Protein Complexes for Prevention and Treatment of Diseases with Angiogenesis Disorders

Field of the Invention

5 The invention is related to the medical field, and in particular, to protein complexes and the application thereof for the correction of autoimmune processes in human or animal bodies and also to a biochemical research for discovering the biochemical mechanisms of progressing diseases with angiogenesis disorders

Prior Art

I O For better understanding of the particular elements of the invention further there is given the description of biological mechanisms accompanying angiogenesis Angiogenesis is a process of formation of new vessels that is inherent to various activities of normal human or animal bodies (Folkman, J , Cancer Research, 46 467473 (1986) Angiogenesis is most strongly characteristic of such processes as reproduction, embryogenesis and wound healing Although the molecular mechanisms of angiogenesis

15 have not been studied in full, its general principles consist in stimulation and inhibition of the activity of endothelial cells Under normal conditions these cells could be inactive for continuous periods of time from several weeks to a number of decades However, should the necessity arise (as for example in wound healing), the same cells become a subject to fast reproduction and are grown into capillaries within 5 days 0 Although angiogenesis is a highly monitored process under normal conditions, many so-called angiogenic diseases are progressing as a result of chronic angiogenesis disorders

Various angiogenesis regulators have been discovered, both positive and negative ones The division into positive and negative ones turns out to be sufficiently conditional, since one and the same factor under various experimental conditions could have quite a different influence that is often related to proteolytic 5 cleavage or to modification of an original molecule when new structures having a reverse physiological direction are formed

The positive regulators or pro-angiogenic factors include as follows members of family of fibroblast growth factors (FGF), in particular, basic fibroblast growth factor (bFGF), fibroblast growth factor-2 (FGF-2) and keratinocyte growth factor (KGF), 0 members of family of vascular endothelial growth factors, in particular, placental growth factor

(PIGF) and vascular endothelial growth factor A (VEGF-A), members of family of interleukines, in particular, ιnterleukιnes-6 and 8 (IL-6, IL-8), members of family of matrix metalloproteinases, in particular, metalloproteιnase-3 (MMP-3) members of family of colony stimulating factors, in particular, granulocyte-colony stimulating 5 factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), members of family of platelet-derived growth factors, in particular, platelet-derived endothelial cell growth factor (PDEGF) and platelet-derived growth factor-BB, members of family of angiopoietins, in particular, angιopoιetιn-1 and -2 (Ang 1 , 2) members of family of insulin-like growth factors, in particular, insulin-like growth factor 1 (IGF-1 ), 0 members of family of tumor-necrosis factors, in particular, tumor-necrosis factor-alpha (TNF- alpha), intercellular adhesion molecule factor (ICAM-1 ), nerve growth factor (NGF) .transforming growth factor beta 1 (TGF-beta 1 ), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) tenascin-R (TN-R), prolactin, growth hormone, placental lactogen, angiogenine, thymidine phosphorylase 5 (TP) etc

The negative regulators or anti-angiogenic factors include members of family of inteferons, in particular, interferon alpha, members of family of tissue inhibitors of matrix metalloproteinases (TIMPs), platelet derived growth factor-4 (PDGF-4), transforming growth factor-b (TGFb) RGD-peptides 50 proteolytic fragments of growth hormone, placental lactogen and prolactin

Many angiogenesis inhibitors originate from intercellular matrix proteins circulating in blood such as fibronectin, plasminogen, collagen, secreted protein acidic and rich in cysteine (SPARC), thrombospondιn-1 (TSP-1 ), serpine-antithrombine etc

Angiogenic factors such as kanstatin and tumstatin are fragments of collagen IV, restin of collagen XV while endostatins are fragments of C-terminal domain of collagen-XVIII

With account to angiogenic factors the most studied ones are angiostatins that are proteolytic fragments of plasminogen

Most angiogenesis regulators are to be found in an intercellalar matrix in a bioactive form The receptors corresponding to these regulators are located on the surface of endothelial cells Under physiological conditions the addition of an angiogenic factor results in a cascade of reactions leading either to activation or inhibition of endothelial cells

Actually the receptors of angiogenic factors are an indispensable part of angiogenesis system and they themselves could be related to angiogenic factors

Should the components interfering with the activity of angiogenic factors appear at the site of a physiological process, they will cause the disturbance of normal angiogenesis proceeding and will result in a new angiogenesis mediated pathology The inventors have discovered that such interfering components are pathologic autoantibodies against various angiogenic factors These autoantibodies under pathologic conditions in a subject's body start to be produced in the increased quantities causing a number of physiologic disorders that are a basic pathogenesis mechanism The most unexpected was the discovery of elevated concentrations of autoantibodies to various angiogenic factors on the early stages of neoplasic diseases in blood and tissues of experimental animals and cancer patients, suggesting the autoimmune origin of malignant growth

At the same time it is quite often when particular angiogenic factors are produced in increased quantities thus also causing pathologic disorders In this case the stimulation of the production of autoantibodies against these angiogenic factors could also decrease the intensity of pathologic disorders and would have a curing effect

Therefore it is necessary to correct the disorders of angogenesis processes by influencing the activity of various angiogenic factors with the help of monitoring autoimmune processes This correction could be performed both by means of decreasing the content of pathologic autoantibodies in a subject's body or by inhibiting their production in the immunocompetent cells and by means of decreasing the activity of angiogenic factors by stimulating the production of autoantibodies used for their neutralization

