The invention relates to the field of immunology, more specifically to the field of immune-mediated disorders such as allergies, auto-immune disease, transplantation-related disease or inflammatory disease. The invention provides among others an immunoregulator (NMPF), use of an NMPF in preparing a pharmaceutical composition for treating an immune-mediated disorder, a pharmaceutical composition and a method for treating an immune-mediated disorder.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides among others an immuno-regulator (NMPF) obtainable or derivable from a urinary metabolite of hCG, in particular from nicked forms of b-hCG, or (synthetic) peptide homologues or analogues thereof. These forms of b-hCG have peptide bond cleavages within the b-subunit (Birken et al, Endocrinology 133:1390-1397, 1993). Surprisingly, it has been found that a range of beta-HCG breakdown products provides a cascade of immunoregulators (NPMF) with a host of functions. Even more surprisingly, said immunoregulators are interrelated and derived from one another. The invention provides use of such an NMPF in preparing a pharmaceutical composition for treating an immune-mediated disorder, a pharmaceutical composition and a method for treating an immune-mediated disorder. Immune-mediated disorders as described herein include chronic inflammatory disease, such as diabetes type I or II, rheumatic disease, Sjogrens syndrome, multiple sclerosis), transplantation-related immune responses such as graft-versus-host-disease, post-transfusion thrombocytopenia, chronic transplant rejection, pre-eclampsia, atherosclerosis, asthma, allergy and chronic auto-immune disease, and acute inflammatory disease, such as (hyper)acute transplant rejection, septic shock and acute autoimmune disease. Autoimmune diseases are a group of disorders of in general unknown etiology. In most of these diseases production of autoreactive antibodies and/or autoreactive T lymphocytes can be found. An autoimmune response may also occur as manifestation of viral or bacterial infection and may result in severe tissue damage, for example destructive hepatitis because of Hepatitis B virus infection.

DETAILED DESCRIPTION OF THE INVENTION

Autoimmune diseases can be classified as organ specific or non-organ specific depending on whether the response is primarily against antigens localised in particular organs, or against wide-spread antigens. The current mainstay of treatment of autoimmune diseases is immune suppression and/or, (because of tissue impairment), substitution of vital components like hormone substitution. However, immunosuppressive agents such as steroids or cytostatic drugs have significant side effects, which limits their application. Now, the use of more specific immunoregulatory drugs is provided by the invention in the treatment of autoimmune disease and other inflammations. Based on the immunoregulatory properties, e.g. the capacities to regulate the Th1/Th2 ratio, to modulate dendritic cell differentiation, their low side-effect profile, and the beneficial clinical effects, etc., it shows these urinary metabolite preparations or synthetic analogues thereof to be very helpful in the treatment of patients with immune-mediated inflammation, such autoimmune disease.

A non-limiting list of an immune diseases includes:

Hashimoto's thyroditis, primary mysxoedema thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, insulin-dependent diabetes mellitus, stiff-man syndrome, Goodpasture's syndrome, myasthenia gravis, male infertility, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune haemolytic anaemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis,

cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, dermatomyositis, polymyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus, and systemic lupus erythematosus.

In one embodiment, the invention provides an immunoregulator capable of down-regulating Th1 cell levels and/or upregulating Th2 cell levels, or influencing their relative ratio in an animal, said immunoregulator obtainable from urine or other sources of bodily products, such as serum, whey, placental extracts, cells or tissues. Obtainable herein refers to directly or indirectly obtaining said NMPF from said source, NMPF is for example obtained via chemical synthesis or from animal or plant sources in nature.

In a preferred embodiment, the invention allows regulating relative ratios and/or cytokine activity of lymphocyte, dendritic or antigen presenting cell subset-populations in a diseased animal (e.g. human), preferably where these lymphocyte subset-populations comprise Th1 or Th2, or DC1 or DC2 populations. In general, naive CD4.sup.+ helper T lymphocytes (Th) develop into functionally mature effector cells upon stimulation with relevant antigenic peptides presented on the major histocompatibility complex (MHC) class II molecules by antigen-presenting cells (APC). Based on the characteristic set of cytokines produced, Th cells are commonly segregated into at least two different subpopulations: Th1 cells producing exclusively interleukin-2 (IL-2), interferon-gamma (IFN-.gamma.) and lymphotoxin, while Th2 cells produce IL-4, IL-5, IL6, IL10 and IL-13. These Th1 and Th2 subsets appear to be extremes in cytokine production profiles and within these polarized subsets, individual Th cells exhibit differential rather than co-ordinated cytokine gene expression. These subsets develop from common Th precursor cells (Thp) after triggering with relevant peptides into Th0 cells producing an array of cytokines, including IL-2, IL-4, IL-5 and IFN-.gamma.. These activated Th0 cells subsequently polarize into the Th1 or Th2 direction based on the cellular and cytokine composition of their microenvironment. Antigen-presenting cells like the various subsets of dendritic cells besides subsets of macrophages largely determine this polarization into Th1 or Th2 subset development. The Th1-TH2 subsets appear to cross-regulate each other's cytokine production profiles, mainly through IFN-.gamma. and IL-10, and from this concept it was rationalized that disturbances in the balance between these two subsets may result in different clinical manifestations [5]. IL-12 is a dominant factor promoting Th1 subset polarization and dendritic cells and macrophages produce IL-12. Moreover, IL-12 induces IFN-.gamma. production by T cells and natural killer (NK) cells. Recently, it was reported that IL-18 acts synergistically with IL-12 to induce Th1 development. Polarization of Th2 cells is critically dependent on the presence of IL-4 produced by T cells or basophils and mast cells. APC-derived IL-6 has also been shown to induce small amounts of IL-4 in developing Th cells. IL-10 and APC-derived prostaglandin E.sub.2 (PGE.sub.2) inhibit IL-12 production and Th1 priming.

