Treating diseases related to insulin resistance
SUBSTANCE: invention refers to medicine, namely to treating diseases related to insulin resistance. A method of treating involves administering an effective amount of IL-17A and/or IL-17F antagonist, wherein the above agonist represents an antibody or its antigen-binding fragment. The group of inventions also refers to a pharmaceutical composition containing the IL-17A and/or IL-17F antagonist with a pharmaceutically acceptable additive added, and to a set containing the above agonist, and an administration instruction.
EFFECT: using the given group of inventions enables reducing the insulin resistance in an individual by administering pro-inflammatory factors IL-17A and/or IL-17F antagonists.
20 cl, 3 ex, 14 dwg
The technical FIELD
The invention relates to the treatment of disorders associated with insulin resistance. In particular, the present invention relates to the treatment of disorders associated with insulin resistance, the introduction of antagonists of IL-17, such as IL-17A and/or IL-17F, such as antibodies against IL-17A and/or IL-17F and/or IL-17Rc or antibody fragments.
The LEVEL of TECHNOLOGY
The family of IL-17
Interleukin-17A (IL-17A) is produced by T cells Pro-inflammatory molecule that stimulates epithelial, endothelial cells and fibroblasts to the formation of other excitatory (inflammatory) cytokines and chemokines, including IL-6, IL-8, G-CSF and MCP-1 (see Yao, Z. et al., J. Immunol., 122(12):5483-5486 (1995); Yao, Z. et al., Immunity, 3(6):811-821 (1995); Fossiez, F., et al., J. Exp. Med., 183(6):2593-2603 (1996); Kennedy, J., et al., J. Interferon Cytokine Res., 16(8):611-7 (1996); Cai, X. Y., et al., Immunol. Lett, 62(l):51-8 (1998); Jovanovic, D. V., et al., J. Immunol., 160(7):3513-21 (1998); Laan, M., et al., J. Immunol., 162(4):2347-52 (1999); Linden, A., et al., Eur Respir J, 15(5):973-7 (2000); and Aggarwal, S. and Gurney, A. L., J Leukoc Biol. 71(1):1-8 (2002)). IL-17 also has a synergistic effect with other cytokines, including TNF-α and IL-1β, further induction of the expression of chemokines (Chabaud, M., et al., J. Immunol. 161(1):409-14 (1998)). IL-17A has pleiotropic biological activities against various types of cells. IL-17A also able to induce the expression on the cell surface ICAM-1, the proliferation of T-cells and the growth and differentiation of predestin the Cove cells CD34 +human neutrophils. Also shown the involvement of IL-17A in bone metabolism, and it is assumed that it plays an important role in pathological conditions characterized by the presence of activated T-cells and production of TNF-α, such as rheumatoid arthritis and loosening of bone implants (Van Bezooijen et al., J. Bone Miner. Res., 14:1513-1521 (1999)). Found that activated T cells from synovial tissue obtained from patients with rheumatoid arthritis, secrete higher amounts of IL-17A than cells obtained from healthy individuals or patients with osteoarthritis (Chabaud et al., Arthritis Rheum., 42:963-970 (1999)). It has been suggested that this proinflammatory cytokine actively contributes to the inflammation of the synovial bags in rheumatoid arthritis. In addition to its proinflammatory role of IL-17A, seems to be making a contribution to the pathology of rheumatoid arthritis using another mechanism. For example, it was shown that IL-17A induces the expression of mRNA of factor differentiation of osteoclasts (ODF) in osteoblasts (Kotake et al., J. Clin. Invest., 103:1345-1352 (1999)). ODF stimulates the differentiation of precursor cells into osteoclasts, the cells involved in bone resorption. Since the level of IL-17A in the synovial fluid of patients with rheumatoid arthritis significantly increased, suppose that induced by IL-17A formation of osteoclasts plays kriticheskie the role in bone resorption in rheumatoid arthritis. It is also believed that IL-17A plays a key role in other autoimmune disorders such as multiple sclerosis (Matusevicius et al., Mult. Scler., 5:101-104 (1999); Kurasawa, K., et al., Arthritis Rheu 43(11):2455-63 (2000)) and psoriasis (Teunissen, M. B., et al., J Invest Dermatol 111(4):645-9 (1998); Albanesi, C., et al., J Invest Dermatol 115(1):81-7 (2000); and Homey, B., et al., J. Immunol. 164(12):6621-32 (2000)).
In addition, it was shown that IL-17A through intracellular signaling stimulates the flow in the cell Ca2+and the decrease in the concentration campneorg.in human macrophages (Jovanovic et al., J. Immunol., 160:3513 (1998)). Fibroblasts treated with IL-17A, induce the activation of NFkB (Yao et al., Immunity, 3:811 (1995), Jovanovic et al., above), while the treated macrophages activate NF-kB and mitogen-activated protein kinase (Shalom-'barek et al., J. Biol. Chem., 273:27467 (1998)). In addition, IL-17A also has sequence similarity with cytogenetically factor-7 mammals that is involved in the growth of bones and cartilage. Other proteins with which the polypeptides of IL-17A have sequence similarity, are embryonic related interleukin factor (EDIRF) and interleukin-20.
In accordance with a wide range of effects of IL-17A found that the receptors of IL-17A on the cell surface is widely expressed in many tissues and cell types (Yao et al., Cytokine, 2:794 (1997)). Although the amino acid sequence of the receptor for IL-17A human IL-R) (866 amino acid is) predicts a protein with a single transmembrane domain and a long, containing 525 amino acid intracellular domain sequence of the receptor is unique and has no similarities with any other receptors of the family of receptors cytokines/growth factors. This fact and the lack of affinity of the IL-17A with other known proteins indicate that IL-17A and its receptor may be part of a new family of signaling proteins and receptors. It is shown that the activity of IL-17A mediated through its binding to a unique receptor on the cell surface (denoted in this document as IL-17R person), while previous studies have shown that exposure of T-cells with a soluble form of the polypeptide receptor IL-17A inhibited the proliferation of T-cells and the production of IL-2, induced by phytohemagglutinin, concanavalin and monoclonal antibodies against TCR (Yao et al., J. Immunol., 155:5483-5486 (1995)). In fact, there is considerable interest in the identification and characterization of new polypeptides having homology to known receptors of cytokines, in particular receptors IL-17A.
Currently interleukin 17A is the prototype member of a new family of cytokines. Large-scale sequencing of genomes of humans and other vertebrates have revealed the presence of additional genes encoding proteins that have an explicit relationship with IL-17A, setting, therefore clicks the zoom, a new family of cytokines. There are at least 6 members of the family of IL-17 in humans and mice, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, as well as new receptors IL-17RH1, IL-17RH2, IL-17RH3 and IL-17RH4 (see WO01/46420, published June 28, 2001). It is shown that one such member of the IL-17 (designated IL-17F) binds to the receptor of IL-17 man (IL-17R) (Yao et al., Cytokine, 9(11):794-800 (1997)). Initial characterization indicates that, like IL-17A, some of these newly discovered molecules have the ability to modulate immune function. A strong stimulating effect found for some of these factors, and to identify associations with serious human diseases indicate that these proteins may play a significant role in inflammatory processes and may provide opportunities for therapeutic intervention.