Known are different methods of effecting angiogenesis processes with the aim of prevention and treatment of a number of diseases Known is a method and a composition for immunotherapy of inflammatory and autoimmune diseases described in US application No 20060140936A 1 published June 29, 2006 Methods and compositions for immunotherapy of inflammatory and immune-dysregulatory diseases, using multispecific antagonists that target at least two different markers are disclosed The different targets include (ι) proinflammatory effectors of the innate immune system, (ιι) coagulation factors, and (in) targets specifically associated with an inflammatory or immune-dysregulatory disorder, with a pathologic angiogenesis or cancer, or with an infectious disease, wherein the targets included in group (in) are neither a proinflammatory effector of the immune system nor a coagulation factor When the multispecific antagonist reacts specifically with a target associated with an inflammatory or immune- dysregulatory disorder, with a pathologic angiogenesis or cancer, or with an infectious disease, it also binds specifically with at least one proinflammatory effector of the immune system or at least one coagulation factor Thus, the multispecific antagonist contains at least one binding specificity related to the diseased cell or condition being treated and at least one specificity to a component of the immune system, such as a receptor or antigen of B cells, T cells, neutrophils, monocytes and macrophages, and dendritic cells, a modulator of coagulation, or a proinflammatory cytokine The multispecific antagonists are used in the treatment of various diseases that are generated or exacerbated by, or otherwise involve proinflammatory effectors of the innate immune system or coagulation factors Such diseases more particularly include acute and chronic inflammatory disorders, autoimmune diseases, giant cell arteritis septicemia and septic shock, coagulopathies (including diffuse intravascular coagulation), neuropathies, graft versus host disease, infectious diseases, acute respiratory distress syndrome, granulomatous diseases, transplant rejection, asthma, cachexia, myocardial ischemia, and atherosclerosis Other diseases also responsive to these therapies include cancers and conditions with pathological angiogenesis

According to this method it is proposed to use a multispecific antagonist that reacts specifically with at least two different targets The targets are selected from the group consisting of (A) proinflammatory effectors of the innate immune system, (B) coagulation factors, (C) complement factors and complement regulatory proteins, and (D) targets specifically associated with an inflammatory or immune-dysregulatory disorder or with a pathologic angiogenesis or cancer, wherein the latter target is not (A) ₁ (B) or (C) When the multispecific antagonist comprises a single multispecific antibody, then CD74 is excluded as a target of the antagonist Furthermore, when the multispecific antagonist comprises a combination of separate antibodies, combinations are excluded where one of the antibodies targets a B-cell antigen and the other antibody targets a T-cell, plasma cell, macrophage or inflammatory cytokine Combinations of separate antibodies are also excluded where one of the antibodies targets CD20 and the other antibody targets C3b or CD40

A multispecific antagonist is used in the treatment of diseases caused by inflammatory immune system effectors or by blood coagulation factors These diseases include inter alia septicemia or septic shock, an infectious disease, a neuropathy, graft versus host disease or transplant rejection, acute respiratory distress syndrome, a granulomatous disease, asthma, atherosclerosis, cachexia, a pathologic angiogenesis, cancer, a coagulopathy, acne, giant cell arteritis, or myocardial ischemia However, the above-described immunotherapy method and composition are intended for functional suppression of the target pathological cells with no account to as their specific contribution to a pathogenesis autoimmune mechanism of particular disease Therefore the main disadvantage of the above method and composition is their low efficiency for diseases with angiogenesis disorders related to autoimmune processes

Known is a method disclosed in US patent 6,863,896 published March 8 2005 which consists of producing an immune response in a mammal, comprising the step of administering mucosally to a mammal an admixture comprising an immunogen and a plant lectin, whereby the mammal produces an immune response to the immunogen which is greater relative to the immune response to the immunogen produced in the absence of the plant lectin However, the above composition and a method of application thereof also do not provide for the correction of the states related to angiogenesis disorders

Known is a protein conjugate and also a method of application thereof described in US patent No 6,451 ,312 This protein conjugate comprises pro-angiogenic factor VEGF and a plant toxin gelonin wherein VEGD is bound to tumour-associated receptor VEGFR expressed on a cell surface of tumour blood vessels and wherein the tumour types to be effected by said conjugate include inter aha squamous cell carcinoma, adenocarcinoma, small-celled carcinoma, glioma or neuroblastoma Any growth factor could be used for its intended application, should it be bound to the tumour-associated endothelial cells and to be more precise, to the growth factor receptors on the surface of the cells in question and with FGF and TGF beta mentioned in this connection, these also being the pro-angiogenic factors It is strongly emphasized that a growth factor receptor should be present in higher concentrations on the surface of the tumour-associated endothelial cells than on any other kind of cells being not tumour- associated The above conjugate was successfully applied for growth suppression of a mouse experimental tumour However, the conjugate in question was used for the purposeful elimination of endothelial cells of tumour microvessels, which is not directly related to autoimmune processes And it is the autoimmune mechanisms that form the pathogenesis basis of spontaneous tumours and of many other angiogenic diseases in human and mammal bodies

Therefore the main disadvantage of above conjugate and a method of application thereof is their low efficiency for diseases with angiogenesis disorders caused by autoimmune processes

Also known is an antigen conjugate and artificial carriers in the treatment of autoimmune diseases by means of biospecific sorption of autoantibodies and immunocompetent cells described in Russian patent No 2167705, published May 27, 2001

However, said conjugate and said method of application thereof do not make it possible to efficiently correct the states related to angiogenesis autoimmune disorders

Known is an application of intravenous immunoglobulin (IVIG) to treat the states of immunity disorders IVIG presents a specially separated and processed immunoglobulin fraction which is made of collected plasma pool from several thousand of donors having a vast distribution of antibodies as to their subclasses and specificity The antibodies in question often include the ones that with high specificity identify

the areas on the pathologic autoantibodies or on the receptors of subject's pathologic modified immunocompetent cells Therefore, when administered to the patients with autoimmune diseases, IVIG often results in a pronounced clinical effect