The Th1-Th2 paradigm has been useful in correlating the function of Th1 cells with cell-mediated immunity (inflammatory responses, delayed type hypersensitivity, and cytotoxicity) and Th2 cells with humoral immunity. In general, among infectious diseases, resistance to intracellular bacteria, fungi, and protozoa is linked to mounting a successful Th1 response. Th1 responses can also be linked to pathology, like arthritis, colitis and other inflammatory states. Effective protection against extracellular pathogens, such as helminths, mostly requires a Th2 response, and enhanced humoral immunity may result in successful neutralisation of pathogens by the production of specific antibodies.

In yet another preferred embodiment, the invention provides an immunoregulator capable of modulating dendritic cell differentiation. The selective outgrowth of Th1 vs. Th2 type cells is dependent on the interaction of precursor Th cells with antigen-presenting cells (APC) carrying the relevant peptide in conjunction with their MHC class II molecules. Cytokines released by the APC and present during the initial interaction between dendritic cells and the pertinent T cell receptor carrying T cells drive the differentiation in to Th1 vs. Th2 subsets. Recently, two different precursors for DC (myeloid vs. lymphoid) have been described in man. Selective development of DC1 from myeloid precursors occurs after stimulation with CD40 Ligand or endotoxin, and results in high production of IL-12. Lymhoid precursors give rise to DC2 cells after CD40 Ligand stimulation, and produced IL-1, IL-6 and IL-10. These cytokines are of prime importance in driving the development of the activated Th cell: IL-4 is required for the outgrowth of Th2 type cells which can be greatly enhanced by the presence of IL-10, while selective differentiation to Th1 type cells is exclusively dependent on the presence of IL-12. Since DC1 are characterized by the production of IL-12, they will primarily induce outgrowth of Th1 type cells, while DC2 produce IL-10 and selectively promote Th2 development in the presence of exogenous IL-4. It is shown herein that an NMPF as provided by the invention is capable of regulating or modulating DC activity and differentiation, thereby allowing selective differentiation and activity of Th1 and/or Th2 cells.

In one embodiment, the invention provides an immunoregulator comprising an active component obtainable from a mammalian chorionic gonadotropin preparation said active component capable of stimulating splenocytes obtained from a non-obese diabetes (NOD) mouse, or comprising an active component functionally related to said active compound, for example allowing regulating or modulating DC activity and differentiation, or allowing selective differentiation and activity of Th1 and/or Th2 cells, in case of chronic inflammation, such as diabetes or chronic transplant rejection for example as shown in the detailed description herein wherein said stimulated splenocytes are capable of delaying the onset of diabetes in a NOD-severe-combined-immunodeficient mouse reconstituted with said splenocytes, or wherein said active component is capable of inhibiting gamma-interferon production of splenocytes obtained from a non-obese diabetes (NOD) mouse, or wherein said active component is capable of stimulating interleukine-4 production of splenocytes obtained from a non-obesediabetes (NOD) mouse.

In another embodiment, the invention provides an immunoregulator comprising an active component obtainable from a mammalian chorionic gonadotropin preparation said active component capable of protecting a mouse against a lipopolysaccharide induced septic shock, for example allowing regulating or modulating DC activity and differentiation, or allowing selective differentiation and activity of Th1 and/or Th2 cells, in case of acute inflammation, such as seen with shock or (hyper)acute transplantation rejection wherein said active component is capable of reducing ASAT or other relevant plasma enzyme levels after or during organ failure, as commonly seen with shock.

Although said immunoregulator according to the invention is easily obtained as urinary gonadotropin metabolite or break down product from urine, for example wherein said mammalian chorionic gonadotropin preparation is derived from urine, other sources, such as serum, cells or tissues comprising gonadotropin are applicable as well. Also from said sources an immunoregulator according to the invention capable of for example regulating Th1 and/or Th2 cell activity, and/or capable of modulating dendritic cell differentiation, is provided. In particular, as immunoregulator a (synthetic) peptide is provided obtainable of derivable from beta-HCG, preferably from nicked beta-HCG. Of course, such a peptide, or functional equivalent thereof is obtainable or derivable from other mammalian gonadotropins, as explained herein earlier. Said peptide is for example capable of protecting against septic shock or other immune-mediated disorders. Preferably, said peptide immunoregulator is obtained from a peptide having at least 10 amino acids such as a peptide having an amino acid sequence MTRVLQGVLPALPQVVC (SEQ ID NO:1) or functional fragment (e.g., a breakdown product) or functional analogue thereof. Functional fragments herein relates to the immunoregulatory effect or activity as for example can be measured in the septic shock or NOD mouse experimental model. Fragments can be somewhat (i.e. 1 or 2 amino acids) smaller or larger on one or both sides, while still providing functional activity.