The gene encoding IL-17F human, is located next to the gene of IL-17A (Hymowitz, S. G., et al., Embo J, 20(19):5332-41 (2001)). IL-17A and IL-17F have about 44% identity on amino acid sequence, while the other members of the family of IL-17 have a more limited amino acid sequence identity in 15-27%, which indicates the formation of IL-17A and IL-17F separate subgroups within the family of IL-17 (Starnes, T., et al., J Immunol. 167(8):4137-40 (2001); Aggarwal, S. and Gurney, A. L., J. Leukoc Biol, 71(1):1-8 (2002)). Apparently, IL-17F has biological activity that is similar to that of IL-17A, and is capable of stimulating the production of IL-6, IL-8 and G-CSF in a wide variety of cells. Like IL-17A, it is able to induce the release of cartilage matrix and to inhibit the synthesis of new cartilage matrix (see U.S. patent 2002-0177188-A1, published on 28 November 2002). Thus, as IL-17A, IL-17F could potentially participate in the pathology of inflammatory disorders. It was reported that both IL-17A and IL-17F induced in T-cells under the action of interleukin 23 (IL-23) (Aggarwal, S., et al., J. Biol. Chem., 278(3):1910-4 (2003)). In particular, both IL-17A and IL-17F have been identified as factors in the progression and pathology of various inflammatory and autoimmune diseases in humans and in models of human diseases in mice. Indeed, IL-17A and, to a lesser extent, IL-17F have been identified as effector cytokines that trigger inflammatory responses and thus participate in some automobiletechnik (autoimmune) diseases, including multiple sclerosis (MS), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). This line was designated as Th17and the number of these cells clearly correlates with the progression and severity of the disease in models of human autoimmune disease in mice. Although the involvement of IL-17A and IL-17F in inflammatory diseases seems obvious (see, for example, Kolls, J. K., A. Linden. Immunity 21:467-476 (2004)), tile and target these cytokines were not identified, in particular, due to the fact that was identified in the receptor for IL-17F. IL-17A has an affinity to IL-17RA. Amino acid sequence of IL-17RA person has in NCBI GenBank under the access number NP_055154.3. To date, at least four additional receptor family, IL-17R were identified on the basis of sequence homology in relation to IL-17RA (IL-17Rh1, IL-17Rc, IL-17RD, and IL-17RE), and among them, as it has been shown that IL-17Rc physically interacts with the IL-17RA, which indicates that he may be a functional component of the complex of IL-17R (Toy, D. et al., J. Immunol. 177:36-39 (2006)). It was recently reported that IL-17Rc is a receptor for both IL-17A and IL-17F (Presnell et al., J. Immunol. 179(8):5462-73 (2007)).
Inflammation and obesity
An important new contribution to our understanding of obesity is the emergence of the concept that inflammation and diabetes are characterized by chronic weak inflammation. The basis for this view is that obesity appear elevated circulating concentrations of some markers of inflammation such as proinflammatory cytokines and acute phase proteins; such markers include IL-6, system TNFα, C-reactive protein (CRP) and haptoglobin. However, it is not clear involvement in terms of the area of the inflammation, whether it is systemic or local.
Insulin resistance, defined as bol is e weak, than expected biological response to a given dose of insulin, is a common correlate of obesity. Indeed, I believe that many of the pathological consequences of obesity include insulin resistance. These include hypertension, hyperlipidemia, and, most importantly, non-insulin dependent diabetes mellitus (NSSD). The majority of patients with NISSL obese, and most important early component of the development NSSD is insulin resistance (Moller et al., New Eng. J. Med., 325:938 (1991)). It is shown that when insulin resistance develops anomaly post-receptor components, in addition to the suppression of the function of insulin receptors during the initial stages of this disease (Olefsky et al., in Diabetes Mellitus, Rifkin and Porte, Jr., Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp. 121-153).
The present invention is based, at least in part, on the fact that the members of the family of IL-17 and, in particular, IL-17A and IL-17F play a role in obesity, insulin resistance and other disorders associated with obesity, such as hyperlipidemia and metabolic syndrome, and that antagonists of IL-17, especially the antagonists of IL-17A and IL-17F may be used to treat these conditions.
In one aspect the present invention relates to a method of treatment of mammalian disorders associated with insulin resistance, which includes the maintenance of the needy in the mammal an effective amount of an antagonist of IL-17A and/or IL-17F.
In another aspect, the present invention relates to a pharmaceutical composition comprising an antagonist of IL-17A and/or IL-17F with the addition of pharmaceutically acceptable excipients, for the treatment of disorders associated with insulin resistance.
In an additional aspect, the present invention relates to the use of an antagonist of IL-17A and/or IL-17F for the treatment of disorders associated with insulin resistance.
In another aspect, the present invention relates to a kit for the treatment of disorders associated with insulin resistance, the specified set, containing: (a) a container containing an antagonist of IL-17A and/or IL-17F; and (b) the label or instructions for the introduction of this antibody for the treatment of such violations.
In all aspects, in one variation of the embodiment of the present invention, disorder selected from the group consisting of non-insulin dependent diabetes mellitus (NSSD), obesity, giperandrogenii ovarian cancer and hypertension. In another variant embodiment of the present invention violation is NSSD or obese.
In an additional variant embodiment the mammal is a human, and the introduction of a system.
In another additional embodiment, embodiment, the antagonist of IL-17A and/or IL-17F is an antibody or its fragment, such as an antibody selected from the group consisting of and is Titel against IL-17A, against IL-17F, anti IL-l7A/F, against IL-17Rc and against IL-17RA, or its fragment.
Preferably, the antibody is a monoclonal antibody, including a chimeric, humanized antibodies or human antibodies bispecific, multispecific antibodies or antibodies with cross-reactivity.
In another variant embodiment of the present invention the method includes introducing an effective amount of an agent for the treatment of insulin resistance, such as insulin, IGF-1 or a sulfonylurea.
In an additional embodiment, the method includes introducing an effective amount of an additional agent capable of curing the breach associated with insulin resistance, such as Dickkopf-5 (Dkk-5).
BRIEF DESCRIPTION of FIGURES
In Fig.1 shows the nucleotide sequence (TH. ID no:1) cDNA native sequence IL-17A man.
In Fig.2 shows the amino acid sequence (TH. ID no:2) of a native sequence IL-17A person, derived from the coding sequence of TH. ID no:1, shown in Fig.1.
In Fig.3 shows the nucleotide sequence (TH. ID no:3) native cDNA sequence of IL-17F person.
In Fig.4 shows the amino acid sequence (TH. ID no: 4) of a native sequence IL-17F person, derived from the coding sequence of TH. ID no:3, is provided in Fig.3.
In Fig.5 shows the nucleotide sequence (TH. ID no:5) encoding a native sequence polypeptide receptor C IL-17 man (IL-17Rc), which is also known as the clone, designated "DNA164625-2890".
In Fig.6 shows the amino acid sequence (TH. ID no:6) of a native sequence polypeptide IL-17Rc man (also known as the receptor for IL-17RH2).