Officially acknowledged is the use of IVIG in the treatment of thrombocytopenic purpura Kawasaki disease, allogenic transplantation of haemopoietic cells, retinochoroiditis of Birdshot Besides, it is strongly recommended by those keen in the art to use IVIG for some kinds of cytopenia and haemophilia with inhibitory autoantibodies to factor VIII There are also some promising results in the treatment of a number of neurologic disorders like Guillain-Barre syndrom, inflammatory demielinating polyneuropathy, dermatomyositis, myastenia gravis and multiple sclerosis To explain the mechanism of IVIG versatility a number of hypotheses were proposed like disabling the FC-receptors on the surface of phagocytes, monocytes, neutrophils and eosinophils, neutralization of circulating pathologic autoantibodies by antiidiotype antibodies, inhibition of negative processes related to complement activation, - modulation of cytokine network, and selection of immune repertoire by means of interaction with T- and B-lymphocytes

Known is a therapeutic methods for inhibiting tumor metastasis and for treating primary tumors (US patent 5965130, published October 12, 1999) administering to a mammal a preparation of intravenous immunoglobulin (IVIG) However, this method does not consider the specific features of the subject's pathologic process and the inhibiting characteristics of the anti-idiotypic antibodies in the donor IVIG This substantially decreases the therapeutic efficiency of IVIG application for neoplastic diseases

Known is a method for preparing Ig fractions from human polyvalent intravenous Immunoglobulins and its application in the treatment of autoimmune diseases (US patent 6,932,969, published August 23 2005) The method consists in separating a fraction of antibodies which react in an anti-idiotypic way with pathologic autoantibodies against actin, myosin, basic myelin protein and tubulin However the above method does not consider the fact of formation of pathologic autoantibodies against angiogenic factors and therefore it cannot be efficiently applied in the treatment of diseases with angiogenesis disorders

Known are a number of methods to diagnose diseases based on identification of autoantibodies that are specific to particular autoantigens Known is a method of diagnosing or predicting diseases related to angiogenesis such as cancer, arthritis, macular degeneration and diabetic retinopathy (Russian patent application No 96122485 published May 10, 1999)

The above method includes the stage of determining the angiostatin concentration, wherein said angiostatin is a protein having a sufficient activity to inhibit the proliferation of endothelial cells and a molecular weight from 38 to 45 kD which is according to electrophoresis data in polyacrylamide gel under restoration conditions In the above application there is also mentioned a pharmaceutical composition comprising an antibody that is specifically bound to angiostatin

The disadvantage of the above method is its insufficient specificity as to the diagnosis of malignant growths Russian patent application No 2005101643, published June 27, 2005 discloses a device containing at least one carrier for immobilizing one or a number of antigens, a biologic preparation and an element for defining the presence of autoantibodies against said antigens in said preparation

However, the above device does not provide for defining the autoantibodies against angiogenic factors Summary of the Invention The inventors have found out that many kinds of diseases with angiogenesis disorders are accompanied by the appearance and the increase of concentrations of neutralizing antibodies which interact with various angiogenic factors and interfere with their physiologic activity

The inventors have disclosed that elimination of autoantibodies against angiogenic factors from a subject's body and neutralization as well as removal or elimination from a subject's body of the

immunocompetent cells taking part in the production of neutralizing autoantibodies results in restoration of normal angiogenesis, the elimination of pathologic symptoms and complete cure

The inventors have found out that the production of immunological tolerance to angiogenic factors against which at the pre-clinical stage a subject's body starts to produce autoantibodies is an efficient means for prevention and treatment of primary stages of autoimmune diseases related to angiogenesis disorders The use of various methods of producing the immunological tolerance results in a sharp decrease of the content of neutralizing autoantibodies in a subject's body At the pre-clinical stages of disease the production of immunological tolerance has a prevention effect, while at the primary stages of the disease it has a treatment effect i e helps to restore normal angiogenesis, to eliminate pathologic symptoms and to completely cure the disease

The inventors have discovered a so far unknown phenomenon consisting in the appearance and sharp decrease of the concentration of autoantibodies against angiogenic factors in blood of the patients suffering from diseases with angiogenesis disorders This phenomenon is proposed as the basis of a pathogenesis research in relation to such diseases It is the aim of the present invention to provide methods and means for fighting a vast number of diseases with autoimmune angiogenesis disorders and also to provide a new method and a new test-system of monitoring of angiogenesis disorders in a subject's body as a result of an increased content of autoantibodies against angiogenic factors in said body

A protein complex comprising an antigen and an attached thereto effector portion with immunomodulating activity makes it possible to achieve the above aim due to said antigen presenting an angiogenic factor

An immunomodulating portion of said complex could be both an immunostimulator and an immunosuppressor

An angiogenic factor of said complex is made so that it interacts with immunocompetent cells belonging to the specific clones of B-lymphocytes and plasma cells that are the derivatives thereof and also with T- lymphocytes

An antigenic portion of the above complex could be made in the form of an artificial structure copying or resembling the antigenic determinants of natural angiogenic factors

An immunosuppressor of said protein complex presents monoclonal antibodies against surface receptors of immunocompetent cells, or a toxin selected from the group including toxins of bacterial, plant animal, fungal or virus origin, or a medication of cytotoxic action, or a combination of monoclonal antibodies toxins and medications