The invention further provides a method for selecting an immunoregulator comprising determining therapeutic effect of an immunoregulator by subjecting an animal prone to show signs of diabetes to a peptide composition or fraction thereof, and determining the development of diabetes in said animal. Similarly, a method for selecting an immunoregulator comprising determining therapeutic effect of an immunoregulator by subjecting an animal prone to show signs of septic shock to a peptide composition or fraction thereof and determining the development of septic shock in said animal is provided herewith, the septic shock model also being a fast read-out model for the determination of anti-diabetic activity. Preferably, peptide compositions tested in a method according to the invention are obtained from a peptide having at least 10 amino acids such as a peptide having an amino acid sequence MTRVLQGVLPALPQVVC (SEQ ID NO:1) or functional fragment (e.g. a breakdown product) or functional analogue thereof.

Functional fragments herein relates to the immunoregulatory effect or activity as for example can be measured in the septic shock or NOD mouse diabetes experimental model. Fragments can be somewhat (i.e. 1 or 2 amino acids) smaller or larger on one or both sides. Surprisingly, it has been found in the animal test systems as provided herein that a range of beta-HCG breakdown products provides a cascade of peptide immunoregulators with a host of functions. Even more surprisingly, said immunoregulator peptides are interrelated and derived from one another and can also be produced synthetically. The invention provides use of such an immunoregulating peptide in preparing a pharmaceutical composition for treating an immune-mediated disorder, a pharmaceutical composition and a method for treating an immune-mediated disorder. A useful peptide found in a method according to the invention can be further modified or improved for one or more characteristics by peptide synthesis skills known, for example by identification of functional analogues with replacement mapping techniques, by binding-site (PEPSCAN) detection technology and so on, and can comprise D- or L-amino acids or modified amino acids at one or more (or all) places in the desired sequence. Also, peptide derivatives can be made, such as by circularization (for example by providing with (terminal) cysteines, dimerisation or multimerisation, by linkage to lysine or cystein or other side-chains that allow linkage or multimerisation, repeated, brought in tandem configuration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation. Of course, newly developed peptide compositions or derivatives can be tested according to a method as provided herein.

Functional analogue herein not only relates to analogues or homologues peptides from MIF or MIF-like proteins, from LH or PMSG, or gonadotropin-like proteins, be it modified by glycosylation or modification with unidentified amino acids or non-protein amino acids, but also to synthetic peptide analogues that can be made with peptide synthesis skills known, for example by identification of functional analogues with replacement mapping techniques, PEPSCAN detection technology and so on, and can comprise D- or L-amino acids or modified amino acids at one or more (or all) places in the desired sequence. Also, peptides can be circularised (for example by providing with (terminal) cysteines, dimerised or multimerised, by linkage to lysine or cystein or other side-chains that allow linkaage or multimerisation, repeated, brought in tandem configuration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation.

Preferably, an immunoregulator as provided by the invention is obtainable or derivable from a gonadotropin from a pregnant mammal, preferably a human, for example obtainable from a pharmacological preparation prepared to contain (placental) gonadotropins such as pregnant mare serum gonadotropin (PMSG) found in serum of pregnant mares, or pregnant mouse uterus extract (PMUE) extracted from uteri of gravid mice or human chorionic gonadotropin (hCG or HCG) found in blood or urine of pregnant women. An NMPF as provided by the invention can be associated with or without gonadotropin as for example present in the urine of first trimester of pregnancy (NMPF) and in commercial hCG preparations (NMPF) has immune regulatory effects.

In particular, NMPF can inhibit or regulate auto-immune and acute- and chronic-inflammatory diseases. TNF and IFN-gamma are pathologically involved in acute inflammatory disease such as sepsis or septic shock and also in auto-immune and chronic inflammatory diseases. Since NMPF has the ability to regulate T-cell sub-populations and inhibit TNF and IFN-gamma, NMPF can be used to treat, suppress or prevent immune mediator disorders such as sepsis or septic shock (acute inflammatory disease) as well as auto-immune disease or chronic inflammatory diseases such as systemic lupus erythematosus, diabetes, rheumatic disease, Sjogrens syndrome, multiple sclerosis, post-partum thyroid dysfunction and thyroid dysfunction related dementia's such as Alzheimer's disease, auto-immune thromocytopenia and others, such as allergies and chronic inflammatory disease and transplantation related immune responses.

Furthermore, the invention provides detection of genetic predisposition for immune-mediated disorders, whereby individuals with particular isoforms or amino acid variations in HCG or HCG derived peptides or immunoregulators are predisposed for certain disorders. Once known, it is provided by the invention to provide the genetically predisposed individual with the proper peptide immunoregulator via gene therapy

In particular, an immunoregulator according to the invention is provided wherein said functional fragment comprises a peptide having at least 10 amino acids such as having an amino acid sequence, LQGVLPALPQVVC (SEQ ID NO:2) (.beta.45+.beta.48), or VLPALPQVVC (SEQ ID NO:3) (.beta. 48) or LQGVLPALPQ (SEQ ID NO:4) (.beta. 45), or a functional analogue thereof, herein also called NMPF-K. Said immunoregulator comprising said peptide (or mixtures of peptides) having the desired length of about at least 10 amino acids (and especially when bound to a larger molecule such as when bound via its cysteine to another beta-HCG fragment) generally regulates Th1/Th2 balance as well as innate immunity during an immune mediated disorder. For example septic shock, LPS induced proliferation of splenocytes or diabetes is accelerated or aggravated. Similar activity is provided by a relative short-chain peptide (third immunoregulator, 3-5 amino acids long) that comprises MTRV (SEQ ID NO:5) or MTR or QVVC (SEQ ID NO:6) or VVC or CLQG (SEQ ID NO:7) or LQGV (SEQ ID NO:8) or LQG (and especially when bound to a larger molecule such as when bound via its cysteine to another beta-HCG fragment).