Fig.7 - experimental design study model, a diet with high fat diet (HFD) using mice IL-17Rc KO.
Fig.8 - the results of the 8 week study model diet with high fat diet (HFD) using mice IL-17Rc KO.
Fig.9A and 9B - glucose levels in wild-type mice and IL-17Rc KO in the control group and the group with a diet high in fat. Mouse IL-17Rc KO resistant to insulin resistance induced by a diet with a high fat diet (HFD).
Fig.10 - the area under the pharmacokinetic curve at 10 weeks.
Fig.11 - the results on body weight.
Fig.12 - effect of monoclonal antibodies (mAb) against IL-17 and against IL-17F in a model of insulin resistance with diet high in fat.
Fig.13 - test for glucose tolerance (GTT) in the period after 9 weeks of doses.
Fig.14 - ectopic expression of IL-17A by injection of plasmid DNA with subsequent breakdown of glucose tolerance (GTT). The effect IL-17 on the status of insulin resistance, estimated using GTT.
A DETAILED DESCRIPTION of the INVENTION
The term "IL-17" is generally used to refer to members of the family of IL-17, including IL-17A, IL-17, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F and IL-17A/F. Preferred IL-17 in this document are IL-17A, IL-17F and IL-17A/F.
"Polypeptide with a native sequence IL-17" includes a polypeptide having the same amino acid sequence as the polypeptide of IL-17 derived from a natural source. Such polypeptides with a native sequence IL-17 can be isolated from natural sources or can be obtained by recombinant or synthetic methods. The term "polypeptide with a native sequence IL-17" explicitly encompasses naturally occurring truncated or secreted forms of the specific polypeptide of IL-17 (e.g., the sequence of the extracellular domain), naturally occurring variant forms (e.g., forms generated by alternative splicing) and naturally occurring allelic variants of the polypeptide. In various embodiments of the present invention polypeptides with a native sequence IL-17 described in this document are Mature or full-length polypeptide with a native sequence IL-17A, IL-17F and IL-17A/F man, containing panoram rye amino acid sequence, shown in Fig.2 and 4 (TH. ID nos:2 and 4). Start and stop codons are indicated on the figures in bold and underlined.
"Polypeptide with a native sequence IL-17Rc" or "native sequence IL-17Rc" refers to a polypeptide having the same amino acid sequence as the polypeptide of IL-17Rc, obtained from a natural source. Such polypeptides with a native sequence IL-17Rc can be isolated from natural sources or can be obtained by recombinant or synthetic methods. The term "polypeptide with a native sequence IL-17Rc" explicitly encompasses naturally occurring truncated or secreted forms of the specific polypeptide of IL-17Rc, naturally occurring variant forms (e.g., forms generated by alternative splicing) and naturally occurring allelic variants of the polypeptide. In various embodiments of the present invention the polypeptide with a native sequence IL-17Rc described in this document, is full-length IL-17Rc human native sequence containing the full amino acid, shown in Fig.6 (TH. ID no:6).
"Isolated", when used to describe the various polypeptides disclosed in this document, means polypeptide that has been identifiziert is h and isolated and/or extracted from a component of its natural environment. Contaminant components of its natural environment are materials which in the typical case would prevent diagnostic or therapeutic applications of the polypeptide and may include enzymes, hormones and other dissolved substances of protein or non-protein nature. In a preferred embodiment, the polypeptide will be purified (1) to the extent sufficient to determine at least 15 residues of N-terminal or internal amino acid sequence by use of sequencing machine with rotating glass, or (2) to homogeneity in LTO-page in non or reducing conditions using colour, Kumasi blue or, preferably, silver. Isolated polypeptide includes polypeptide in situ within recombinant cells since, when at least one component of the natural environment of the polypeptide of IL-17 will be missing. Ordinarily, however, isolated polypeptide will be prepared by using at least one purification stage.
When used in this document, "obesity" refers to a condition in which the mammal has a body mass index (BMI) calculated as weight (in kg) on the growth of2(in metres) equal to at least 25,9. It is generally accepted that individuals with normal weight have a BMI from 19.9 to less than 25,9. Obesity, associated with insul what nerezidentnosti, explicitly fall under this definition.
"Insulin resistance" or "disease associated with insulin resistance" or "activity associated with insulin resistance" are a disease, condition or disorder resulting from a disruption of normal metabolic response of peripheral tissues (dead) on the action of exogenous insulin, i.e., it is a condition in which the presence of insulin leads to a weaker than normal, the biological response. In clinical terms, insulin resistance is present when the normal or high glucose levels in the blood remain in the presence of normal or elevated levels of insulin. It represents, essentially, the inhibition of glycogen synthesis, in which either basal or insulin-stimulated glycogen synthesis, or both, are reduced to levels below normal. Insulin resistance plays a major role in type 2 diabetes, as demonstrated by the fact that hyperglycemia is present when type 2 diabetes, sometimes can be reversed through diet or weight reduction, sufficient, apparently, to restore the sensitivity of peripheral tissues to insulin. The term includes abnormal glucose tolerance, as well as many violations in which insulinresistance who plays a key role such as obesity, diabetes, ovarian hyperandrogenism and hypertension.
"Diabetes mellitus" refers to a state of chronic hyperglycemia, i.e., the excess sugar in the blood, as a result of relative or complete lack of insulin action. There are three main types of diabetes, type I or insulin-dependent diabetes mellitus (IDDM), type II or non-insulin dependent diabetes mellitus (NSSD) and insulin resistance of type A, while type a is relatively rare. Patients with diabetes type I or type II can purchase insensitivity to the action of exogenous insulin by different mechanisms. Insulin resistance type A occurs as a result of either mutation in the gene for the insulin receptor, or violations in the post-receptor fields of action that are critical for glucose metabolism. Diabetics subjects can be easily identified by a doctor, and they are characterized by hyperglycemia, impaired glucose tolerance, glycosylated hemoglobin and, in some cases, ketoacidosis associated with injury or disease.
"Non-insulin-dependent diabetes mellitus" or "NSSD" refers to type II diabetes. Patients with NISSL have abnormally high concentration of glucose in blood on an empty stomach and slow absorption of glucose by cells after eating, or after a diagnostic test is, known as the test for glucose tolerance. NSSD diagnosed on the basis of the recognized criteria (American Diabetes Association, Physician''s Guide to Insulin-Dependent (Type I) Diabetes, 1988; American Diabetes Association, Physician''s Guide to Non-Insulin-Dependent (Type II) Diabetes, 1988).
Symptoms and complications of diabetes, which will be treated as a violation, when used in this document include hyperglycemia, poor glycemic control, ketoacidosis, insulin resistance, elevated levels of growth hormone, elevated levels of glycosylated hemoglobin and increased content glycosylation end products (AGE), morning hyperglycemia, poor lipid profile, vascular disease (e.g. atherosclerosis), disease of capillaries, abnormalities of the retina (e.g., proliferative diabetic retinopathy), renal dysfunction, neuropathy, complications during pregnancy (e.g., premature birth and developmental defects), and the like. In the determination of treatment included such endpoints as, for example, increase insulin sensitivity, decrease the dosage of insulin while maintaining glycemic control, reduction in HbAlc, improved glycemic control, the weakening of the cardiovascular, renal, neural, retinal, and other diabetic complications, prevention or reduction of morning g is pellicani, improved lipid profile, reduced complications in pregnancy and the reduction of ketoacidosis.