The monoclonal antibodies can be selected from the groups including anti CD3, anti CD4, anti CD19 antιCD20, anti CD21 , anti CD22, anti CD23, anti CD40 and anti CD80

A toxin of a protein complex immunosuppressor is preferably a bacterial toxin A medication of a protein complex immunosuppressor can be specifically selected from groups related to kalicheamycin, thiotepa, taxan, nitrous ypeπte, ethylene imine derivatives, alkyl sulfonate tπazene, taxol, kamptotecin, azaseπne, tamoxifen, dolastine/auπstatine, haemiasterlin and maitanzmod ethoposide, procarbazine, carmustine, phenyl butyrate, brιostatιne-1 and leucovorin, enzymes, e g , protease, lipase, phosphohpase, RNAse, DNAse, vinca alkaloids, e g , vincristine, vinblastine, antibiotics, e g , actinomycin D, mitomycin C, pyramycin, chloramphenicol, cyclosporin, tacrolymus, micophenolate, motephyl, bleomycin, a group of dien antibiotics, e g , dinemycin A, esperamycin, a group of anthracyclines, e g , daunorubicin, doxorubicin, idarubicin epirubicin, 2- pirrolinodoxorubicin, morpholinodoxorubicin and cyanomorpholinodoxorubicin, a group of anthramycins, e g , porothramycin B and pιrrolo-[2, 1 -c][1 ,4]benzodιazepιne4 purine analogues, e g , azathiopnne, mercaptopuπne, chlorambucil, pyπmidine analogues, e g , cytozine arabinozide, immunusuppressor analogues, e g , azathiopnne, cyclospoπne, penicilamine, antimalarial medications, e g , chloroquine, hydroxychloroquine, antimetabolites, e g , methotrexate,

alkylating agents, e g , cyclophosphamide,

Methylhydrazine derivatives, platinum coordinating compositons, e g , cisplatin,

Cortisol derivatives, e g , methylprednisolone, prednisone, dexamethasone, nonsteroid antiinflammatory medications, e g , COX-2 inhibitors, salicylates para-aminophenol derivatives, indole and indene derivatives, heteroarylacetic acids, arylpropionic acids, anthranihc acids enolic acids, alkanons, 2,3-dιphenyltetrahydrofuran, urea derivatives, e g , nitrosourea, folic acid analogues, e g , methotrexate, aminopterin, and also the combinations of substances of several chemical groups, groups of origin and groups according to the mechanism of action

An angiogenic factor of a protein complex could be both a pro-angiogenic factor and an anti-angiogenic factor

Pro-angiogenic factors of a protein complex are selected from the group including the groups of fibroblast growth factors, vascular endothelial growth factors, colony stimulating factors, interleukins, platelet- derived growth factors, angiopoetms, tumor-necrosis factors, matrix metalloproteinases and, in particular from transforming growth factor beta 1 , intercellular adhesion molecule 1 , hepatocyte growth factor, nerve growth factor, connective tissue growth factor, tenascin-R, prolactin, growth hormone, placental lactogen, insulin-like growth factor-1 , thymidine-phosphorylase The above aim is achieved due to use of the protein complexes described above for prevention and treatment of diseases with angiogenesis disorders

A protein complex could be in the form of an immunoglobulin-like structure, in which fragment of variable domen is identical to fragment of an angiogenic factor, while a constant domen of an immunoglobulin-like structure acts as immunosuppressor The donor immunoglobulins or the ones obtained by cell cultuπng actually present multi-functional protein complexes having a structure comprisng at least two domens one of said domen being a variable immunoglobulin domen for the case of donor anti-idiotypic antibodies that reacts with the discovered pathologic autoantibodies against angiogenic factors is actually a structural fragment of said angiogenic factor The second domen of said structure that is an immunoglobulin constant domen when introduced into a subject's body performs a cascade of biological processes leading to the suppression of pathologic autoantibody products by immunocompetent cells i e this essentially being an effector portion of said protein complex Taking into account the availability of the cell cultuπng methods for a discrete production of the variable and constant domens of immunoglobulins with their subsequent combining into a common protein complex the anti-idiotypic antibodies could be related to the protein complexes filed according to the present invention description A second variant of a protein complex comprising an antigen and an effector portion bound thereto to provide the specific influence on a target cell makes it possible to achieve the above aim due to said antigen presenting an angiogenic factor

An anti-angiogenic factor of a protein complex in the above variant could be selected from the group comprising members of families like inteferons, tissue inhibitors of metalloproteinases fibroblast growth factors, endothelial growth factors, vascular endothelial growth factors, plasminogen, collagen fibronectin, prolactin, growth hormone, placental lactogen and thrombospondin and fragments thereof or it could be in the form of an artificial structure copying or resembling the antigen determinants of natural anti-angiogenic factors

An effector portion of a protein complex in the above variant presents a toxin selected from the group including the toxins of bacterial, plant, animal, fungal or virus origin, cytotoxic antibiotics, ions of heavy metals in various forms, photosensitazers, radionuclides or the combinations thereof

A third variant of a protein complex comprising an antigen immobilized on an effector portion in the form of a polymer carrier makes it possible to achieve the above aim due to said antigen comprising an angiogenic factor An angiogenic factor according to this variant could be either a pro- or anti-angiogenic factor

Pro-angiogenic factors in the above variant of a protein complex could be selected from a group including group comprising members of families like fibroblast growth factors, vascular endothelial growth factors, colony stimulating factors, interleukins, platelet-derived growth factors, angiopoietins, tumor-necrosis

factors, matrix metalloproteinases and, in particular, transforming growth factor beta 1 , intercellular adhesion molecule, hepatocyte growth factor, nerve growth factor, connective tissue growth factor tenascin-R, prolactin, growth hormone, placental lactogen, insulin-like growth factor 1 , thymidine- phosphorylase