More in particular, a first immunoregulator is provided comprising a functional fragment comprising an amino acid sequence VLPALPQVVC (SEQ ID NO:3) or LQGVLPALPQ (SEQ ID NO:4) or functional analogue thereof which counteracts the regulatory activities of another, second immunoregulator according to the invention wherein said functional fragment comprises an amino acid sequence of from 9 to 6 amino acids (herein also called NMPF-Kb), such as VLPALPQ (SEQ ID NO:9) or GVLPALPQ (SEQ ID NO:10) or GVLPALP (SEQ ID NO:11) or VLPALP (SEQ ID NO:12) or functional analogue thereof, which for example is capable of regulating Th1/Th2 balance as well as innate immunity during an immune mediated disorder such that it is capable to reduce the clinical symptoms seen with immune-mediated disorders, such as septic shock, LPS induced proliferation of splenocytes or diabetes, instead of accelerating or aggravating these symptoms of immune-mediated disease, as for example is shown in the detailed description where NMPF-Kb is capable of protecting a mouse against a lipopolysaccharide induced septic shock, or other acute or chronic immune-mediated disorder as explained herein. As there is an overlap between .beta.45 and .beta.48 peptide (.beta.45; LQGVLPALPQ (SEQ ID NO:4) .beta.48: VLPALPQVVC (SEQ ID NO:3)), we also tested denaturated .beta.45+.beta.48 (LQGVLPALPQVVC (SEQ ID NO:2)) peptide for its effect on LPS induced proliferation (in vitro) and anti-shock activity (in vivo) in BALB/c mice. Our results showed that denaturated .beta.45+.beta.48 peptide inhibits LPS induced proliferation and in vivo septic shock. Breakdown products are generated via proteolysis, for example by lysis with leucocyte elastate, and can undergo further notification such as by the activity of (glutathion) transferases. One of the possible breakdown product of .beta.45+.beta.48 peptide is LQG which resembles glutathione (tripeptide of G, C, and Q with L-glutamate having an isopeptide bond with the amino moiety of L-cysteine). We have shown that NMPF also inhibits (toxin) streptozotocin (SZ) induced diabetes in mice through destruction of beta-cells. One of the mechanisms involved in the destruction of pancreatic beta cells is the formation of reactive radicals (ROS, NO etc.) that also play an important role in the pathogenesis of many other diseases like nephropathy, obstructive nephropathy, acute and chronic renal allograft rejection, auto-immune diseases (like SLE, rheumatoid arthritis, diabetes, MS), AIDS, diseases related to angiogenesis, atherosclerosis, thrombosis and type II diabetes mellitus. So, it is likely that NMPF also acts as `anti-oxidant`. For example breakdown products of .beta.45+.beta.48 such as LQG or CLQG (SEQ ID NO:7) peptides alone or in combination with certain carbohydrates or modified with unidentified amino acids or with nonprotein amino acids such as .beta.-alanine, .gamma.-Aminobutyric acid, Omithine, etc. posses immunomodulatory activity (NMPF).

Not wishing to be bound by theory, NMPF-K and NMPF-Kb activity can be described as maintaining a Th1/Th2 balance, whereby NMPF-K acts as if binding to an appropriate receptor but not activating it whereas NMPF-Kb is binding to said receptor and activating it to modulate the Th1/Th2 balance in a beneficial way. NMPF-K and NMPF-Kb are therein both ligands of the same or at least a conformationally similar or alike receptor molecule. Said receptor molecule is now also provided, since it and its acitivity are defined herein by said ligands.

For example, our results show that NMPF-Kb inhibits sepsis or septic shock caused by endotoxin or by exotoxin. NMPF-Kb as provided by the invention inhibits or counters immune mediated auto-immune diseases, chronic inflammatory diseases as well as acute inflammatory diseases.

The invention provides a pharmaceutical composition for treating an immune-mediated disorder such as an allergy, auto-immune disease, transplantation-related disease or acute or chronic inflammatory disease and/or provides an immunoregulator (NMPF), for example for stimulating or regulating lymphocyte action comprising an active component said active component capable of stimulating splenocytes obtained from a 20-week-old female non-obese diabetes (NOD) mouse, said stimulated splenocytes delaying the onset of diabetes in a NOD-severe-combined-immunodeficient (NOD.scid) mouse reconstituted at 8 weeks old with said splenocytes, or comprising an active component functionally related thereto.

In one embodiment, the invention provides an pharmaceutical composition or immunoregulator wherein said active component is capable of inhibiting gamma-interferon production or stimulating interleukine-4 production of splenocytes obtained from a 20-week-old female non-obese diabetes (NOD) mouse. Clinical grade preparations of gonadotropins such as hCG and PMSG have since long been used to help treat reproductive failure in situations where follicular growth or stimulation of ovulation is desired. Said preparations are generally obtained from serum or urine, and often vary in degree of purification and relative activity, depending on initial concentration in serum or urine and depending on the various methods of preparation used.

