benefit from administration of lipid-related antigens such as MDA and/or PC is a mammal that has a lower than average level of antibodies against the lipid-related antigens compared with the average level in a given population of the mammal. Even though the present invention is shown for antibodies of the IgM isotype, it is believed that any lipid-related antibodies of the 5 isotypes IgA, IgG, IgM, IgE and IgD and of any subclass thereof i.e. lgA1 , lgA2, lgG1 , lgG2, lgG3 or lgG4 would provide the same results. It is known that approximately 30% of patients receiving methotrexate treatment do not respond to this treatment. Therefore, it is of great importance to reduce the number of non-responders to methotrexate treatment and also to improve the therapeutic effect of patients that do respond to the methotrexate treatment. The present invention provides a solution to this based on the finding that methotrexate decreases the levels of anti- MDA and anti-PC in patients and that non-responders have a lower baseline level of these antibodies compared to responders.

Thus, the present invention provides lipid-related antigens for use in active immunization including subcutaneous, intramuscular, intranasal, oral or otherwise to raise antibody levels against the mentioned antigens in a mammal in order to prevent the following conditions or in order to treat mammals already suffering from one or more of the following diseases: cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammator myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment; optionally for use in combination therapy with an antineoplastic agent in mammals. The present invention further provides compositions comprising such antigens and optionally an antineoplastic agent; use of such compositions for the prevention and/or treatment of rheumatic disease, cancer, psoriasis, SLE or CVD; a method for treating a human suffering from one or more of the abovementioned diseases. The present invention also provides antibodies against lipid-related antigens for use in combination therapy with an antineoplastic agent in mammals; compositions comprising antibodies against lipid-related antigens and an antineoplastic agent; use of such compositions for the prevention and/or treatment of rheumatic disease, cancer, psoriasis, SLE or CVD; a method for treating a human suffering from a rheumatic disease, cancer, psoriasis, SLE or CVD including one or more of the following diseases: atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies ( M), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment; comprising administering to said human lipid-related antigens and an antineoplastic agent or by administering antibodies against lipid-related antigens and an antineoplastic agent. Detailed description of the invention

One important finding herein is that methotrexate treatment of RA patients was associated with a decrease in IgM anti-MDA levels and a decrease in IgM anti-PC levels. Another important finding herein is that non-responders to the methotrexate treatment have low levels of anti-MDA and anti-PC prior to administration of methotrexate.

In RA, dyslipidemia combined with enhanced activity of pro-inflammatory cytokines leads to a pro-oxidative state, which further promotes oxidation of low-density lipoprotein (LDL) to the highly atherogenic oxidized LDL (oxLDL). OxLDL has also pro-inflammatory effects, such as activation of monocytes, endothelial cells, T cells and B cells ¹⁹ . The immunostimulatory and prothrombotic effects of oxLDL appear to be mediated through the platelet activating factor (PAF)-receptor, where phosphorylcholine (PC) is the major ligand ²⁰ . interestingly, oxLDL and foam cells are present in synovia in RA ²¹ . Several studies in the general population reported gradually increasing risk for CVD events with increasing plasma oxLDL ^{22, 23} .

The strength of the present study is in its prospective design, structured collection of blood samples and information about RA status at baseline and 12 months of follow-up. The correlation found between the levels of anti-MDA and/or anti-PC after 12 months of methotrexate treatment shows that the antineoplastic agent had a significant impact on the level of anti-MDA and anti-PC in RA patients.

It is further surprisingly shown that the level of anti-MDA and/or anti-PC is lower among non-responders in therapy of RA with methotrexate than among responders. This finding shows that sufficient levels of anti-MDA and anti-PC increases the therapeutic effect of the methotrexate.

It is believed that the correlation observed here between antibodies against lipid-related antigens will also be present in patients being treated with methotrexate in combination with other medicinal products such, as patients receiving combination therapy with methotrexate, sulfasalazine and chloroquine or treatment with methotrexate, sulfasalazine and chloroquine and one or more corticosteroids such as prednisone. Thus, the invention also includes situations where patients are treated with a combination of methotrexate and sulfasalazine and chloroquine. The present invention thus relates to lipid-related antigens for use in active immunization to raise antibody levels against said antigens in a mammal optionally in combination therapy with an antineoplastic agent. The present invention moreover relates to antibodies against lipid-related antigens for use in combination therapy with an antineoplastic agent in mammals.

The present invention further relates to antibodies against lipid-related antigens or to lipid-related antigens for use in combination therapy with an antineoplastic agent in mammals in order to prevent the development of, or to treat a mammal suffering from one or more of the following diseases: active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases The antigens may be antigens from apoptotic or necrotic cells or oxidized LDL and/or may be selected from one or more of the following: malondialdehyde, malondialdehyde- acetaldehyde (MMA), phosphorylcholine, oxidized cardiolipin, oxidized phosphatidylserine, 4-hydroxynonenal. The antigens may carried by or linked to a compound which can be a protein or part thereof, a carbohydrate, a lipid, a micelle or a nanoparticle.

The antibodies against lipid-related antigens may be monoclonal or polyclonal and be of any of the five isotypes IgA, including the subclasses lgA1 and lgA2, IgD, IgE, IgG, including the subclasses lgG1 , lgG2, lgG3 and lgG4, and IgM antibodies hereunder conjugates and/or bioactive components and/or fragments thereof. Accordingly, one aspect of the invention relates to antibodies against lipid-related antigens selected from the group comprising IgA, lgA1 , lgA2, IgD, IgE, IgG, lgG1 , lgG2, lgG3 and lgG4, and IgM and to conjugates and/or bioactive components and/or fragments thereof for use in combination and/or in combination therapy with an antineoplastic agent.

In one aspect of the invention, the antibodies against lipid-related antigens are IgM antibodies against MDA and/or IgM antibodies against PC; MDA and PC conjugates and/or bioactive components and/or fragments thereof for use in combination and/or in combination therapy with an antineoplastic agent. In a preferred embodiment of the invention the antineoplastic agent is methotrexate.

In another aspect of the invention, the antibodies against lipid-related antigens are IgM antibodies against MDA and/or IgM antibodies against PC; MDA and PC conjugates and/or bioactive components and/or fragments thereof for use in combination and/or in combination therapy with an antineoplastic agent. In a preferred embodiment of the invention the antineoplastic agent is methotrexate administered in combination with sulfasalazine and chloroquine including combinations comprising one or more corticosteroids such as prednisone.

For all of the aspects of the present invention, the antineoplastic agent may be selected from the group comprising antimetabolites, folic acid analogues, methotrexate, cytosine arabinoside. cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5-Fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate.