5 Anti-angeoginic factors in the protein complex of present invention could be selected from the members of families like inteferons, tissue inhibitors of metalloproteinases, fibroblast growth factors placental endothelial growth factors, vascular endothelial growth factors, plasminogens, collagens, fibronectins, prolactins, growth hormones, placental lactogens, thrombospondins and fragments thereof

Angiogenic factors in the above variant of a protein complex could be made in the form of an artificial I O structure coping, resembling, presenting the antigenic determinants of natural pro- or anti-angiogenic factors

The polymer carriers in the above variant of a protein complex could be selected from the group including polymer particles, both magnetic and non-magnetic ones, continuous and hollow fibers, single-layer or multi-layer membranes

15 The above aim is achieved due to use of the second and the third variants of protein complexes in the treatment of diseases with angiogenesis disorders

Filed is a method for prevention and treatment of diseases with angiogenesis disorders consisting in the administration of an effective dose of a pharmaceutically active substance in the form of a protein complex comprising an antigen portion and an immunomodulating portion, wherein the above-set aim is 0 achieved due to a) pre-defining the content of an angiogenic factor/factors in the subject's biologic samples and determining the factors which concentration exceed the normal level , b) defining a kind/kinds of angiogenic factors against which the patient's body produces elevated levels of autoantibodies, 5 c) producing a first variant of a protein complex (according to claims from 1 to 13) containing the identified angiogenic factors as an antigenic portion and an immunomodulating portion and d) administration of said protein complex for modulating of an immune response to the angiogenic factor in question

Said protein complex is administered intravenously, intraarterial, intramusculary, intraosseously 0 intrafascialy, intralumbaly, hypodermally, intraorganically and also to the surface of mucous membranes e g , intraconjunctival, intranasal, intratracheal, intrabronchial, intraalveolar, intravaginal, intrauteral, per os, per rectum

The administration of said protein complex is performed regularly with the periodicity from 1 to 48 hours, preferably with the periodicity of 24 hours 5 A filed method for prevention and treatment of diseases with angiogenesis disorders is used for the following groups of diseases including cardiovascular diseases, e g , embolism, thrombosis, stroke, infarction arteriosclerosis aneurism, apoplexy, stenocardia, myocardial angiogenesis insufficiency, angiostenosis teleangiectasia, restenosis, 0 respiratory diseases, e g , chronic pneumonia, pulmonary fibrosis, pulmonary emphysema bronchial asthma, urinary tract diseases, e g , glomerulonephritis, cystitis, kidney bladder stone kidney stone gastrointestinal truct diseases, e g , gastritis, duodenitis, pancreatitis, hepatitis, chronic appendicitis, ulcerative colitis, celiac disease, Crohn's disease, intestinal absorption 5 reproductive pathology, e g , endometriosis, frequent fetal loss, abnormal placentation preeclampsia, intrauterine growth contraction, nervous system diseases, e g , multiple sclerosis, chronic fatigue syndrome, encephalitis, myasthenia gravis, mental health disorders, e g , Alzheimer's disease, schizophrenia, mania and depression 0 psychosis, musculoskeletal system diseases, e g , osteoporosis, osteochondrosis, Paget ^' s disease rheumatoid arthritis, osteoarthritis, osteoarthrosis, endocrine disorders, e g , hyperparathyroidism, Grave's disease, Hashimoto's disease, Addison ^' s disease, Cushing's syndrome, diabetes mellitus,

55 connective tissue diseases, e g , pathologic proliferation of connective tissue in organs, in particular, pulmonary fibrosis, cystic fibrosis, dermatofibrosis, hepatic fibrosis hepatic cirrhosis, urethrofibrosis, renal fibrosis, cardiac fibrosis, infantile endocardial fibrosis, pancreafibrosis collagenoses,

skin diseases, e g , atopic dermatitis, derma cornification disorders leukoplakia, lichen planus psoriasis, wound granulation, hypertrophic scars, scleroderma, pemphigus, cat scratch disease, neoplastic diseases, e g , progression of non-malignant growths, malignant growths under remission and acute conditions, tumour angiogenesis, tumour metastases, transplantation diseases, e g , inconsistent blood transfusion, graft rejection reaction, "graft- versus-host" disease, diseases related to sense organs, e g , diabetic retinopathy, macular degeneration, myopia, corneal transplantation, rubeothic glaucoma,, infectious diseases, e g , virus, bacterial, fungal infections and the complications thereof

An angeoginic factor of a protein complex used in the present invention and treatment method is produced so that it interacts with immunocompetent cells belonging to the specific clones of B- lymphocytes and to the plasma cells that are derivatives thereof, and also to T-lymphocytes

Another method of treatment diseases with angiogenesis disorders consisting in the administration of an effective dose of a pharmaceutically active substance in the form of a protein complex containing an antigen portion and an effector portion makes it possible to achieve the above-set aim due to pre-defining a kind/kinds of angiogenic factors against which a subject's body overgenerates autoantibodies producing a protein complex containing an identified angiogenic factor and using said protein complex to completely remove from said subject's body said autoantibodies and/or the cells-producers thereof A protein complex according to this variant of a treatment method is produced so that its effector portion is intended to specifically influence the target cell, while said influence is provided by introducing said complex into a subject's body