"Therapeutic composition" or "composition", when used in this document, is defined as a composition comprising a Dkk-5 and a pharmaceutically acceptable carrier, such as water, inorganic compounds, proteins and other excipients known to specialists in this field.
The term "mammal" for purposes of treatment refers to any animal classified as a mammal, including, as non-limiting examples, humans, rodents, animals used in sport contained in zoos and homes, and domesticated or farm animals, such as dogs, cats, cattle, sheep, pigs, horses and not anthropoid primates, such as monkeys. The preferred rodents are mice or rats. Preferably, the mammal is a human, also called in this document the patient.
When used in this document, "treatment" means the treatment and care of mammals to struggle with any of the diseases or conditions to treatment which applies the present invention, including, as non-limiting examples, insulin resistance, diabetes mellitus, hyperinsulinemia, hypoinsulinemia or obese, and includes the introduction of cartonnage means for preventing the onset of symptoms or complications, mitigate the symptoms or complications, or eliminating the target diseases or conditions.
For the purposes of this invention, beneficial or desired clinical results "treatment" to reduce insulin resistance include, as non-limiting examples, the mitigation of symptoms associated with insulin resistance, decrease the severity of symptoms of insulin resistance, stabilization (i.e., no deterioration of symptoms of insulin resistance (e.g., reduction in insulin requirements), increased sensitivity to insulin and/or insulin secretion to prevent damage to the cells of the islets and delay or slow the progression of insulin resistance, for example, the progression of diabetes.
In relation to obesity, "treatment" generally means a reduction in BMI of the mammal to less than about 25,9 and maintaining this weight for at least 6 months. The suitability of treatment results in reduction in the consumption of mammals of the food or calories. In addition, in this context, the treatment is to prevent the occurrence of obesity, if the treatment is carried out until the condition of obesity. Treatment includes inhibition and/or complete inhibition of lipogenesis in mammals with obesity, i.e., excessive accumulation of lipids in fat cells, which one is camping one of the main characteristics of obesity in humans and animals, as well as a decrease in overall body mass.
Those who are "in need of treatment", are mammals, have a violation, as well as those who have the propensity to purchase violations, including those violations should be prevented.
"Agent for the treatment of insulin resistance" is the agent that is different from the antagonist of IL-17, which is used for the treatment of insulin resistance, such as, for example, Dickkopf-5 (Dkk-5) (see, for example, the publication of the patent application U.S. No. 2005/0170440) and hypoglycemic agents. Examples of such pharmaceutical agents include insulin (one or several different insulin), insulin mimetics, such as low molecular weight insulin, for example, L-783,281; insulin analogs (e.g., insulin HUMALOG® (Eli Lilly Co.), insulin LysB28insulin ProB29or insulin AspB21or those described, for example, in U.S. patent No. 5149777 and 5514646), or their physiologically active fragments; related insulin peptide (C-peptide, GLP-1, insulin-like growth factor-I (IGE-1) or a complex of IGF-1/IGFBP-3), or their analogues, or fragments; Argosy; pramlintide; leptin; BAY-27-9955; T-1095; antagonists of inhibitor of the tyrosine kinase of the insulin receptor; antagonists of the function of TNF-α; agent to release growth hormone; Amylin or antibodies to Emilio; to insulin sensitizing agent, such as connections family glitazone, including those described the data in U.S. patent No. 5753681, such as troglitazone, pioglitazone, englitazone and related compounds; linalool alone or with vitamin E (U.S. patent No. 6187333); amplifiers insulin secretion, such as nateglinide (AY-4166), calcium (2S)-2-benzyl-3-(CIS-hexahydro-2-isoindoline)propionate dihydrate (mitiglinide, KAD-1229) and Repaglinide; medicines on the basis of sulfonylureas, for example acetohexamide, hlorpropamid, tolazamide, tolbutamide, glyclopyramide and its ammonium salt, glibenclamide, glyburide, gliclazide, 1-butyl-3-methylinosine, carbutamide, glipizide, glikvidon, glisoxepide, globaliza, glybuzole, glyhexamide, glymidine, pipename, fenbutatin, tolciclate, glimepiride, and so on; biguanides (such as phenformin, Metformin, buformin, and so on); inhibitors of α-glucosidase (such as acarbose, voglibose, miglitol, emiglitate and so on) and such unusual methods of treatment, as transplantation of the pancreas or autoimmune reagents.
"Agent for weight reduction" refers to a molecule, useful for the treatment or prevention of obesity. Such molecules include, for example, hormones (catecholamines, glucagon, ACTH, and growth hormone in combination with IGF-1); Ob protein; clofibrate; halogenate; cinchocaine; chlorpromazine; suppress the appetite medicines acting on noradrenergic neurotransmitters, such as mazindol, and derivatives panatela the ina, for example phenylpropanolamine, diethylpropion, phentermine, phendimetrazine, benzphetamine, amphetamine, methamphetamine and phenmetrazine; drugs that act on serotonin neurotransmitters, such as fenfluramine, tryptophan, 5-hydroxy-tryptophane, fluoxetine and sertraline; medicines Central actions, such as naloxone, neuropeptide Y, Galanin, corticotropin-releasing hormone, cholecystokinin; cholinergic agonists such as pyridostigmine; sphingolipid, such as liposterolic or its derivative; thermogenic medications, such as thyroid hormone; ephedrine; beta-adrenergic agonists; drugs, affecting the gastrointestinal tract, such as enzyme inhibitors, for example, tetrahydrolipstatin, nevereverever foods, such as sucrose polyester, and inhibitors of gastric emptying, such as trichlorosilane acid or its derivatives; beta-adrenergic agonists, such as isoproterenol and yohimbine; aminophylline to enhance the effects yohimbine, such as the beta-adrenergic, the medication that blocks α2-adrenergic transmission, such as clonidine alone or in combination with a peptide that stimulates growth hormone medications from absorption in the intestine, such as biguanidine, for example, Metformin and phenformin; the volume of mnye fillers, such as methylcellulose; drugs that block the metabolism, such as hydroxycitrate; progesterone; cholecystokinin agonists; small molecule mimics under the keto acid; antagonists of corticotropin-releasing hormone; derived from ergot prolactin inhibitory connection to reduce the fat reserves of the body (U.S. patent No. 4783469, issued November 8, 1988); beta-3 agonists; parlodel; antagonists of opioid peptides; antagonists of neuropeptide Y receptor antagonists glucocorticoids; agonists of growth hormone; combinations thereof, and so on
When used in this document, "insulin" refers to any substance having the action of insulin and is represented, for example, the insulin of animal origin, extracted from the pancreas of the ox or pig, a semisynthetic human insulin, which is synthesized enzymatically from insulin extracted from the pancreas of pigs, and human insulin synthesized using techniques of genetic engineering, usually with the use of E. coli or yeast, etc. Additionally, insulin may include a complex of insulin with zinc, containing from about 0.45 to 0.9 wt.% zinc, Protamine-insulin-zinc derived from zinc chloride complex of Protamine sulfate and insulin, etc. Insulin can be represented in the form of its fragments is whether derivatives, for example, INS-1. Insulin may also include insulin-like substance, such as L83281 and agonists of insulin. Because insulin is available in a variety of types, such as superseeding actions, immediate actions, bimodal action, interim action, long action, and so on, these species can be properly selected in accordance with the patient's condition.