Another aspect of the present invention relates to antibodies against lipid-related antigens selected from the group comprising IgA, lgA1 , lgA2, IgD, IgE, IgG, IgG 1 , lgG2, lgG3 and lgG4, and IgM as risk marker(s) in the prevention and/or treatment of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (P ), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases; wherein low antibody levels is a risk marker, and high levels as a protective marker, so individuals at risk of developing one or more of the above-mentioned diseases could be identified and be eligible for treatment with any of the above-mentioned antibodies against lipid-related antigens prior to or together with treatment with an antineoplastic agent.

In one embodiment of the invention anti-MDA and/or anti-PC are risk marker(s) in the prevention and/or treatment of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases; wherein low antibody levels is a risk marker, and high levels as a protective marker, so individuals at risk of developing one or more of the above- mentioned diseases could be identified and be eligible for treatment with any of the above-mentioned antibodies against lipid-related antigens prior to or together with treatment with an antineoplastic agent.

In a preferred embodiment of the invention anti-MDA and/or anti-PC are risk marker(s) in the prevention and/or treatment of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck, cancer and breast cancer and/or other cancer diseases; wherein low antibody levels is a risk marker, and high levels as a protective marker, so individuals at risk of developing one or more of the abo e- mentioned diseases could be identified and be eligible for treatment with any of the above-mentioned antibodies against lipid-related antigens prior to or together with treatment with methotrexate.

In a further preferred embodiment of the invention anti-MDA and/or anti-PC are used in the prevention and/or treatment of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis or severe psoriatic arthritis; wherein low antibody levels is a risk marker, and high levels as a protective marker, so individuals at risk of developing one or more of the above-mentioned diseases could be identified and be eligible for treatment with any of the above-mentioned lipid-related antigens or antibodies against lipid-related antigens prior to or together with treatment with methotrexate.

In all aspects of the invention wherein antibodies against lipid-related antigens from the group comprising IgA, lgA1 , lgA2, IgD, IgE, IgG, lgG1 , lgG2, lgG3 and lgG4, and IgM such as anti-MDA and/or anti-PC are used as risk marker(s) prior to or simultaneous with the treatment of one or more of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases with an antineoplastic agent; said antibody level is below the mean average, or below a particular percentile value determined with reference to the wider population, such as below the 5th, 10th, 20th or 25th percentile, such as to a level, wherein the odds ratio is above one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of high risk.

In one preferred aspect of the invention IgM antibodies against anti-MDA and/or anti-PC are used as risk markers) prior to or simultaneous with the treatment of one or more of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis or severe psoriatic arthritis; where said antibody level is below the mean average, or below a particular percentile value determined with reference to the wider population, such as below the 5th, 10th, 20th or 25th percentile, such as to a level, wherein the odds ratio is above one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of high risk.

Another aspect of the present invention is use of antibodies against lipid-related antigens selected from the group comprising IgA, lgA1 , lgA2, IgD, IgE, IgG, lgG1 , lgG2, lgG3 and igG4, and IgM or bioactive components and/or parts thereof for the preparation of a pharmaceutical composition to be used in a treatment, prophylaxis and/or prevention of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases: prior to or together with the treatment with an antineoplastic agent. A preferred aspect of the present invention is use of MDA conjugates or DA or bioactive components and/or parts thereof for the preparation of a pharmaceutical composition to be used in a treatment, prophylaxis and/or prevention of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases; prior to or together with the treatment with an antineoplastic agent. In a more preferred aspect of the present invention the treatment, prophylaxis and or prevention is related to active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis antineoplastic and the antineoplastic agent is methotrexate.

A preferred aspect of the present invention is use of PC conjugates or PC or bioactive components and/or parts thereof for the preparation of a pharmaceutical composition to be used in a treatment, prophylaxis and/or prevention of active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases; prior to or together with the treatment with an antineoplastic agent. In a more preferred aspect of the present invention the treatment, prophylaxis and or prevention is related to active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis, severe psoriatic arthritis antineoplastic and the antineoplastic agent is methotrexate.

In all aspects of the invention, the lipid-related antigens such as MDA conjugate and/or the PC conjugate can be linked to a pharmaceutically acceptable protein, carbohydrate, or polymer. The pharmaceutical composition is preferably given by injection, but can in practice be administered by any suitable means that allows the MDA conjugate and/or the PC conjugate to provoke an immune response in the subject to which it is administered such as via subcutaneous, intramuscular, intranasal or oral formulations or by other means to raise antibody levels against the mentioned antigens in a mammal The proposed method of active immunization will modulate the titre of the anti-MDA and/or the anti-PC antibodies, which in turn will have a positive effect on the development of autoimmune diseases, chronic inflammatory diseases, and cancerous diseases. Thus, active immunization may be used to increase the titre of anti-MDA and/or anti-PC antibodies to a level that, when assessed by the methods of diagnosis according to the present application, would not be said to be "low" or indicative of an increased risk of development, or progression, of autoimmune diseases, chronic inflammatory diseases, and cancerous diseases. Thus, a method of active immunization according to the present invention may be used to increase the levels of lipid-related antibodies such as the anti-MDA and/or anti-PC levels, such as IgM anti-MDA levels and/or anti-PC levels, in an individual to a level that is greater than the average baseline level in a representative group of patients when tested by the methods described in the Examples. Accordingly, the method of active immunization according to the present invention may be used to increase the anti-MDA and/or the anti-PC levels to a level that is above the mean average, or above a particular percentile value determined with reference to the wider population, such as above the 5th, 10th, 20th or 25th percentile, such as to a level, wherein the odds ratio is below one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk.

Another embodiment of the invention concerns the use of an antibody preparation, for example a monoclonal or polyclonal antibody, recognizing MDA conjugates or MDA or bioactive components and/or parts thereof for the preparation of a pharmaceutical composition to be used in a treatment, prophylaxis and/or prevention prior to treatment of a mammal with an antineoplastic agent such as methotrexate for curing autoimmune diseases, chronic inflammatory diseases, and cancerous diseases such as active rheumatoid arthritis, active juvenile idiopathic arthritis, osteoarthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases.

Another embodiment of the invention concerns the use PC conjugates or PC or bioactive components and/or parts thereof for the preparation of a pharmaceutical composition to be used in a treatment, prophylaxis and/or prevention prior to treatment of a mammal with biologic agents for curing autoimmune diseases, chronic inflammatory diseases, and cancerous diseases. Another embodiment of the invention concerns the use of an antibody preparation, comprising a combination of antibodies recognizing both MDA conjugates or MDA or bioactive components and/or parts thereof and PC conjugates or PC or bioactive components and/or parts thereof for the preparation of a pharmaceutical composition to be used in a treatment, prophylaxis and/or prevention prior to treatment of a mammal with an antineoplastic agent such as methotrexate for curing autoimmune diseases, chronic inflammatory diseases, and cancerous diseases such as active rheumatoid arthritis, active juvenile idiopathic arthritis, osteoarthritis, psoriasis, severe psoriatic arthritis, cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases.