Said protein complex is injected intravenous, intraarterial, intramascular, intraosseous hypodermic, intraorganic, intrafascial, intralumbar, per os, per rectum A filed method for treatment of diseases with angiogenesis disorders is used for the following groups of diseases including cardiovascular diseases, respiratory diseases, urinary tract diseases, gastrointestinal truct diseases, reproductive pathology, nervous system diseases, mental health disorders, musculoskeletal system diseases, endocrine disorders, connective tissue diseases, skin diseases, diseases related to sense organs neoplastic diseases, transplantation diseases, infectious diseases and the complications thereof

A protein complex according to a method of treatment described below is produced so that its effector portion presents a polymer carrier with an antigen immobilized thereon, while its influence is effected by means of bio-specific sorption of a subject's biologic fluid An angiogenic factor according to this variant of a treatment method could be either a pro- or aπti- angiogenic factor

A treatment method according to the variant in question could be performed either simultaneously or after the sorption process is completed

Blood, blood plasma, lymph, cerebrospinal fluid and/or bone marrow could be used as a biologic fluid A third variant of a treatment method for diseases with angiogenesis disorders in animals consisting in effecting the pathologic autoantibodies present in the animal's body or the cell-producers of said autoantibodies by a medication of intravenous immunoglobulin, and further consisting in the selection of intravenous immunoglobulins reacting with the autoantibodies characteristic of a specific disease and the

administration of the selected immunoglobulins to the animal makes it possible to achieve the aim set by the invention due to said pathologic autoantibodies being the antibodies against the angiogenic factors while the presence of said pathologic autoantibodies and/or of respective immunocompetent cells is defined directly in a biologic sample of said animal, and the anti-idiotypic antibodies that are used as immunoglobulins are taken from a donor/donors of the same biologic type so that their anti-idiotypic antibodies would most efficiently react with the pathologic autoantibodies and the donor anti-idiotypic antibodies received from said donor/donors are selectively concentrated

The presence of autoantibodies could be defined by analyzing the interaction of a wide range of antigens immobilized on carries with a sample/samples of the animal's biologic samples with further analysis of the quantity and quality of said antoantibodies having bound to said antigens

The antigens immobilized on carriers are mainly related to angiogenic factors

The structures that are immunoidentical to antigens could be used as antigens immobilized on hard carriers

Blood samples and its constituent parts, cerebrospinal fluid, saliva, phlegm mucus could be used as the animal's biologic samples

The analysis of the quantity and quality of autoantibodiesis performed by the following methods enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) immunofluorescence assay electrophoresis, chromatography, mass spectrometry, light microscopy atomic-force microscopy plasmon resonance or the combinations thereof The choice of a donor/donors is performed on the basis of maximum inhibition by the donor anti-idiotypic antibodies of binding and interaction of the autoantibodies with the antigen

A selective concentration of donor anti-idiotypic antibodies could be provided by means of an affinity sorption on the animal's pathologic autoantibodies immobilized on a hard carrier while said immobilization of said pathologic autoantibodies on said hard carrier could be performed by a covalent or non-covalent method either immediately after the sorption on an antigen is completed or after the de- sorption from an antigen

A selective concentration of the donor anti-idiotypic antibodies could also be performed by means of separation of immunocompetent cells from the donor biologic samples, generation of hybπdoma and its further cultivation with separation of the donor anti-idiotypic antibodies The hybπdoma cultivation is performed either outside the animal's body or actually therein

A selective concentration of the donor anti-idiotypic antibodies could also be performed by means of genetic engineering and introducing the genetic structure into the animal's cells

The efficiency of binding the donor immunoglobulins with the pathologic autoantibodies or receptors on the surface of immunocompetent cells is improved by their chemical modification which is preferably provided either by means of binding the additional chemical groups to an antigen molecule or by binding the antibodies to a bio-compatible soluble polymer or by removing a portion of an antibody molecule

The decrease of the content of pathologic autoantibodies in the subject's blood or cerebrospinal fluid is performed by means of an immunosuppressive therapy or by means of selective sorption of autoantibodies from blood or from cerebrospinal fluid By animals there is meant fish, amphibias, reptiles, birds and mammals

To accomplish the above-described methods of treatment necessitating the determination of the presence and of the content of angiogenic factors, a method of investigation of the origins of a malignant process in a subject's body was designed, said method consisting in immobilizing one or a number of antigens, making a biologic preparation, providing the interaction of said preparation with said antigens and defining the presence of autoantibodies against said antigens in said preparation wherein the angiogenic factors are used as antigens

The angiogenic factors with a normal or modified due to pathology structure are selected from the group family members like plasminogen and its fragments, e g , angiostatin, collagen-XVIII and its fragments, e g , endostatin, a group of fibroblast growth factors (FGF), placental growth factor of endothelial cells and vascular growth factor of endothelial cells (vascular permeability factor), fibronectin, prolactin, 5 thrombospondin-l and fragments thereof

The artificial structures copying or resembling the antigenic determinants of natural angiogenic factors could be generated for use as an antigen

A biologic preparation could be selected from the group including tissue samplings, blood and its constituents, lymph, cerebrospinal fluid, urea, saliva, lacrimal fluid

I O To accomplish the above-described investigating method a test-system to investigate the origins of pathologic angiogenesis in a subject's body was designed, said system comprising at least one carrier for immobilzation of one or a number of antigens, a biologic preparation and an element for defining the presence of autoantibodies against said antigens in said preparation, wherein the angiogenic factors are used as antigens

15 A test-system element for defining the presence in said preparation of autoantibodies against said antigens is preferably made in the form of revealing reagents or in the form of a direct detection device

Detailed Description of the Invention

Implementation of any of the variants according to claims from 27 to 70 starts from defining the specific angiogenic factors against which the antibodies are produced or from defining the levels that exceed the 0 normal values by using a method of research filed according to claims from 71 to 76 and a test system filed according to claims from 77 to 81