"Therapeutic composition" when used in this document is defined as a composition comprising the antagonist of IL-17 (including antagonists of IL-17A and IL-17F) and a pharmaceutically acceptable carrier, such as water, inorganic compounds, proteins and other excipients known to specialists in this field.
It is understood that the expression "antagonist, antagonist to the IL-17 (A and/or F)", "antagonist of IL-17 (A and/or F)" and the like within the present invention include any molecule that inhibits the function of IL-17, such as IL-17A and/or IL-17F, or blocks or neutralizes the corresponding activity of IL-17 (such as IL-17A and/or F) in any way, depending on the indications for treatment. It can prevent the interaction between IL-17 (including IL-17 and IL-17F) and one or more of its receptors. These agents carry out this action in a variety of ways. For example, the class of antagonists that "neutralize" Akti is the ability of IL-17, will be contacted with IL-17 or its receptor IL-17 with sufficient affinity and specificity, creating an obstacle for the action of IL-17, as described below. An antibody "which binds" with IL-17 or its receptor IL-17 (e.g., IL-17Rc), is an antibody that is able to communicate with that antigen with sufficient affinity such that the antibody is useful as a therapeutic agent to target cells expressing IL-17 or the receptor for IL-17. The term "antagonist of IL-17 is used to refer to any of the antagonists of IL-17A, IL-17F and IL-17A/F.
The composition of this group of antagonists include, for example, antibodies produced against IL-17 or its parts, reactive against IL-17, or receptor, IL-17, or parts thereof, including antibodies against IL-17A and/or IL-17F and IL-17Rc. The term also includes any agent which will prevent overproduction IL-17A and/or IL-17F or to have antagonistic activity against receptor, at least one IL-17 (e.g., IL-17A and/or IL-17F), such as IL-17Rc. Such antagonists can be represented in the form of chimeric hybrids, useful for combining the functions of the agent with the protein carrier to increase the half-life of serum therapeutic agent or to provide cross-species transferability. Therefore, examples of such antagonists include Bioorganic mo is ecoli (for example, peptidomimetics, antibodies, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, pharmaceutical agents and their metabolites, transcriptional and translational regulatory sequences and the like. In a preferred embodiment, the antagonist is represented by antibody having the required properties of binding IL-17A and/or IL-17F and prevents its interaction with the receptor, preferably with IL-17Rc.
The term "antibody" is used in its broadest sense and specifically includes, for example, single monoclonal antibodies against IL-17A/F or against IL-17A, or anti-IL-17F (including agonist, antagonist and neutralizing antibodies), the composition of the respective antibodies polyepitopic specificity, polyclonal antibodies, single-chain antibodies and fragments of antibodies (see below), if they exhibit the desired biological or immunological activity.
The basic unit 4-chain antibodies is heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (IgM antibody consists of 5 main heterotetrameric units and an additional polypeptide called J chain, and therefore contains 10 antigenspecific sites, at the same time, secreted IgA antibodies can Polym resultsa with the formation of multivalent complexes, consisting of 2-5 basic 4-chain units and J-chain). In the case of IgG, 4-chain unit usually has a weight of approximately 150,000 daltons. Each L-chain is linked to the H-chain by one covalent disulfide bond, and the two H-chains are connected to each other by one or more disulfide bonds, depending on the isotype of the H-chain. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H-chain N-end has a variable domain (VH), followed by three conserved domain (CHfor each of the α and γ chains and four CHdomain in the case of μ and ε isotypes. Each L-chain N-end has a variable domain (VL), followed by conservative domain (CL) at the other end. VLlocated next to the VHand CLlocated next to the first conservative domain of the heavy chain (CH1). It is believed that individual amino acid residues form the surface interaction between the variable domains of the light chain and heavy chain. Pairing VHand VLleads to the formation of a single antigennegative site. Structure and properties of different classes of antibodies are presented, for example, in Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
L-chain of any species of vertebrate vividly the data can be attributed to one of two clearly differentiated types, called Kappa and lambda, based on the amino acid sequences of their conservative domains. Depending on the amino acid sequence of the conservative domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, which have heavy chains designated α, δ, ε, γ and μ, respectively. Classes γ and α are further divided into subclasses on the basis of relatively minor differences in the sequence CHand functions; for example, expressed the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
The term "variable" refers to the fact that certain segments of the variable domains differ greatly in sequence among antibodies. V-domain mediates binding to the antigen and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the length of the 110-amino acid of the variable domain. In contrast, the V-region consist of relatively invariant sections, called frame regions (FR) with a length of 15-30 amino acids separated by shorter areas of high variability called "hypervariable regions", which have a length of 9-12 amino acids each. Vari is belinya domains of native heavy and light chains consist each of the four FR, generally having the configuration of a beta-sheet, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of the structure of beta-sheet. Hypervariable region in each chain are closely contiguous to each other with the help of FR and together with the hypervariable regions of the other circuit involved in the formation antigennegative site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Conservative domains are not involved directly in binding the antibody to the antigen, but perform various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).
The term "hypervariable region", when used in this document, refers to the amino acid residues of an antibody which are responsible for binding to the antigen. Hypervariable region generally contains amino acid residues from a "region that defines complementarity" or "CDR" (e.g., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VLand about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and/or residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the Ostrava V Land 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
The term "monoclonal antibody" when used in this document, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations of natural origin that may be present in small quantities. Monoclonal antibodies of vysokospetsifichnymi, as produced against a single antigenic site. Additionally, in contrast to the preparations of polyclonal antibodies, which contain different antibodies generated against different determinants (epitopes), each monoclonal antibody generated against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without admixture of other antibodies. This monoclonal antibody typically includes the antibody containing the variable region that binds to the target, when this antibody was obtained by the method, including the selection of antibodies from a variety of antibodies. For example, the method of selection may be a selection of unique clone of many clones, such as a pool of hybridoma clones, phage clones or clones with recombinant DNA. While SL is blowing to understand that the selected antibody can be further modified, for example, to improve the affinity to the target, for humanitarian antibody to improve its products in cell cultures, to reduce its immunogenicityin vivoto create multispecific antibodies, etc. and that the antibody containing the modified sequence of the variable regions, also is a monoclonal antibody of the present invention. In addition to their specificity, the preparations of monoclonal antibodies have the advantage that they usually do not contain impurities of other immunoglobulins. The definition of "monoclonal" indicates the property of the antibodies that it was obtained from a substantially homogeneous population of antibodies, and should not be construed as a requirement to obtain antibodies specific way. For example, monoclonal antibodies that will be applied in accordance with the present invention, can be obtained using various methods, including hybridoma method (for example, Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, N. Y., 1981)), the methods of recombinant DNA (see, for example, U.S. patent No. 4816567), methods of phage display (see, for example, Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J Mol. Biol.340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1-2):119-132 (2004)) and methods for obtaining human antibodies or antibodies, such antibodies man from animals that contain part or whole immunoglobulin loci of human or genes, coding sequences of human immunoglobulins (see, for example, WO98/24893, WO/9634096, WO/9633735 and WO/9110741, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Measurement., 7:33 (1993); U.S. patent No. 5545806, 5569825, 5591669 (all owned by GenPharm); 5545807; WO 97/17852, U.S. patents№№ 5545807; 5545806; 5569825; 5625126; 5633425; and 5661016, and Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).