In all aspects of the invention relating to treatment of patients, said treatment may be in the form of either passive immunization or active immunization or a combination of both. Any form of administration that increases the antibody levels directly or indirectly (i.e. by inducing the immune response of the patient) known in the art may be applied such as injection, transdermal, oral or nasal administration which are increasingly used in other contexts. Recent examples where oral immunization is used include polio and cholera ²⁴ .

The monoclonal antibody can be produced using methods known in the art. Other antibody preparations may be used, such as enriched preparations of antibodies against lipid-related antigens selected from the group comprising IgA, lgA1 , lgA2, IgD, IgE, IgG, lgG1 , lgG2, lgG3 and lgG4, and IgM or bioactive components and/or parts thereof obtained from intravenous immunoglobulin preparations, recombinantly produced antibodies and/or other artificially created antibody derivatives.

In one aspect of the invention anti-MDA and/or anti-PC enriched preparations may be obtained from intravenous immunoglobulin preparations, recombinantly produced anti- MDA and/or anti-PC antibodies and/or other artificially created anti-MDA and/or anti-PC antibody derivatives. Thus, passive immunization may be used to increase the titre of antibodies against lipid-related antigens selected from the group comprising IgA, lgA , lgA2, IgD, IgE, IgG, lgG1 , lgG2, lgG3 and lgG4, and IgM anti-MDA such as anti-MDA and/or anti-PC antibodies in an individual to a level that, when assessed by the methods according to the present application, would not be said to be "low" or indicative of an increased risk of development, or progression, of autoimmune diseases, chronic inflammatory diseases, and cancerous diseases. Thus, a method of passive immunization according to the present invention may be used to increase antibody levels such as anti-MDA levels and/or anti-PC levels, such as IgM anti-MDA levels and/or IgM anti-PC levels, in an individual to a level that is greater the average baseline level in a representative group of patients when tested by the methods described in the examples. Further, a method of passive immunization according to the present invention may be used to increase anti-PC levels, such as IgM anti-PC levels, in an individual to a level that is greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 85 U/ml when tested by the methods described above. Moreover, a method of passive immunization according to the present invention may be used to increase both anti-MDA levels and anti-PC levels simultaneously to a level that is greater than the above- mentioned levels for each antibody, respectively.

Accordingly, the method of active immunization according to the present invention may be used to increase the level(s) of antibodies against lipid-related antigens selected from the group comprising IgA, lgA1 , lgA2, IgD, IgE, IgG, lgG1 , lgG2, lgG3 and lgG4, and IgM to a level that is above the mean average, or above a particular percentile value determined with reference to the wider population, such as above the 5th, 10th, 20th or 25th percentile, such as to a level wherein the odds ratio is below one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk.

Also, the method of active immunization according to the present invention may be used to increase the level of anti-MDA to a level that is above the mean average, or above a particular percentile value determined with reference to the wider population, such as above the 5th, 10th, 20th or 25th percentile, such as to a level wherein the odds ratio is below one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk.

Further, the method of active immunization according to the present invention may be used to increase anti-PC levels to a level that is above the mean average, or above a particular percentile value determined with reference to the wider population, such as above the 5th, 10th, 20th or 25th percentile, such as to a level wherein the odds ratio is below one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk.

Also, the method of active immunization according to the present invention may be used to increase both anti-MDA levels and anti-PC levels to levels that are above the mean average, or above a particular percentile value determined with reference to the wider population, such as above the 5th, 10th, 20th or 25th percentile, such as to levels wherein the odds ratio is below one, the p-value is < 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk. In all aspects of the invention relating to antibodies against MDA, MDA conjugate or bioactive components and/or fragments thereof for use in combination therapy with one or more antineoplastic agents, the antibodies can be used for curing, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, or cancerous diseases in a mammal, and can be monoclonal, polyclonal or chimeric antibodies of isotype IgA, IgD, IgE, IgG, IgM, optionally in combination with any suitable excipients or adjuvants.

In all aspects of the invention relating to antibodies against PC, PC conjugate or bioactive components and/or fragments thereof for use in combination therapy with one or more biologic agents and/or stem cells in a mammal, the antibodies can be used for curing, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, or cancerous diseases in a mammal, and can be monoclonal, polyclonal or chimeric antibodies of isotype IgA, IgD, IgE, IgG, IgM, optionally in combination with any suitable excipients or adjuvants.

In one embodiment the antibodies against MDA and PC, respectively, may be administered together. That is, the antibodies against MDA and PC, respectively, may be administered simultaneously by the same composition or by two or more compositions. The antibodies against MDA and PC, respectively, may in another embodiment be administered separately at different time-points prior to or during the treatment with one or more antineoplastic agents.

Another embodiment according to the invention relates to antibodies against MDA, MDA conjugate or bioactive components and/or fragments thereof for immunization, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, and cancerous diseases in a mammal, said mammal being in therapy with one or more neoplastic agents such as methotrexate. Yet another embodiment according to the invention relates to compositions comprising antibodies against MDA, MDA conjugates or bioactive components and/or fragments thereof in combination with one or more neoplastic agents such as methotrexate, particularly for use as a medicament, especially for curing, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, or cancerous diseases in a mammal. Another embodiment according to the invention relates to antibodies against PC, PC conjugate or bioactive components and/or fragments thereof for immunization, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, and cancerous diseases in a mammal, said mammal being in therapy with one or more neoplastic agents such as methotrexate. Yet another embodiment according to the invention relates to compositions comprising antibodies against PC, PC conjugates or bioactive components and/or fragments thereof in combination with one or more neoplastic agents such as methotrexate, particularly for use as a medicament, especially for curing, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, or cancerous diseases in a mammal.