The implementation examples of a test system filed are illustrated in Fig 1 and 2, while the concept of a research method is disclosed in detail in the following non-limiting examples

Fig 1 illustrates an implementation example of a test system in the form of a flexible immune kit (Titertek) 5 with one well of said kit shown as an example Molecules 1 of antigen i e angiogenic factor immobilized on a well 2 of said kit are shown virtually Molecules of just one angiogenic factor could be immobilized throughout the whole of the kit or of various angiogenic factors in the groups of neighboring wells, or of one factor per well

Molecules 3 of autoantibodies contained in a biologic preparation and molecules of secondary (revealing) 0 autoantibodies 4 having a marker are also shown virtually

Fig 2 illustrates an implementation example of a test-system in the form of a set of carrier particles 5 Molecules 1 of an antigen i e angiogenic factor immobilized on particles 5 are shown virtually Molecules of just one angiogenic factor could be immobilized on all particles or of various angiogenic factors on different particles or of several kinds of angiogenic factors on just one particle Molecules 3 of 5 autoantibodies contained in a biologic preparation are also shown virtually Numeral 6 designates antibodies that are non-sorbing on a specific antigen Numerals 7 and 8 designate respectively a tip and a magnetic rod of a magnetic manipulator A reaction is performed in a laboratory vessel 9

Example 1

The wells of a flexible immune kit (Titertek) (See Fig 1 ) were saturated with endostatin (See Fig 1a) and 0 incubated for 30 mm with the samples of the subjects' urea (See Fig 1 b) After thorough rinsing from the urea residues the kits were incubated for 3 hrs at 37° with dissolved in the buffer ^{1 5} l-labelled goat ^' s polyclonal antibodies against human immunoglobulins in dilution 1 50000 (1 x10 ⁶ c/min" ml, 50 μl of solution per well) (See Fig 1 c) The kits were 4-5 times rinsed with distilled water After drying during the night at a room temperature the wells were cut out and radioactivity was measured on a Gamma Trac 5 counter To determine the total amount of ¹²⁵ I- antibodies an aliquot having the volume of 50 μl was calculated from each dilution

Should a radioactivity level exceed at least by 50% the level of control samples, the subject was diagnosed to have angiogenesis disorders related, in particular, to malignant growth

Since in the case described an increased content of autoantibodies to endostatin was discovered, it was endostatin that was further used for generating a protein complex in accordance with the present invention

Further use of a prepared protein complex to correct the identified disorders was provided in accordance with the methods filed

Example 2

Preparation of Anqiostatin Complex with Gelonin

Angiostatin (Sigma) (50mg) dissolved in 10 ml of 0,1 M sodium phosphate buffer was mixed with 0 1 ml of 2%gelonιn and then added to the mixture of 0,5 ml of 2% activated copolymer of polyvinyl pyrrolidone and acrolein A conjugation reaction was performed at a room temperature with constant stirring for 2 hrs Separation of low-molecular products was made by dialysis against 0,9% solution of NaCI A protein complex thus produced was used for intravenous injection as a component of a pharmaceutical composition

Generation of Pharmaceutical Composition A solution of a complex produced as described in Example 2 was added with up to 10% of a glucose solution, up to 500 units of heparin and further sterilized by filtration through disposable membrane filters Amicon A sterile solution in sealed bottles was stored at +4 ⁰ C for maximum 5 days until the actual use

The above protein complex could be used in the treatment of diseases with angiogenesis disorders according to claims from 44 to 47 A protein complex thus produced was used for implementing one of the embodiments of the invention filed according to claims from 27 to 43, wherein should an increased content of neutralizing autoantibodies against some angiogenic factor or a group of factors be discovered in a subject's body, a protein complex is prepared and applied, said protein complex comprising the above factor (factors) and means causing the production of immunological tolerance against the above factor (factors) with an immunosuppressive agent used as an immunomodulating portion To achieve the above-set aim it is feasible to generate in a subject's body the state of immunological unresponsiveness (a common name of this state is immunological tolerance) that is using non-toxic methods and substances to selectively suppress the synthesis of autoantibodies against inherent natural protein as in the case described against anti-angiogenic factors or a number of such factors The production of immunological tolerance as a means of suppressing an autoimmune process is widely popular A distinguishing feature of a method filed is that it is proposed to use this customary practice in a new way i e in the prevention and treatment of diseases accompanied by the production of anti-angiogenic antibodies, in particular, malignant ones by the subject's immune system It is proposed to use for this purpose the most optimal combinations of the means of generating the immunological tolerance that have already been successfully used in the experiments for fighting various autoimmune diseases like e g autoimmune encephalomyelitis, diabetes with autoimmune components etc

Immunological tolerance could be achieved either by eliminating the specific clones or by introducing the latter into the state of anergy and not only at the early stages of ontogeny The tolerance depends on the appearance of new kinds of activated lymphocytes and it does not depend on the elimination of the old active ones

According to another embodiment of a method filed, should an increased content of some angiogenic factor/factors be discovered in a subject's body, a protein complex is prepared and applied said protein complex comprising the above factors and means causing the production of neutralizing autoantibodies wherein an immunostimulating agent is used as an immunomodulating portion Example 3

Preparation of Anqiostatin Complex with polymer carπer(Nanopartιcles)