In a particular embodiment, the invention provides a immunoregulator comprising an active component obtainable or derivable from a mammalian CG preparation said active component capable of stimulating splenocytes obtained from a non-obese diabetes (NOD) mouse, or comprising an active component functionally related to said active compound, for example wherein said stimulated splenocytes are capable of delaying the onset of diabetes in a NOD-severe-combined-immunodeficient mouse reconstituted with said splenocytes.

The invention also provides an immunoregulator wherein said active component is capable of inhibiting gamma-interferon production obtained from a non-obese diabetes (NOD) mouse. The invention also provides an immunoregulator wherein said active component is capable of stimulating interleukine-4 production of splenocytes obtained from a non-obese diabetes (NOD) mouse.

An immunoregulator as provided by the invention (NMPF) has immune regulatory effects. In particular, NMPF can inhibit or regulate auto-immune and acute- and chronic-inflammatory diseases. TNF and IFN-gamma are pathologically involved in acute inflammatory disease such as sepsis or septic shock and also in auto-immune and chronic inflammatory diseases. Since NMPF has the ability to regulate T-cell sub-populations and inhibit TNF and IFN-gamma, NMPF can be used to treat, suppress or prevent immune mediator disorders such as sepsis or septic shock (acute inflammatory disease) as well as auto-immune disease or chronic inflammatory diseases such as systemic lupus erythematosus, diabetes, rheumatoid arthritis, post-partum thyroid dysfunction, auto-immune thromocytopenia and others, such as allergies and chronic inflammatory disease (i.e. rheumatic disease, Sjogrens syndrome, multiple sclerosis) and transplantation related immune responses. Our results for example show that NMPF-Kb inhibit sepsis or septic shock caused by endotoxin or by exotoxin. NMPF-Kb as provided by the invention inhibits or counters immune mediated auto-immune diseases, chronic inflammatory diseases as well as acute inflammatory diseases.

The invention thus provides use of an immunoregulator according to the invention for the production of a pharmaceutical composition for the treatment of an immune-mediated-disorder, for example wherein said immune-mediated disorder comprises chronic inflammation, such as diabetes, multiple sclerosis or chronic transplant rejection, wherein said immune-mediated disorder comprises acute inflammation, such as septic or anaphylactic shock or acute or hyper acute transplant rejection, wherein said immune-mediated disorder comprises auto-immune disease, such as systemic lupus erythematosus or rheumatoid arthritis, wherein said immune-mediated disorder comprises allergy, such as asthma or parasitic disease, in particular wherein said immune-mediated disorder comprises an overly strong immune response directed against an infectious agent, such as a virus or bacterium or wherein said immune-mediated disorder comprises pre-eclampsia or another pregnancy related immune-mediated disorder. Use of NMPF-K as contraceptive (e.g. as morning-after-pill or contraceptive vaccine eliciting contraceptive or sterilising antibodies in the vaccinated female mammal) is also provided. Use of NMPF-Kb is provided for facilitating fertility, especially in case where improved implantation is required. Especially, use is provided wherein said treatment comprises regulating innate immunity and/or relative ratios and/or cytokine activity of lymphocyte, dendritic or antigen presenting cell subset-populations in a treated individual, in particular wherein said subset populations comprise Th1 or Th2, or DC1 or DC2 cells. Thus the invention provides a method for treating an immune-mediated-disorder comprising subjecting an animal to treatment with at least one immunoregulator according to the invention, in particular wherein said disorder comprises diabetes or sepsis.

The invention provides also a method for diagnosing or determining the risk of non-pregnancy related immune disorders associated with Th1/Th2 misbalance as demonstrable by a misbalance between NMPF-K and NMPF-Kb, as for example produced or derived from pituitary derived gonadotropin, especially in age-related disease such as auto-immune and chronic inflammatory disease, such as type II diabetes, rheumatic disease, thyroid dysfunction related mental disease such as dementia's like Alzheimers and others, and atherosclerosis and related disease, said method comprising determining in a sample, preferably a blood or urine sample, the relative ratio of a relative long-chain peptide versus a relative short-chain peptide, said peptides derivable from breakdown of beta-HCG, in particular comprising determining the relative ratio of a relative long-chain peptide versus a relative short-chain peptide derived from breakdown a peptide having an amino acid sequence MTRVLQGVLPALPQVVC (SEQ ID NO:1), for example wherein said relative long-chain peptide comprises an amino acid sequence LQGVLPALPQ (SEQ ID NO:4) or GVLPALPQ (SEQ ID NO:10) or VLPALPQ (SEQ ID NO:9) or GVLPALP (SEQ ID NO:11) or VLPALP (SEQ ID NO:12), in particular wherein said relative short-chain peptide comprises MTRV (SEQ ID NO:5) or MTR or PALP (SEQ ID NO:13) or QVVC (SEQ ID NO:6) or VVC or LQGV (SEQ ID NO:8) or LQG. Detection of said long-chain peptides and short chain peptides, be it modified by glycosylation or modification with unidentified amino acids or non-protein amino acids is preferably achieved by immunological detection methods as known in the art.