Yet another embodiment according to the invention relates to MDA conjugates, MDA or bioactive components and/or parts/fragments thereof for use in activation immunotherapy prior to or in combination (combination therapy) with the treatment of a mammal with one or more antineoplastic agents such as methotrexate, for curing, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, and cancer diseases. Yet another embodiment according to the invention relates to PC conjugates, PC or bioactive components and/or parts/fragments thereof for use in activation immunotherapy prior to or in combination (combination therapy) with the treatment of a mammal with one or more antineoplastic agents such as methotrexate for curing, treating, preventing, and/or reducing the risk of developing autoimmune diseases, chronic inflammatory diseases, and cancer diseases

Definitions

In accordance with the present invention, the term "mammal" means all known mammals, and in particular mice, rats, rabbits, dogs, cats, cattle, horses and humans. In accordance with the present invention, the medicament may be administered by any suitable means known by the skilled person and be formulated with excipients and adjuvants normally employed for such medicaments and their corresponding formulations. In particular, the medicament may be intended for administration by injection, optionally together with any suitable excipients and adjuvants employed for such formulations. Alternatively, the medicament may be given in the form of a pill, tablet, capsule or spray. The medicament may be administered in a way so as to be compatible with the dosage formulation and in such amount as will be therapeutically effective and/or immunogenic. In accordance with the present invention, any agent that is suitable for increasing the antibody response towards lipid-related antigens such as anti-MDA and/or the anti-PC response, in particular MDA conjugates and/or PC conjugates, DA or bioactive components and/or parts thereof, optionally in combination with any suitable adjuvants and PC or bioactive components and/or parts thereof, optionally in combination with any suitable adjuvants, is to be considered as part of this invention. Such lipid-related antigens including MDA which can be exposed on OxLDL and on apoptotic necrotic and damaged cells and/or PC-exposing compounds also include platelet activating factor (PAF), PAF-like lipids or lysophosphatidylcholine, which have pro-inflammatory effects where PC is an important mediator. In accordance with the present invention, monoclonal, polyclonal or chimeric antibodies of isotype IgA, IgD, IgE, IgG, IgM, raised against MDA, MDA conjugate or bioactive components and/or fragments thereof or raised against PC, PC conjugate or bioactive components and/or parts/fragments thereof (e.g. where PC is a component, such as is the case in PAF or PAF-like lipids), refer to any monoclonal or polyclonal antibody produced by immunization of a suitable mammal, including, but not limited to, mouse, rabbit, goat, sheep, or horse.

In accordance with the present invention, the term "bodily fluid" means any natural bodily fluid or secretion of fluid including, but not limited to, plasma, serum, blood, urine, or saliva.

In accordance with the present invention, the term "combination therapy" means the individual or simultaneous administration of two or more medications to treat a single disease and/or diseases that may develop due to unwanted medical side- effects or lack of satisfactory positive pharmaceutical response from the administration of one of the medications, e.g. unwanted medical side-effects due to the administration of biologic agents to treat chronic inflammatory diseases, autoimmune disease and cancer diseases.

In accordance with the present invention, "chronic inflammatory diseases, autoimmune and cancer diseases" means any of - including, but not limited to - the following diseases: cardiovascular disease (CVD), such as atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis (including Wegeners granulomatosus, Takayasu arteritis, temporal arteritis), polymyalgia rheumatics, bowel disease including Crohn's disease and colitis of different kinds, nephritis, asthma, or cancer diseases, such as, but not limited to, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment. Immunotherapy is normally defined within medicine as "treatment of disease by inducing, enhancing, or suppressing an immune response". Passive immunity can be achieved through the transfer of ready-made antibodies into the affected individual or mammal. Immunotherapies designed to elicit or amplify an immune response are normally termed "activation immunotherapies", and include, for example, immunization. Immunotherapies designed to reduce, suppress or more appropriately direct an existing immune response, as in cases of autoimmunity or allergy, are normally termed "suppression immunotherapies". The active agents of immunotherapy are collectively called "immunomodulators", and include a wide variety of natural, synthetic and recombinant substances, many of which are biological agents as defined above. Examples of immunomodulators include medicinal mushrooms and herbs, various plant and microorganism extracts, vaccines, adjuvants, hormones, various drugs, interleukins, interferons and other cytokines.

Further, a detailed description of antibodies against PC conjugate, PC or bioactive components and/or fragments can be found in W010/003602, Thus, based on this it is clear that monoclonal antibodies reactive against PC conjugate, anti-PC or bioactive components and/or fragments can be produced using any standard method known in the art. See for example Briles et al., 1982. J Exp Med 156, 1177- 1185 or Spira et al., 1988. J. Immunology 140, 2675-2680. Other antibodies against a phosphorylcholine and/or its conjugate can be prepared using methods well known to those skilled in the art. For example, a sub-fraction with anti-PC activity of a human immunoglobulin preparation can be prepared as described below by affinity purification using a PC conjugate. Further, intravenous immunoglobulin preparations {e.g., 1GIV; Baxter and others) are highly purified preparations of IgG commercially available and are used in the treatment of patients who have no, or very low levels, of antibody production. Immunoglobulin preparations include those available from the following manufacturers: Baxter (US), e.g., Gammagard ®, Isiven (Antimo Naples, Italy), Omri (Tel-Hashomer, Israel), Miles (Biological Products Division, West Heaven, CT), Sclavo (Lucca, Italy), Sandoz (Novautis, Basel, Swizeriand), e.g., Sandoglobulin ®, Biotest Diagnostic Corporation (Deville, NJ). Examples of immunoglobulin preparations are GammagardS/D ®, GammarlV ®, Gaimnar-PIV ®, Gammimune N®, Iveegam®, Panglobulin ®, Polygam S/D®, Sandoglobulin ®, Venoglobulin ®. Immunoglobulin preparations typically contain some IgM as well as IgG. Trace amounts of IgM are present in Gammagard ®. Pentaglobin (Biotest) is an enriched IgM preparation, which has been used for treatment of SARS. The subtraction with anti-PC activity may comprise both IgG and IgM, or may be selected to comprise mainly IgG (for example by starting with an IgG-rich preparation, such as Gammagard ® and/or by selecting for IgG); or mainly IgM (for example by starting with an IgM-rich preparation such as Pentaglobin and/or by selecting for IgM). Further, an antibody preparation with specificity to a PC conjugate binds to unconjugated PC and may also bind to PC present in PC-containing compounds, in which PC is exposed, for example in lysophosphatidylcholine (see for example, Kim et ai., 2002 J Exp Med. 196, 655-65). Thus, an antibody preparation with specificity to a PC conjugate may also bind to lysophosphatidylcholine or other phospholipids or phospholipid-containing compounds as platelet activating factor (PAF) or PAF- like lipids which have PC as a major component.