Superparamagnetic nanopaticles Fersar of carbonyl iron (having the diameter from 10 to 50 nm) (Saratov, Russia) covered with polyvinyl alcohol were incubated for 1 hr at a room temperature in the solution of 0, 1 % glutaraldehyde in 0,1 n HCI After thorough rinsing from the acid residues and unbounded

aldehyde the particles were added to the solution of angiostatin (Sigma) (100 μg/ml) in 0,02 M sodium phosphate buffer, pH 7,4 and incubated at constant stirring for 12 hrs at +4 ⁰ C A complex with magnetic particles after rinsing from unbounded angiostatin was used for extracorporeal bio-specific hemosorption according to a method as in claims from 48 to 51

5 After an extracorporeal hemosorption an intravenous injection course of a pharmaceutical composition produced as in the Example 3 could be administered

A method filed according to claims from 52 to 70 is implemented in the following way

Firstly the presence of pathologic autoantibodies and/or immunocompetent cells producing said autoantibodies is defined as described e g in the example 1

I O Then the biologic samples of the donors are analyzed, said donors being of the same kind that the animal in question The analysis consists in defining the inhibiting influence of the donor anti-idyotypic antibodies on the interaction of the animal's pathologic autoantibodies with angiogenic factors The choice of a donor/donors is performed on the basis of maximum inhibition by the donor anti-idyotypic antibodies of binding and interaction of the autoantibodies with an antigen The angiogenic factors according to this

15 variant of a treatment method could be either pro- or anti-angiogenic factors

Only anti-idyotypic antibodies could be selected from a biologic sample/samples of the donors chosen

This process could be performed in three different ways

I The concentration of antι-ιdyotypιc antibodies is provided by means of preparative affinity sorption on mammal pathologic autoantibodies immobilized on a carrier 0 The immobilization of pathologic autoantibodies is performed on angiogenic factor already immobilized on a carrier Immobilization of both angiogenic factor and pathologic autoantibodies is performed either by covalent or y non-covalent binding method, however, the combination of the above methods is possible Synthetic ligands could be immobilized on an affinity column instead of autoantibodies with anti-idyotypic antibodies exhibiting an enhanced binding activity in relation to said ligands The substances included into 5 the following groups could be used as such ligands chemical dyes, in particular, dinitrophenol, procion blue and derivatives thereof amino acids, in particular, lysine, arginine, aspartic acid, tyrosine, phenylalanine tryptophan cysteine in D- and L-forms, in the form of monomers and polymers, linear and branched structures and derivatives thereof, 0 - synthetic peptides and derivatives thereof,

- lectins, in particular, lectins from Phaseolus vulgaris, Canavalia ensiformis, πcin, abπn phytohemagglutinin, , clearance lectins,

- nucleic acids, in particular, aptamers and derivatives thereof, oligo or polysaccharides, in particular, heparin, heparan sulpfate dermatan sulphate 5 hyaluronic acid, chitin and derivatives thereof, lipids, in particular, phospholipids and derivatives thereof

The combinations of the above substances are also possible A number of ligands to be used for concentration of anti-idyotypic antibodies is not limited by those listed above

II A selective concentration of anti-idyotypic antibodies could also be performed by means of separating 0 immunocompetent cells producing said anti-idyotypic antibodies from the donors' biologic samples generating a hybridoma and cultivating said hybπdoma with further separation of anti-idyotypic antibodies

In case of antigenic compatibility the cultivation of hybridoma is performed actually inside the animal s body

In other cases the cultivation of hybridoma is performed outside the animal s body The cultivation is performed either in the body of an experimental animal or in a cell cultivation reactor

III It is possible to perform a selective concentration of anti-idyotypic antibodies by means of engineering a genetic structure providing a bio-synthesis of a structure having the features of anti-idyotypic antibody and introducing said structure into the animal's cells

Should it be the case, the anti-idyotypic antibodies are actually produced inside the animal's body by its own cells

Anti-idyotypic antibodies could be subjected to chemical modification after their separation with the aim of improving the efficiency of binding with pathologic autoantibodies or receptors on the surface of immunocompetent cells Said modification could be performed as follows binding the additional chemical groups to an antigen molecule, binding the antibodies to a biocompatible soluble polymer, removing a potion of an antigen molecule

The chemical groups bound thereto could belong to various classes, in particular, acyl, ether sulphohydryl, hydroxy/, sulphate, sulphite, carbohydrate, peptide, nucleotide The combinations of chemical groups bound thereto are possible A number of chemical groups bound thereto to increase the efficiency of binding to pathologic autoantibodies or receptors on the surface of immunocompetent cells is not limited by those listed above

Bio-compatible soluble polymers to which antibodies would be bound to could belong to various chemical groups, in particular, starch, inulin, hyaluronic acid, chitin, polylactate, polyglycolate, polyvinyl pyrrolidone copolymer of polyvinyl pyrrolidone and acrolein, polyethylene glycol, polyvinyl alcohol, dextran, and modifications thereof

Binding of anti-idyotypic antibodies to polymers could be both covalent and non-covalent one A number of bio-compatible soluble polymers to which the anti-idyotypic antibodies would be bound to increase the efficiency of binding to pathologic autoantibodies or receptors on the surface of immunocompetent cells is not limited by those listed above

The application of anti-idyotypic antibodies is provided on the background of decreasing the content of pathologic autoantibodies in subject's blood or cerebrospinal fluid The decrease of the content of pathologic autoantibodies is provided either by means of an immunosuppressive therapy or by means of selective sorption of autoantibodies from blood or cerebrospinal fluid

Thus, a method filed provides for a specific treatment of diseases with angiogenesis disorders The indications for application of specific anti-idyotypic antibodies are extending, and, moreover, the specific anti-idyotypic antibodies thus produced are characterized by increased efficiency and improved tolerance on the part of the patient