The invention provides also a method for diagnosing or determining the risk of a pregnancy related immune-mediated disorder such as pre-eclampsia, or other immune-mediated disorder and the outcome of pregnancy and/or pregnancy related immune disease (such as gestation diabetes mellitus (GDM)) comprising determining in a sample, preferably a urine sample, the relative ratio of a relative long-chain peptide versus a relative short-chain peptide, said peptides derivable from breakdown of beta-HCG, in particular comprising determining the relative ratio of a relative long-chain peptide versus a relative short-chain peptide derived from breakdown a peptide having an amino acid sequence MTRVLQGVLPALPQVVC (SEQ ID NO:1), for example wherein said relative long-chain peptide comprises an amino acid sequence LQGVLPALPQ (SEQ ID NO:4) or GVLPALPQ (SEQ ID NO:10) or VLPALPQ (SEQ ID NO:9) or GVLPALP (SEQ ID NO:11), in particular wherein said relative short-chain peptide comprises MTRV (SEQ ID NO:5) or MTR or QVVC (SEQ ID NO:6) or VVC, or LQGV (SEQ ID NO:8) or LQG.

Anecdotal observations and laboratory studies indicated previously that hCG might have an anti-Kapos's sarcoma and anti-human-immunodeficiency-virus effect (Treatment Issues, July/August 1995, page 15). It has been observed that hCG preparations have a direct apoptotic (cytotoxic) effect on Kaposi's sarcoma (KS) in vitro and in immunodeficient patients and mice and a prohematopoetic effect on immunodeficient patients (Lunardi-Iskandar et al., Nature 375, 64-68; Gill et al., New. Eng. J. Med. 335, 1261-1269, 1996; U.S. Pat. No. 5,677,275), and a direct inhibitory antiviral effect on human and simian immunodeficiency virus (HIV and SIV) (Lunardi-Iskandar et al., Nature Med. 4, 428-434, 1998, U.S. Pat. No. 5,700,781). Said cytotoxic and anti-viral effects have also been attributed to an unknown hCG mediated factor (HAF), present in clinical grade preparations of hCG. However, commercial hCG preparations (such as CG-10, Steris Profasi, Pregnyl, Choragon, Serono Profasi, APL), have various effects. Analysis of several of these, (AIDS, 11: 1333-1340, 1997) for example shows that only some (such as CG-10, Steris Profasi) are KS-killing whereas others (Pregnyl, Choragon, Serono Profasi) were not. Secondly, recombinant subunits of (a or .beta.) hCG were killing but intact recombinant hCH not. It was also found that the killing effect was also seen with lymphocytes. Therapy of KS has recently been directed at using beta-hCG for its anti-tumour effect (Eur. J. Med Res. 21: 155-158, 1997), and it was reported that the beta-core fragment isolated from urine had the highest apoptotic activity on KS cells (AIDS, 11: 1713-721, 1997).

Recently, Gallo et. al. reported anti-Kaposi's Sarcoma, anti-HIV, anti-SIV and distinct hematopoietic effects of clinical grade crude preparations of human chorionic gonadotropin (hCG) (Lunardi-Iskandar et al. 1995, Gill et al. 1996, Lunardi-Iskandar et al. 1998). In contrast to their previous studies, it is also claimed that the anti-tumour and anti-viral activity of hCG preparation is not due to the native hCG heterodimer, including its purified subunits or its major degradation product, the .beta.-core; instead the active moiety resides in an as yet unidentified hCG mediated factor (HAF). Whatever the true factor may be, these unidentified factors in several hCG preparations have anti-tumour activity through the selective induction of apoptosis, besides direct cytotoxic effects on the tumour cells. Furthermore, they postulated that the anti-tumour activity could not be due to an immune-mediated response, since there was no infiltration of the tumour with mononuclear cells.

Moreover, the reported pro-hematopoietic effect of clinical grade hCG was noted in clinical studies in humans infected with HIV, (Lunardi-Iskandar et al. 1998) indicating that the hematopoietic effect is indirect, and caused by rescuing CD4+cells otherwise killed by HIV through the anti-HIV activity of hCG.

The invention provides an immunoregulator or a pharmaceutical composition for treating an immune-mediated disorder obtainable from a hCG preparation or a fraction derived thereof. The effects of said immunoregulator include a stimulating effect on lymphocyte populations (such as found in peripheral lymphocytes, thymocytes or splenocytes), instead of cytotoxic or anti-viral effects. The invention provides a method for treating an immune-mediated-disorder comprising subjecting an animal to treatment with at least one immunoregulator obtainable from a pregnant mammal. Said treatment can be direct, for example treatment can comprise providing said individual with a pharmaceutical composition, such as a hCG or PMSG preparation, comprising an immunoregulator as provided by the invention. It is also possible to provide said pharmaceutical composition with a fraction or fractions derived from a pregnant animal by for example sampling urine or serum or placental (be it of maternal or foetal origin) or other tissue or cells and preparing said immunoregulator comprising said active component from said urine or serum or tissue or cells by fractionation techniques known in the art (for example by gel permeation chromatograpy) and testing for its active component by stimulating a NOD mouse or its splenocytes as described. In particular, said preparation or component is preferably derived from a pregnant animal since an embryo has to survive a potentially fatal immunological conflict with its mother: developing as an essentially foreign tissue within the womb without triggering a hostile immune attack. So, to prevent this rejection "allograft" the immunological interaction between mother and fetus has to be suppressed, either for instance through lack of fetal-antigen presentation to maternal lymphocytes, or through functional "suppression" of the maternal lymphocytes. If fetal antigens are presented, maternal immune responses would be biased to the less damaging, antibody-mediated T helper 2 (Th2)-type. This would suggest that pregnant women are susceptible to overwhelming infection, which is not the case. Female individuals during pregnancy maintain or even increase their resistance to infection. Moreover, while said individuals normally are more susceptible to immune diseases than male individuals, especially autoimmune diseases, during pregnancy they are more resistant to these diseases.