As used herein "Combination therapy" comprises any kind of therapy wherein one or more antibodies against lipid-related antigens (such as anti-MDA and/or anti-PC) is administered to a patient either prior to, simultaneous with or subsequent to administration of an antineoplastic agent to the same patient. As used herein ''Combination therapy" also comprises any kind of therapy wherein one or more lipid- related antigens (such as MDA and/or PC) is administered to a patient either prior to, simultaneous with or subsequent to administration of an antineoplastic agent to the same patient. Combination therapy may also relate specifically to the situation where methotrexate is administered in combination with sulfasalazine and chloroquine including combinations comprising one or more corticosteroids such as prednisone. As used herein, the term "antineoplastic agents" comprises cell cycle phase specific agents and cell cycle phase non-specific agents. Cell cycle specific agents act on the cells in a specific phase. They are most effective against tumors that have a large proportion of cells actively moving through the cell cycle and cycling at a fast rate. Rapid cycling ensures that the cell passes through the phase in which it is vulnerable to the drugs' effects. Cell cycle phase non-specific agents are not dependent on the cell being in a particular phase of the cell cycle for them to work - they affect cells in all phases of the cell cycle. Resting cells (phase GO) are as vulnerable as dividing cells to the cytotoxic effects of these agents. As a result, phase non-specific agents are effective drugs against slow-growing tumors. Antineoplastic agents are traditionally divided by their origin or mechanism of action. The main groups include: alkylating and alkylating-like agents, antimetabolites, antitumor antibiotics, plant alkaloids, miscellaneous agents and Hormonal agents.

As used herein the term "antimetabolite" refers to a chemical that inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid. Antimetabolites includes Main categories of these drugs include: base analogs (altered nucleobases): purine analogues, pyrimidine analogues; nucleoside analogues: nucleosides with altered nucleobases, nucleosides with altered sugar component (e.g. Cytarabine); nucleotide analogues and antifolates. The antimetabolite group includes the following drugs: cytosine arabinoside, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5- fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate

Herein, the wording "folic acid analogues" means any pharmaceutical substance that has similar, if not identical, properties to folic acid. Folic acid is a water-soluble vitamin belonging to the B-complex group of vitamins. These vitamins break down complex carbohydrates into simple sugars to be used for energy. Folic acid works together with vitamin B12 and vitamin C to metabolize protein in the body. It is important for the formation of red and white blood cells. It is necessary for the proper differentiation and growth of cells and for the development of the fetus. It is also used to form the nucleic acid of DNA and RNA. It increases the appetite and stimulates the production of stomach acid for digestion and it aids in maintaining a healthy liver. A deficiency of folic acid may lead to anemia, in which there is decreased production of red blood cells. This reduces the amounts of oxygen and nutrients that are able to get to the tissues.

To summarize, the following issues are included as aspects of the present invention:

1. Lipid-related antigens for use in active immunization to raise antibody levels against said antigens in a mammal

2. Lipid-related antigens for use in according to aspect 1 to prevent one or more of the following conditions or in order to treat a mammal already suffering from one or more of the following diseases: cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Retter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment

3. Lipid-related antigens for use in according to aspect 1 or 2 in combination therapy with an antineoplastic agent in mammals

4. Lipid-related antigens for use in according to any of aspects 1 to 3 wherein said lipid-related antigens are administered via a subcutaneous, intramuscular, intranasal or oral formulation or by other means to raise antibody levels against the mentioned antigens in a mammal

5. Lipid-related antigens for use according to any of aspects 1 to 4 wherein said antigens are selected from one or more of the following: malondialdehyde, malondialdehyde-acetaldehyde (MMA), phosphorylcholine, oxidized cardiolipin, oxidized phosphatidylserine, 4-hydroxynonenal

6. Lipid-related antigens for use according to any of aspects 1 to 5 wherein said antigens are carried by or linked to a compound which can be a protein or part thereof, a carbohydrate, a lipid, a micelle or a nanoparticle 7. Lipid-related antigens for use according to any of aspects 3 to 6 wherein said antineoplastic agent is a folic acid analogue

8. Lipid-related antigens for use according to any of aspects 3 to 7 wherein said antimetabolite is selected from the group comprising cytosine arabinoside, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate

9. Lipid-related antigens for use according to aspect 8 wherein said antineoplastic agent is methotrexate

10. Lipid-related antigens for use according to aspect 9 wherein said methotrexate is administered in combination with sulfasalazine and chloroquine with or without one or more corticosteroids

11. Lipid-related antigens for use according to any of aspects 1 to 9 wherein said mammal is a human

12. Antibodies against lipid-related antigens for use in in combination therapy with an antineoplastic agent in mammals in order to prevent or treat one or more diseases

13. Antibodies against lipid-related antigens for use according to aspect 12 wherein said antibodies are selected from the group comprising IgA, including lgA1 and lgA1 , IgG, including lgG1 , lgG2, !gG3, lgG4, IgM, IgE and IgD

14. Antibodies for use according to aspect 12 wherein said antibodies are selected from the group comprising IgA, including lgA1 and lgA1 , IgG, including lgG1 , lgG2, lgG3, lgG4, IgM, IgE and IgD and where said one or more antibodies are against malondialdehyde

15. Antibodies against lipid-related antigens for use according to aspect 12 wherein said antibodies are one or more IgM antibody against malondialdehyde

16. Antibodies against lipid-related antigens for use according to aspect 12 or 13 wherein antibodies are said antibodies are selected from the group comprising IgA, including lgA1 and lgA1 , IgG, including lgG1 , lgG2, lgG3, lgG4, IgM, IgE and IgD and where said one or more antibodies are against phosphorylcholine

17. Antibodies against lipid-related antigens for use according to aspect 12 or

13 wherein said antibodies are one or more IgM antibody against phosphorylcholine 18. Antibodies against lipid-related antigens for use according to aspect 12 or

13 wherein said antibodies are one or more antibodies against malondialdehyde and one or more antibodies against phosphorylcholine

19. Antibodies against lipid-related antigens for use according to any of aspects 12 to 18 wherein said antineoplastic agent is an antimetabolite 20. Antibodies against lipid-related antigens for use according to aspect 19 wherein said antimetabolite is a folic acid analogue

21. Antibodies against lipid-related antigens for use according to aspect 19 wherein said antimetabolite is selected from the group comprising cytosine arabinoside, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate

22. Antibodies against lipid-related antigens for use according to aspect 21 wherein said antineoplastic agent is methotrexate

23. Antibodies against lipid-related antigens for use according to any of aspects 12 to 22 wherein said antibodies are one or more antibody against malondialdehyde and said antineoplastic agent is methotrexate

24. Antibodies against lipid-related antigens for use according to any of aspects 12 to 19 wherein said antibodies are one or more antibody against phosphorylcholine and said antineoplastic agent is methotrexate

25. Antibodies against lipid-related antigens for use according to any of aspects 12 to 21 wherein said antibodies are one or more antibodies against malondialdehyde and one or more antibodies against phosphorylcholine and wherein said antineoplastic agent is methotrexate

26. Antibodies against lipid-related antigens for use according to any of aspects 22 to 25 wherein methotrexate is administered in combination with other drugs

27. Antibodies against lipid-related antigens for use according to aspect 26 wherein methotrexate is administered in combination with and sulfasalazine and chloroquine with or without one or more corticosteroids

28. Antibodies against lipid-related antigens for use according to any of aspects 12 to 27 wherein said mammal is a human