Monoclonal antibodies in this document include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, if they possess the desired biological activity (see patent is SHA No. 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies described in this document include "primaryservername" antibodies that contain antigennegative sequence of the variable domains derived from primates, non-human (e.g., old world monkeys, apes, and so on), and sequences of conserved regions of the person.
"Intact" antibody is an antibody that contains antigennegative site, and CLand, at least, conservative domains of the heavy chain CH1, CH2 and CH3. Conservative domains can be conservative domains with the native sequence (e.g., conservative domains with native sequence human) or their variants amino acid sequence. Preferably, the intact antibody has one or more effector functions.
"Fragments of the antibodies contain a portion of an intact antibody, preferably antigennegative or variable region of the intact antibody. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')2and Fv; dyatel; linear antibodies (see U.S. patent No. 5641870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 ); molecules of single-chain antibodies; and multispecific antibodies formed from fragments of antibodies. In a preferred variant embodiment of ragment is "functional", i.e. qualitatively preserves the ability of the corresponding intact antibodies to communicate with the target polypeptides of IL-17A and IL-17F and, if intact antibody also inhibits the biological activity or function of IL-17A/F is also of high quality stores such inhibitory property. Quality means that there is the same activity, but the degree of affinity of binding and/or activity may differ.
Cleavage of antibodies with papain leads to the formation of two identical antigenspecific fragments, called "Fab"fragments, and a residual "Fc"fragment, whose designation reflects its ability to easily crystallize. Fab-fragment consists of an entire L chain, connected with the domain variable regions of the H-chain (VH), and the first conservative domain of one heavy chain (CH1). Each Fab fragment of monovalent relative to the binding of antigen, i.e. it has a single antigennegative website. Processing of antibodies with pepsin yields a single large F(ab')2the fragment, which corresponds approximately to two crosslinked by a disulfide bond Fab-fragments having bivalent antigennegative activity, and retains the ability of cross-linking antigen. Fab'fragments differ from Fab fragments by the presence of several additional residues at the carboxyl end of the domain CH1in the second number of one or more cysteines from the hinge region of the antibody. Fab'-SH in this document refers to Fab' in which the residue (remainder) of cysteine conservative domains bear a free Tilney group. Antibody fragments F(ab')2originally given as pairs of Fab'-fragments, which are interconnected cysteine hinge region. Also other known methods of chemical cross-linking of fragments of antibodies.
Fc-fragment contains a carboxy-terminal part of the two H-chains linked together by disulfide bonds. Effector functions of antibodies are determined by sequences in the Fc region, and this area is also part of, recognized by Fc receptors (FcR), found in certain types of cells.
"Fv" is the minimum antibody fragment which contains a complete site recognition and binding of the antigen. This fragment consists of a dimer of the variable regions of the domain of one heavy and one light chain, which is in strong non-covalent interaction. As a result of folding of the two domains are formed six hypervariable loops (3 loops of H - and L-chains), which provide amino acid residues for the binding of the antigen and the antibody binding specificity of the antigen. However, even a single variable domain (or half of an Fv, containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower the affinity, than the entire binding site.
"Single-chain Fv", also abbreviated label "sFv" or "scFv" - fragments of antibodies, which contain domains VHand VLantibodies, United in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between domains VHand VLwhich enables the sFv to form necessary for binding of the antigen structure. A review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, below.
The term "diately" denotes small fragments of antibodies, prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between domains VHand VLthus, what is achieved megamachine, but not intrachain, the pairing of the V domains, resulting in a bivalent fragment, i.e. a fragment with two antihistamine sites. Bispecific of diately are heterodimeric two "cross" sFv fragments, in which the domains of the VHand VLtwo antibodies are present on different polypeptide chains. Diately described in more detail, for example, in EP 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
"Humanized" forms of antibodies belonging not to the person (e.g., rodents), is a chimeric antibodies that contain min the normal sequence, with the origin of the antibodies, not owned by the person. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a species other than human (donor antibody) such as mouse, rat, rabbit or non-human Primate having the desired specificity, affinity and capacity of the antibodies. In some cases, remnants of the framework region (FR) of a human immunoglobulin replace the corresponding residues from species other than human. Additionally, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications produced to further improve the operating characteristics of the antibody. In General, humanitariannet antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of immunoglobulin that does not belong to man, and all or substantially all of the FR are those with the sequence of human immunoglobulin. Humanitariannet antibody optionally also will comprise at least part of the conservative region of the immunoglobulin (Fc), in the typical case, that of a human immunoglobulin. For a more detailed description, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The term "multispecific antibody" is used in its broadest sense and, in particular, includes the antibody containing the variable domain of the heavy chain (VH) and the variable domain of the light chain (VLduring this unit of VHVLhas polyepitopic specificity (i.e., able to bind two different epitopes on a single biological molecule or each epitope on other biological molecule). Such multispecific antibodies include, as non-limiting examples, full-length antibodies, antibodies that have two or more domains VLand VH, fragments of antibodies, such as Fab, Fv, dsFv, scFv, diately, bispecific of diately and Triatel, fragments of antibodies, covalently or ecovalence stitched.
"Polyepitopic specificity" refers to the specific ability to communicate with two or more different epitopes on the same or another(their) target(s).
"Monospecificity" means the ability to bind to only one epitope. According to one variant, multispecific antibody in the form of IgG1 is associated with each epitope with an affinity of from 5 μm to 0.001 PM, from 3 μm to 0.001 PM, from 1 μm to 0.001 PM, from 0.5 μm to 0.001 PM, or from 0.1 m to the M to 0.001 PM.
"Antibody cross-reactivity" is an antibody that recognizes an identical or similar epitopes on more than one antigen. Thus, antibodies with cross-reactivity of the present invention recognize identical or similar epitopes present on IL-17A and IL-17F. In one embodiment, the antibody cross-reactivity using the same or substantially the same Pratap to bind both IL-17A and IL-17F. Preferably, antibodies with cross-reactivity in this document also block the function (activity) as IL-17A and IL-17F.
The term "pretop" is used in this document to refer to parts of an antibody that binds to the target antigen.