The invention also provides a method for in vitro stimulation of lymphocytes and transferring said stimulated lymphocytes as a pharmaceutical composition to an animal for treating said animal for an immune mediated disorder. In a particular embodiment of the invention a pharmaceutical composition is provided comprising lymphocytes stimulated in vitro with an immunoregulator provided by the invention.

In a preferred embodiment of the invention, said disorder comprises diabetes, yet other immune mediated disorders, such as acute and chronic inflammation, can also be treated. In yet another preferred embodiment, said disorder comprises sepsis or septic shock. The invention provides a method of treatment for an animal, preferably wherein said animal is human.

In a particular embodiment, a method provided by the invention is further comprising regulating relative ratios and/or cytokine activity or cytokine expression or marker expression of lymphocyte, dendritic or antigen presenting cell subset-populations in said animal, such as subset-populations that comprise Th1 or Th2 cells, or Th3 or Th8 cells, or DC1 or DC2 cells or other effector or regulatory T-cell populations.

The invention also provides an immunoregulator for use in a method according to the invention, and use of said immunoregulator, preferably obtainable from a pregnant mammal, for the production of a pharmaceutical composition for the treatment of an immune-mediated-disorder, preferably selected from a group consisting of allergies, auto-immune disease (such as systemic lupus erythematosus or rheumatoid arthritis), transplantation-related disease and acute (such as septic or anaphylactic shock or acute or hyper acute transplant rejection) and chronic inflammatory disease (such as atherosclerose, diabetes, multiple sclerosis or chronic transplant rejection). Furthermore, the invention provides a use according to the invention wherein said immune-mediated disorder comprises allergy, such as asthma or parasitic disease, or use according to the invention wherein said immune-mediated disorder comprises an overly strong immune response directed against an infectious agent, such as a virus or bacterium. Often in most of these diseases production of autoreactive antibodies and/or autoreactive T lymphocytes can be found mounting or being part of a too strong immune response. This is for example seen with parasitic disease, where IgE production is overly strong or which disease is Th2 dependent, and detrimental for the organism, but also with (myco)bacterial infections such as TBC or leprosy. An autoimmune response may also occur as manifestation of viral or bacterial infection and may result in severe tissue damage, for example destructive hepatitis because of Hepatitis B virus infection, or as seen with lymphocytic choriomeningitis virus (LCMV) infections. Said overly strong immune response is kept at bay with an immunoregulator as provided by the invention. Yet other use as provided by the invention relates to treatment of vascular disease, whereby radical damage (damage caused by radicals) to cells and tissue is prevented or repaired by treatment with NMPF according to the invention; whereby NMPF also acts as anti-oxidant directly or indirectly. For example, a determining event in the pathogenesis of diabetes I is the destruction of insulin-producing pancreatic beta cells. There is strong evidence that the progressive reduction of the beta-cell mass is the result of a chronic autoimmune reaction. During this process, islet-infiltrating immune cells, islet capillary endothelial cells and the beta cell itself are able to release cytotoxic mediators. Cytokines, and in particular nitric oxide (NO), are potent beta-cell toxic effector molecules. The reactive radical NO mediates its deleterious effect mainly through the induction of widespread DNA strand breaks, other radicals, such as oxygen, through their effects on lymfocyte sub-populations such as Th1 and Th2 cells. This initial damage triggers a chain of events terminating in the death of the beta cell and disarray of the immune response.

Furthermore, an immunoregulator according to the invention is capable of regulating radical induced or directed cell-cell interactions or cell responses, specifically those interactions or responses of an immunological nature, e.g. related to regulating interactions of the innate or adaptive immune system. Not wishing to be bound by theory, there are two arms of the immune system: the innate (non-specific) and adaptive (specific) systems, both of which have cellular and humoral components. Examples of cellular components of the innate immune system are monocytes, macrophages, granulocytes, NK cells, mast cells, gd T cell etc, while, examples of humoral components are lysozyme, complement, acute phase proteins and mannose-binding lectin (MBL). The major cellular components of the adaptive immune system are T and B cells, while examples of humoral components are antibodies. The adaptive system has been studied most because of its specificity, effectiveness at eliminating infection and exclusive presence in higher multicellular organisms. The innate system is often considered primitive and thought to be `unsophisticated`. However, the innate system not only persists but could also play a critical role in one of the most fundamental immune challenges--viviparity. The innate system instigates an immune response by processing and presenting antigen in association with major histocompatibility complex (MHC) class I and II molecules to lymphocytes. Full response often requires adjuvant (such as endotoxin), which, through interaction with the innate immune system, produce costimulatory surface molecules or cytokines. This determines the biological significance of antigens and communicates this information to the adaptive system. So it instructs the adaptive system to either respond or not. So these two great arms of immune system not only influence each other but also regulate each other at least at the cellular level through for example cytokines and co-stimulatory molecules etc.