29. Antibodies against lipid-related antigens for use according to aspect 28 wherein said human is suffering from one or more of the following diseases: cardiovascular disease (CVD), atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, cancer diseases, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment

30. Antibodies against lipid-related antigens for use according to aspect 29 wherein said human is suffering from one or more of the following diseases: a rheumatic disease, cancer, psoriasis, SLE or CVD

31. Antibodies against lipid-related antigens for use according to aspect 29 wherein said human is suffering from active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis or severe psoriatic arthritis

32. Composition comprising lipid-related antigens

33. Composition according to aspect 32 wherein said lipid-related antingens are selected from one or more of malondiaidehyde, malondialdehyde-acetaldehyde (MMA), phosphorylcholine, oxidized cardiolipin, oxidized phosphatidylserine, 4- hydroxynonenal

34. Composition according to any of aspects 32 to 33 further comprising an antineoplastic agent

35. Composition according to aspect 34 wherein said antineoplastic agent is an antimetabolite

36. Composition according to aspect 35 wherein said antimetabolite is a folic acid analogue

37. Composition according to aspect 34 wherein said antineoplastic agent is selected from the group comprising: cytosine arabinoside, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate

38. Composition according to aspect 37 wherein said antineoplastic agent is methotrexate

39. Composition according to aspect 38 further comprising sulfasalazine and chloroquine with or without one or more corticosteroids

40. Composition comprising antibodies against lipid-related antigens and an antineoplastic agent

41. Composition according to aspect 21 wherein said antibodies against lipid- related antigens are selected from the group comprising IgA, including IgAl and lgA1 , IgG, including lgG1 , lgG2, lgG3, lgG4, IgM, IgE and IgD 42. Composition according to aspect 40 wherein said antibodies are one or more antibody against malondialdehyde and/or one or more antibody against phosphorylcholine

43. Composition according to aspect 41 wherein said antibodies is one or more IgM antibody against malondialdehyde

44. Composition according to aspect 41 wherein said antibody is one or more IgM antibody against phosphorylcholine

45. Composition according to aspect 41 wherein said antibodies are one or more IgM antibody against malondialdehyde and one or more IgM antibody against phosphorylcholine

46. Composition according to any of aspects 40 to 25 wherein said antineoplastic agent is an antimetabolite

47. Composition according to aspect 46 wherein said antimetabolite is a folic acid analogue

48. Composition according to aspect 47 wherein said antineoplastic agent is selected from the group comprising: cytosine arabinoside, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate

49. Composition according to aspect 48 wherein said antineoplastic agent is methotrexate

50. Composition according to any of aspects 40 to 43 wherein said antibodies are one or more antibody against malondialdehyde and said antineoplastic agent is methotrexate

51. Composition according to any of aspects 40, 41 or 44 wherein said antibodies are one or more antibody against phosphorylcholine and said antineoplastic agent is methotrexate

52. Composition according to aspect 40 wherein said natural IgM antibodies are one or more antibody against malondialdehyde and one or more antibody against phosphorylcholine and wherein said antineoplastic agent is methotrexate

53. Composition according to any of aspects 49 to 52 further comprising sulfasalazine and chloroquine with or without one or more corticosteroids

54. Use of a composition according to any of aspects 32 to 53 in the prevention of development of rheumatic disease, cancer, psoriasis, SLE or CVD or for treatment of a human suffering from a rheumatic disease, cancer, psoriasis, SLE or CVD 55. Use of a composition according to aspect 54 in the prevention of development of or in the treatment of a human suffering from active rheumatoid arthritis, active juvenile idiopathic arthritis, psoriasis or severe psoriatic arthritis

56. Use of a composition according to aspect 54 in the prevention of development of or in the treatment of a human suffering from one or more of the following diseases, atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment

57. Use according to any of aspects 54 to 56 wherein the level of anti- DA and/or anti-PC in said human prior to treatment with an antineoplastic agent is below the mean average or below a particular percentile value selected from the 5th, 10th, 20th or 25th percentile, determined with reference to the wider population.

58. Method for preventing the development of, or for treating a human suffering from a rheumatic disease, cancer, psoriasis, SLE or CVD including one or more of the following diseases: atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment; comprising administering to said human lipid-related antigens 59. Method according to aspect 58 comprising administering to said human antibodies against lipid-related antigens and an antineoplastic agent

60. Method according to any of aspects 58 to 59 wherein said antineoplastic agent is an antimetabolite

61. Method according to aspect 60 wherein said antimetabolite is a folic acid analogue

62. Method according to aspect 60 wherein said antimetabolite is selected from cytosine arabinoside, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, raltitrexed, pemetrexed and pralatrexate

63. Method according to aspect 62 wherein said folic acid analogue is methotrexate

64. Method according to aspect 62 wherein said folic acid analogue is methotrexate administered in combination with sulfasalazine and chloroquine with or without one or more corticosteroids

65. Method according to any of aspects 59 to 64 wherein said one or more lipid-related antigens are administered to a patient prior to administration of said antineoplastic agent

66. Method according to any of aspects 58 to 65 wherein the level of anti-MDA and/or anti-PC in said human prior to treatment with an antineoplastic agent is below the mean average or below a particular percentile value selected from the 5th, 10th, 20th or 25th percentile, determined with reference to the wider population.

67. Method according to any of aspects 58 to 66 wherein said one or more lipid-related antigens are administered to a patient that does not respond to treatment with an antineoplastic agent

68. Method according to any of aspects 58 to 67 wherein said one or more lipid-related antigens are administered to a patient in order to improve the therapeutic effect of an antineoplastic agent that have been administered previously to said patient

69. Method for preventing the development of, or for treating a human suffering from a rheumatic disease, cancer, psoriasis, SLE or CVD including one or more of the following diseases: atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris; rheumatic diseases, acute and/or chronic inflammatory conditions, rheumatoid arthritis, osteoarthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, encephalitis, inflammatory bowel disease, arthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, spondylopathies, vasculitis, Wegeners granulomatosus, Takayasu arteritis, temporal arteritis, polymyalgia rheumatica, bowel disease, Crohn's disease and colitis of different kinds, nephritis, asthma, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer and/or other cancer diseases where immunomodulation is used as treatment; comprising administering to said human antibodies against lipid-related antigens and an antineoplastic agent

70. Method according to aspect 69 wherein said antibodies is one or more antibody selected from the group comprising IgA, including lgA1 and lgA1 , IgG, including lgG1 , lgG2, lgG3, lgG4, IgM, IgE and IgD against malondialdehyde and/or against phosphorylcholine