"Species-specific (dependent) antibody, such as antibody of a mammal against IL-17A/F antibody, which has a stronger binding affinity of the antigen from the first species of mammals than to the homologue of that antigen from a second mammal species. Normally, the species-specific antibody "specific associated with antigen person (i.e., has a value of affinity binding (Kd) is not more than about 1×10-7M, preferably no more than about 1×10-8M and most preferably no more than about 1×10-9M) but has a binding affinity of with a homologue of the antigen from the showing of the species of mammal, other than man, which at least about 50-fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity of the antigen person. Species-specific antibody may belong to any of various types of antibodies mentioned above, but preferably is a humanized antibody or a human antibody.
An antibody "which binds" the antigen is an antibody that binds to the antigen with a significant affinity so that the antibody is useful as a diagnostic and/or therapeutic agent at the target effect on the cells or tissue expressing the antigen, and does not have significant cross-specificity in relation to other proteins. In such embodiments, the embodiment of the degree of binding of the antibody with the "non-target" protein will be equal to less than about 10% of the binding of the antibody with its specific target protein, as defined by the method of fluorescent-activated cell sorting (FACS) or radioimmunoprecipitation (RIA). In relation to the binding of an antibody to a target molecule, the term "specific binding" or "specific binds to" or is "specific for" a particular polypeptide or epitope on a particular target polypeptide means binding, to the which measurable differs from nonspecific interactions. Specific binding can be measured, for example, by determining binding molecules, in comparison with the binding reference molecule, which is typically a molecule with a similar structure, which does not have binding activity. For example, specific binding can be determined by competition with a control molecule, which is similar to the target, for example, with an excess of unlabeled target molecules. In this case, specific binding indicates the fact that the binding of the labeled target molecule and a probe competitive inhibited by excess unlabeled target molecules. The term "specific binding" or "specific binds to" or is "specific for" a particular polypeptide or epitope on a particular target polypeptide, when used in this document can be used in relation to, for example, a molecule having a Kd for target molecules, at least about 10-4M, alternatively at least about 10-5M, alternatively at least about 10-6M, alternatively at least about 10-7M, alternatively at least about 10-8M, alternatively at least about 10-9M, alternatively at least about 10-10M, alternatively at least about 10 -11M, alternatively at least about 10-12M or more. In one embodiment, the term "specific binding" refers to binding, in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide. In preferred embodiments, embodiments of the affinity of the specific binding equal to at least about 10-10M
"The effector functions of antibodies indicate biological activity characteristic of the Fc region (Fc region with a native sequence Fc region variant amino acid sequence) antibodies, and change depending on the isotype of the antibody. Examples of effects or functions of antibodies include Clq binding and complement-dependent cytotoxicity; the binding of the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; the suppression of the activity of receptors on the cell surface (for example, receptor B-cells); and activation of B-cells.
"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins associated with Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) is ledah, neutrophils and macrophages), give these cytotoxic effector cells specific ability to communicate with bearing the antigen of the target cell and subsequently kill the target cell by means of cytotoxins. Antibodies arm cytotoxic cells and are absolutely necessary for such destruction. The primary cells for mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess activity in ADCC studied molecules can be carried out analysis of ADCC in vitro, such as analysis, described in U.S. patent No. 5500362 or 5821337. Useful for such analyses effector cells include mononuclear cells of peripheral blood (MCPC) and natural killer (NK) cells. In accordance with another variant, or additionally, activity in ADCC studied molecules can be assessed in vivo, e.g., in an animal model, such as described in Clynes et al. Proc. Natl. Acad. Sci. U. S. A. 95:652-656 (1998).
"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of antibodies. Preferred FcR FcR is human native sequence. Moreover, a preferred FcR is one such receptor, which binds with the antibody IgG (g is MMA-receptor) and includes receptors of the subclasses of FcγRI, FcγRII and FcγRIII, including allelic variants and forms of these receptors, resulting from alternative splicing. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. Activating receptor FcγRIIA contains immunoreceptor that contains tyrosine, an activator motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains immunoreceptor containing tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). Review of FcR is given in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcR, including those that will be identified in the future are also included in the term "FcR" in this document. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
"Effector cells" are leukocytes which Express one or more FcR and perform effector functions. Preferably, the cells Express at least FcγRIII and perform effector function in ADCC. Examples of human leukocytes which mediate ADCC include Mononoke rnie cells of peripheral blood (MCPC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; however, preferred are MCPC and NK-cells. The effector cells may be isolated from a natural source, for example from the blood.
"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of the target cells in the presence of complement. Activation of the classical complement cascade begins with the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) that are associated with their respective antigens. To assess activation of complement may be conducted by CDC analysis, for example, as described in Gazzano-three-bet et al., Immunol. Methods 202:163 (1996).
The terms "neutralize" and "neutralizing activity" are used in this document to refer to, for example, block, prevent, reduce, counteract the activity or conversion of IL-17 (e.g., IL-17A and/or IL-17F) in inefficient molecule using any mechanism. Therefore, the antagonist can prevent the act of binding is required for activation of IL-17.
By "neutralizing antibody" is understood molecule antibodies described in this document, which is able to block or significantly reduce effector function of IL-17 (including IL-17A and/or IL-17F). For example, a neutralizing antibody may inhibit or reduce the ability of IL-17 for example, IL-17A and/or IL-17F) to interact with the receptor, IL-17, such as IL-17Rc. In accordance with another variant of a neutralizing antibody may inhibit or reduce the ability of IL-17 to block the signaling pathway of the receptor for IL-17. The neutralizing antibody may also immunospecific contact with IL-17 when the immunoassay on the activity of IL-17. "Neutralizing antibody" according to the invention differs in that it retains its functional activity in the situation both in vitro and in vivo.
B. Detailed description of the invention
1. Therapeutic use
Insulin resistance is a condition in which the presence of insulin causes reduced compared to normal biological response. In clinical terms, insulin resistance is present when the normal or high glucose levels in the blood remain in the presence of normal or elevated levels of insulin. It represents, essentially, the inhibition of glycogen synthesis, in which either basal or insulin-stimulated glycogen synthesis, or both of these types are reduced to levels below normal. Insulin resistance plays a major role in type 2 diabetes, as demonstrated by the fact that hyperglycemia is present when type 2 diabetes, sometimes can be reversed through diet or weight reduction, sufficient evidence is about, to restore the sensitivity of peripheral tissues to insulin.
The present invention relates to the treatment of insulin resistance or type 2 diabetes by administration of antagonists of IL-17A and/or IL-17F. As previously discussed, the antagonist of IL-17A and/or IL-17F may be any molecule that inhibits the function of IL-17A and/or IL-17F, or blocks or neutralizes the corresponding activity of IL-17A and/or F in any way, depending on the indications for treatment. It can prevent the interaction between IL-17A and/or IL-17F and one or more of its receptors, in particular IL-17Rc. These agents carry out this action in a variety of ways. For example, the class of antagonists that neutralize the activity of IL-17A and/or IL-17F, will be contacted with IL-17A and/or IL-17F, or with the receptor of IL-17A and/or IL-17F, in particular with IL-17Rc, with significant affinity and specificity for disturbing influence on IL-17A and/or IL-17F.