There are many physiological conditions and immune pathologies where these two systems are involved separately or in combination. For example, it has been shown that in pregnancy the maternal innate immune system is more stimulated, or for it has been proposed that type II diabetes mellitus is a disease of a chronic hyperactive innate immune system. Another example is the involvement of the innate immune system in listeriosis. Dysregulation in the adaptive immune system may also lead to immune diseases like systemic or organ-specific autoimmunity, allergy, asthma etc, but it can also play a role in the maintenance of pregnancy and in the prevention of "allograft" rejection.

As mentioned above, the adaptive system has been studied most because of its specificity, effectiveness at eliminating infection, and exclusive presence in higher multicellular organisms. Its regulation has also been studied most. For example, it well known that the cytokine micro-environment plays a key role in T helper cell differentiation toward the Th1 or Th2 cell type during immune responses. IL-12 induces Th1 differentiation, whereas IL-4 drives Th2 differentiation. Recently it has also been shown that subsets of dendritic cells (DC1, DC2) provide different cytokine microenvironments that determine the differentiation of either Th1 or Th2 cells. In addition, negative feedback loops from mature T helper cell responses also regulate the survival of the appropriate dendritic cell subset and thereby selectively inhibit prolonged Th1 or Th2 responses. Moreover, development of Th1 responses can be antagonized directly by IL-4 and indirectly by IL-10, which inhibits the production of IL-12 and interferon-g-inducing factor (IGIF) by macrophages stimulated by the innate immune response. Th2 cells dependent on IL-4 to proliferate and differentiate have been implicated in allergic and atopic manifestations, and in addition through their production of IL-4 and IL-10, have been suggested to play a role in tolerance. Specifically, it has been suggested that Th1 to Th2 switch may prevent the development of organ-specific autoimmune pathologies and required for the maintance of pregnancy. Recently it has become clear that distinct subsets of regulatory T cells are responsible for regulating both Th1 and Th2 responses and prevent the development of immune pathologies. One of the common features of many of these regulatory T cells is that their function is at least in part due the action of TGF-beta; this would be in keeping with the ability of TGF-beta to inhibit both Th1 and Th2 development while IL-10 could preferentially inhibit Th1 alone.

The selective outgrowth of Th1 vs. Th2 type cells is dependent on the interaction of precursor Th cells with antigen-presenting cells (APC) carrying the relevant peptide in conjunction with their MHC class II molecules. Cytokines released by the APC and present during the initial interaction between dendritic cells and the pertinent T cell receptor carrying T cells drive the differentiation in to Th1 vs. Th2 subsets. Recently, two different precursors for DC (myeloid vs. lymphoid) have been described in man. Selective development of DC1 from myeloid precursors occurs after stimulation with CD40 Ligand or endotoxin, and results in high production of IL-12. Lymhoid precursors give rise to DC2 cells after CD40 Ligand stimulation, and produced IL-1, IL-6 and IL-10. These cytokines are of prime importance in driving the development of the activated Th cell: IL-4 is required for the outgrowth of Th2 type cells which can be greatly enhanced by the presence of IL-10, while selective differentiation to Th1 type cells is exclusively dependent on the presence of IL-12. Since DC1 are characterized by the production of IL-12, they will primarily induce outgrowth of Th1 type cells, while DC2 produce IL-10 and selectively promote Th2 development in the presence of exogenous IL-4.

NMPF as provided by the invention is able to regulate the Th1/Th2 balance in vivo (BALB/c, NOD) and in vitro. In dominant Th1 phenotype models like NOD, NMPF (like NMPF-P and its fractions) amongst others down-regulates the IFN-gamma production (in vivo/in vitro) and promote the IL-10 and TGF-beta production, in contrast to IL-4 production, which indicates the induction of regulatory cells like Th3 and Tr1 by NMPF. These regulatory cells may play role in the therapeutic effects of NMPF in immune and inflammatory diseases and immune tolerance. Furthermore, the invention provides an immunoregulator selected by a method according to the invention, a pharmaceutical composition comprising such a selected immunoregulator, and the use of said for the preparation of a pharmaceutical composition for the treatment of an immune-mediated disorder.

Purified NMPF is used to produce monoclonal antibodies and/or other specific reagents thereby facilitating the design of an NMPF-specific quantitative immuno-assay. Also single chain F.sub.v fragments are isolated by using the phage display technology with the use of a phage library containing a repertoire comprising a vast number of different specificities.

The invention further provides a method and a pharmaceutical composition for modulating cardiovascular or circulatory disorders, such as heart failure, brain infarctions, Alzheimer's disease, thrombosis, arteriosclerosis, pregnancy related cardiovascular or circulatory disorders and the like. It has been found that an immunoregulator as described supra has a very beneficial effect on animals, including humans, suffering from a cardiovascular disorder.

An immunoregulator according to the invention also widens the scope of possibilities of dotter treatments. In cases where conventionally such a treatment could not be performed because of risks of an oxygen tension becoming too low, a dotter treatment is now feasible when combined with treatment with an immunoregulator described above. Accordingly, expensive and difficult bypass surgery may in many cases be avoided.
 

Claim 1 of 5 Claims

1. A purified immunoregulator consisting of LQGV (SEQ ID NO:8).

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