71. Method according to any of aspects 69 to 70 wherein said antineoplastic agent is an antimetabolite

72. Method according to aspect 71 wherein said antimetabolite is a folic acid analogue

73. Method according to aspect 72 wherein said folic acid analogue is methotrexate

74. Method according to aspect 69 wherein said antibodies are one or more antibody against malondialdehyde and said antineoplastic agent is methotrexate

75. Method according to aspect 69 wherein said antibodies are one or more antibody against phosphorylcholine and said antineoplastic agent is methotrexate

76. Method according to aspect 69 wherein said antibodies are one or more antibody against malondialdehyde and one or more antibody against phosphorylcholine and wherein said antineoplastic agent is methotrexate

77. Method according to any of aspects 73 to 76 wherein sulfasalazine and chloroquine with or without one or more corticosteroids are also administered to the patient

78. Method according to any of aspects 69 to 77 wherein said one or more antibodies are administered to a patient prior to administration of said antineoplastic agent

79. Method according to any of aspects 69 to 78 wherein the level of anti-MDA and/or anti-PC in said human prior to treatment with an antineoplastic agent is below the mean average or below a particular percentile value selected from the 5th, 10th, 20th or 25th percentile, determined with reference to the wider population. 80. Method according to any of aspects 69 to 79 wherein said one or more natural IgM antibodies are administered to a patient that does not respond to treatment with an antineoplastic agent

81. Method according to any of aspects 69 to 80 wherein said one or more natural IgM antibodies are administered to a patient in order to improve the therapeutic effect of an antineoplastic agent that have been administered previously to said patient

Brief description of the drawings

Figure 1 Comparison of anti-MDA IgM levels at baseline and after 3 months, 6 months and 12 months, respectively, after initiation of methotrexate treatment in RA patients. Based on samples from 60 RA patients.

Figure 2. Comparison of anti-PC IgM levels at baseline and after 3 months, 6 months and 12 months, respectively, after initiation of methotrexate treatment in RA patients. Based on samples from 60 RA patients.

Examples

EXAMPLE 1

Levels of IgM antibodies against MDA in RA patients at baseline and after 12 months of methotrexate treatment

47 patients with rheumatoid arthritis (RA) participated in this study. The patients were diagnosed with rheumatoid arthritis less than 2 years prior to initiation of the study. They were all below 75 years old and both males and females were included in the study. The disease activity of the patients was determined by the disease activity score (DAS 28). The DAS28 is a measure of disease activity in RA. DAS stands for 'disease activity score' and the number 28 refers to the 28 joints that are examined in this assessment.

The method is commonly used and the measures of disease activity in RA includes:- •examination of joints for swelling and tenderness,

•global scores of pain and overall status,

•blood markers of inflammation (e.g. ESR and CRP)

'questionnaires (e.g. the HAQ which assesses function),

•X-rays, and other imaging techniques such as ultrasound and MRI. The DAS28 is a composite score derived from 4 of these measures. The '28' version is a simplification of the original DAS score, which requires 44 joints to be counted. Other versions of the DAS28 allow the CRP to be used instead of the ESR, or the omission of either. To calculate the DAS28 the following parameters are measured:

1. The number of swollen joints (out of the 28),

2. The number of tender joints (out of the 28),

3. The erythrocyte sedimentation rate (ESR) or C reactive protein (CRP),

4. A 'global assessment of health' (indicated by marking a 10 cm line between very good and very bad) based on questioning of the patients.

These results are then fed into a mathematical formula to produce the overall disease activity score. A DAS28 of greater than 5.1 implies active disease, less than 3.2 low disease activity, and less than 2.6 remission.

Blood samples were collected from the patients at baseline, i.e. immediately prior to the first administration of methotrexate and again 12 months later. During the 12 months, the patients were treated with methotrexate 15 - 20 mg once weekly.

The Ig anti-MDA antibody levels in the blood samples were determined by ELISA using arbitrary units (AU) defined as percentage of the absorbance to the plasma pool.

Determination of IgM antibodies against MDA with ELISA

IgM antibodies to MDA were determined by enzyme-linked immunosorbent assay (ELISA). Serum from donors with an anti-MDA level above median level was used as internal standard and tested on every plate. This thus represents a standard in the ELISA, allowing for reproducibility as compared to other cohorts tested using this method. The plateau of antibody binding was reached with the antigen concentration of 10 pg/mL. Nunc Immuno microwell plates (Thermo Labsystems, Franklin, MA, USA) were coated with MDA conjugated with BSA. Coated plates were incubated overnight at 4°C. After five washings with PBS, the plates were blocked with 2% BSA-PBS for 90 min at room temperature and washed as described above. Serum samples were diluted in 0.2% BSA-PBS and added at 50 pL/well.

Plates were incubated at room temperature for 2 h and washed as described above. Alkanine phosphatase conjugated goat anti-human IgM (diluted 1 :2,000 for PC-BSA and 1 : 1500 for MDA-HSA in the 1 % BSA) was added at 100 pL/well and incubated at room temperature for 2 h. After four washings, the plate was incubated with 200ul/well of pNPP(p-Nitrophenyl phosphate) substrate (Sigma, P7988) for 75 min. We then added 3M NaOH to stop further colour development. Finally, plates were read in an ELISA Multiscan Plus spectrophotometer (Spectra Max 250, Molecular Devices, CA, USA) at 450 nm. All samples were measured in duplicates within a single assay and the coefficient of variation between the duplicates was below 15%. In order to investigate the specificity of these antibodies, competition assays were performed. At a dilution giving 50% of maximal binding to antibodies, sera were preincubated with different concentrations of antigen overnight in glass tubes. After vortexing, the tubes were incubated at 4°C overnight and centrifuged at 13000 r.p.m. for 30 min (4°C). The supernatants were tested for antibody binding to antigens as described above. The method demonstrated competition above 60% (data not shown), similar to the method we described previously2, 3. The percentage of inhibition was calculated as follows: % inhibition = (OD without competitor - OD with competitor)/ OD without competitor X100 Results:

Anti-MDA levels below 140 AU were associated with increased risk of not being in remission, under this 2 patients were in remission and 21 were not in remission.

With anti DA levels above 140 AU, 9 patients were in remission and 15 patients were not.

With anti MDA levels below 120, there was also an association, with 1 in remission and 15 not, while above 120 AU, 10 were in remission and 21 not.

These differences were significant statistically (using Fischer ^' s test), with p<0.05. These findings thus support the notion that the greater the amount of antibodies to MDA that are present in a patient at baseline, i.e. prior to treatment with methotrexate, the better is the chance of remission in the patient. Thus, the level of anti-MDA is associated with better outcome during Methotrexate treatment. EXAMPLE 2

Levels of IgM antibodies against MDA in RA patients at baseline and after 3, 6, 9 and 12 months of methotrexate treatment

60 patients with rheumatoid arthritis (RA) participated in this study. The patients were diagnosed with rheumatoid arthritis less than 2 years prior to initiation of the study. They were all below 75 years old and both males and females were included in the study. The disease activity of the patients was determined by the disease activity score (DAS 28) in the same way as described in Example 1 ,

Blood samples were collected from the patients at baseline, i.e. immediately prior to the first administration of methotrexate and again every 3 moths i.e. after 3 months, 6 months, 9 months and 12 months after treatment with methotrexate. During the 12 months, the patients were treated with methotrexate 15 - 20 mg once weekly.

The IgM anti-MDA antibody levels in the blood samples were determined by ELISA using arbitrary units (AU) defined as percentage of the absorbance to the plasma pool.

Determination of IgM antibodies against MDA was performed with ELISA in the same way as described in Example 1.

Results:

The results are shown in Figure 1. A comparison of the anti-MDA levels at baseline and at 3 months, 6 months and 12 months, respectively, showed that the anti-MDA levels decreased significantly during the methotrexate treatment period.

EXAMPLE 3

Levels of IgM antibodies against PC in RA patients at baseline and after 12 months of methotrexate treatment

47 patients with rheumatoid arthritis (RA) participated in this study. The patients were diagnosed with rheumatoid arthritis less than 2 years prior to initiation of the study. They were all below 75 years old and both males and females were included in the study.

The disease activity of the patients was determined by the disease activity score (DAS 28). The DAS28 is a measure of disease activity in RA. DAS stands for 'disease activity score' and the number 28 refers to the 28 joints that are examined in this assessment. The method is commonly used and the measures of disease activity in RA includes:-

•examination of joints for swelling and tenderness,

•global scores of pain and overall status,

•blood markers of inflammation (e.g. ESR and CRP)

•questionnaires (e.g. the HAQ which assesses function),

-X-rays, and other imaging techniques such as ultrasound and MRI. The DAS28 is a composite score derived from 4 of these measures, The '28' version is a simplification of the original DAS score, which requires 44 joints to be counted. Other versions of the DAS28 allow the CRP to be used instead of the ESR, or the omission of either one. To calculate the DAS28 the following parameters are measured:

1. The number of swollen joints (out of the 28),

2. The number of tender joints (out of the 28),

3. The erythrocyte sedimentation rate (ESR) or C reactive protein (CRP),

4. A 'global assessment of health' (indicated by marking a 10 cm line between very good and very bad) based on questioning of the patients.

These results are then fed into a mathematical formula to produce the overall disease activity score. A DAS28 of greater than 5.1 implies active disease, less than 3 2 low disease activity, and less than 2.6 remission. Blood samples were collected from the patients at baseline, i.e. immediately prior to the first administration of methotrexate and again 12 months later. During the 12 months, the patients were treated with methotrexate 15 - 20 mg once weekly.

The IgM anti-MDA antibody levels in the blood samples were determined by ELISA using arbitrary units (AU) defined as percentage of the absorbance to the plasma pool.

Determination of IgM antibodies against PC with ELISA

IgM antibodies to PC were determined by enzyme-linked immunosorbent assay (ELISA). Serum from donors with an anti-PC level above median level was used as internal standard and tested on every plate. This thus represents a standard in the ELISA, allowing for reproducibility as compared to other cohorts tested using this method. The plateau of antibody binding was reached with the antigen concentration of 10 pg/mL. Nunc Immuno microwell plates (Thermo Labsystems, Franklin, MA, USA) were coated with PC conjugated with BSA. Coated plates were incubated overnight at 4°C. After five washings with PBS, the plates were blocked with 2% BSA-PBS for 90 min at room temperature and washed as described above. Serum samples were diluted in 0.2% BSA-PBS and added at 50 pL/well.

Plates were incubated at room temperature for 2 h and washed as described above. Alkanine phosphatase conjugated goat anti-human IgM (diluted 1 :2,000 for PC-BSA and 1 : 1500 for MDA-HSA in the 1 % BSA) was added at 100 pUwell and incubated at room temperature for 2 h. After four washings, the plate was incubated with 200ul/well of pNPP(p-Nitrophenyl phosphate) substrate (Sigma, P7988) for 75 min. We then added 3M NaOH to stop further colour development. Finally, plates were read in an ELISA ultiscan Plus spectrophotometer (Spectra Max 250, Molecular Devices, CA, USA) at 450 nm. All samples were measured in duplicates within a single assay and the coefficient of variation between the duplicates was below 5%. In order to investigate the specificity of these antibodies, competition assays were performed. At a dilution giving 50% of maximal binding to antibodies, sera were preincubated with different concentrations of antigen overnight in glass tubes. After vortexing, the tubes were incubated at 4°C overnight and centrifuged at 13000 r.p.m. for 30 min (4°C). The supernatants were tested for antibody binding to antigens as described above. The method demonstrated competition above 60% (data not shown), similar to the method we described previously^?, 3. The percentage of inhibition was calculated as follows: % inhibition = (OD without competitor - OD with competitor)/ OD without competitor X 00 Results:

Anti-PC levels below 120AU were associated with increased risk of not being in remission. Under this level, 3 patients were in remission whereas 24 patients were not in remission. In patients where the baseline level was above 120 AU, 8 patients were in remission and 12 patients were not in remission.

These differences were significant statistically (using Fischer ^' s test), with p<0.05.

These findings thus support the notion that the greater the amount of antibodies to PC that are present in a patient at baseline, i.e. prior to treatment with methotrexate, the better is the chance of remission in the patient. Thus, the level of anti-PC is associated with better outcome during Methotrexate treatment.

EXAMPLE 4

Levels of IqM antibodies against PC in RA patients at baseline and after 3, 6, 9 and 12 months of methotrexate treatment

60 patients with rheumatoid arthritis (RA) participated in this study. The patients were diagnosed with rheumatoid arthritis less than 2 years prior to initiation of the study. They were all below 75 years old and both males and females were included in the study. The disease activity of the patients was determined by the disease activity score (DAS 28) in the same way as described in Example 3. Blood samples were collected from the patients at baseline, i.e. immediately prior to the first administration of methotrexate and again every 3 moths i.e. after 3 months, 6 months, 9 months and 12 months after treatment with methotrexate. During the 12 months, the patients were treated with methotrexate 15 - 20 mg once weekly.

The IgM anti-PC antibody levels in the blood samples were determined by ELISA using arbitrary units (AU) defined as percentage of the absorbance to the plasma pool. Determination of IgM antibodies against PC was performed with ELISA in the same way as described in Example 1.

Results:

The results are shown in Figure 2. A comparison of the anti-PC levels at baseline and at 3 months, 6 months and 12 months, respectively, showed that the anti-PC levels decreased significantly during the methotrexate treatment period.

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