2. Introduction and recipe
The antagonist of IL-17 may be introduced by any suitable means, including parenteral injection, such as, as non-limiting examples, intravenous (IV), intramuscular (IM), subcutaneous (SC) and intraperitoneally (IP), as well as transdermal, buccal, sublingual, intrarectal, intranasal and inhalation methods. IV, IM, SC and IP introduction can be implemented as a shock to the s or infusion, and in the case of SC injection can also be applied to implantable device for gradual release, including, as non-limiting examples, pumps, formulations with a gradual release and mechanical devices. Preferably, the introduction of a system.
One particularly preferred method of administration of the antagonist of IL-17 is a subcutaneous infusion, in particular, with the use of dosing infusion device, such as a pump. This pump can be reusable or disposable and implantable or externally attached. Pumps for infusion of drugs successfully used for this purpose include, for example, the pumps described in U.S. patent No. 5637095; 5569186; and 5527307. The composition can be entered from such devices permanently or periodically.
Therapeutic formulations of antagonists of IL-17, suitable for storage, include a mixture of antagonists having the desired degree of purity, with pharmaceutically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilised compositions or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used dosages and concentrations and include b is very, such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecylsilane ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; complexes with metals (such as Zn complexes with proteins); and/or nonionic surfactants such as TWEENT™, PLURONICS™ or polyethylene glycol (PEG). Preferred formulations of lyophilized antibodies against IL-17 is described in WO 97/04801. These compositions contain an antagonist of IL-17, comprising from about 0.1 to 90% by weight of active antagonist, preferably in a soluble form, and generally from about 10 to 30%.
Akti is these ingredients may also be enclosed in microcapsules, prepared, for example, using methods koatservatsii or by polymerization on the phase boundary, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methylmetacrylate) microcapsules, respectively, in colloidal drug delivery system tools (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in Remington's Pharmaceutical Sciences, above.
Antagonists of IL-17A and/or IL-17F, such as antibodies against IL-17 described in this document can also be prepared in the form of immunoliposome. Liposomes containing the antibody are prepared by methods known in this field, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. patent No. 4485045 and 4544545; and WO97/38731, published on 23 October 1997 Liposomes with enhanced circulation time is described in U.S. patent No. 5013556.
Particularly useful liposomes can be obtained by the method of reverse-phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and modified PEG the phosphatidylethanolamine (PEG-PE). Liposomes push through filters with defined pore size to obtain liposomes with the desired diameter. Fab'-fragments of the antibodies of the present invention can be conjugated to the liposomes as described is in Martin et al., J. Biol. Chem., 257:286-288 (1982), by reaction of disulfide exchange.
Can be cooked recipes prolonged action. Suitable examples of sustained-release preparations include semi-permeable the matrices a solid hydrophobic polymers containing the antibody, and these matrices are in the form of decorated particles, e.g. films, or microcapsules. Examples of matrices prolonged action include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or polyvinyl alcohol), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem the ethylene vinyl acetate, degradiruete copolymers of lactic acid and glycolic acid, such as the LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid and glycolic acid and leuprolide acetate), and poly-D-(-)-3-hydroxipropionic acid.
Any specific antagonists can be connected with protein carrier to increase the half-life of serum therapeutic antagonist. For example, a soluble immunoglobulin chimeras, such as described in this document can be obtained for each specific antagonist of IL-17 or its antagonistic parts, as described in U.S. patent No. 5116964. Chimera antibodies can be purified using chromatography aseparate with IgG-binding protein A. Chimeras have the ability to form immunoglobulinemia dimers with a simultaneous increase in the avidity and half-life of serum.
Formulations that will be used for administration in vivo, must be sterile. This is easily achieved by filtering through a sterile membrane filters.
The formulation described in this document may also contain more than one active connection, if it is necessary for the specific indications for treatment, preferably those with complementary activities that do not have each other undesirable effects. Also, this active compound may be administered to a mammal, the treated separately.
For example, in the case of such indications may be desirable to additionally provide an agent for the treatment of insulin resistance. In addition, type 2 diabetes, with no response to diet and weight loss may respond to therapy with the use of sulfonylureas in conjunction with an antagonist of IL-17. The class of medicines on the basis of sulfonylureas include acetohexamide, hlorpropamid, tolazamide, tolbutamide, glibenclamide, glyburide, gliclazide, glipizide, glikvidon and glymidine. Other agents used for this purpose include autoimmune reagent, sensitizing to insulinogenic, such as connection pitsanuloke family, including those described in U.S. patent No. 5753681, such as troglitazone, pioglitazone, englitazone and related compounds, antagonists of inhibitor of the tyrosine kinase of the insulin receptor (U.S. patent No. 5939269 and 5939269), the complex of IGF-1/IGFBP-3 (U.S. patent No. 6040292), antagonists of the function of TNF-alpha (U.S. patent No. 6015558), agent release growth hormone (U.S. patent No. 5939387) and antibodies to Emilio (U.S. patent No. 5942227). Other compounds that can be used include insulin (one or several different insulin), insulin mimetics, such as low molecular weight insulin, insulin analogs, above, or their physiologically active fragments, related to insulin peptides, as described above, or their analogs or fragments. Agents are additionally described in the definitions above.
For the treatment of hypoinsulinemia, for example, insulin can enter singly or together with an antagonist of IL-17.
Such additional molecules properly present or are introduced in combination in amounts that are effective for the intended purpose, usually in smaller amounts than are used when administered separately, without an antagonist of IL-17. If they are in a mixture, they can be included in the mixture in amounts determined in accordance with, for example, the type shown what I the subject, the age and body weight of the subject, current clinical status, time of administration, dosage form, method of administration etc., for Example, in this document the concomitant drug is used preferably in a ratio of from about 0.0001 to 10000 mass fractions of one mass part of the antagonist of IL-17.
The use of an antagonist of IL-17 in combination with insulin can reduce the insulin dose compared to the dose with the introduction of only one insulin. Consequently, reduces the risk of complications on the blood vessels and induction of hypoglycemia, which can be a problem with the introduction of large quantities of insulin. For administering insulin to the adult patient with diabetes (weighing about 50 kg), for example, the daily dose is usually about 10 to 100 E (Units), preferably from 10 to 80, but may be less in accordance with the instructions of the doctor. For the introduction of enhancers of insulin secretion, the same type of patient, for example, the daily dose is preferably from about 0.1 to 1000 mg, more preferably about 1 to 100 mg. For the introduction of biguanides same type of patient, for example, the daily dose is preferably about 10 to 2500 mg, more preferably from about 100 to 1000 mg. For the introduction of inhibitors of α-glucosidase same type of patient, such as the er, the daily dose is preferably from about 0.1 to 400 mg, more preferably from about 0.6 to 300 mg. Introduction ergosta, pramlintide, leptin, BAY-27-9955 or T-1095 such patients can be effective if the dosage is preferably from about 0.1 to 2500 mg, more preferably from about 0.5 to 1000 mg All of the above dose may be administered once or several times a day.
The antagonist of IL-17 can also be administered together with a suitable non-pharmacological treatment of insulin resistance, such as transplantation of the pancreas.
The dosage of the antagonist, administered to the mammal with insulin resistance or hypoinsulinemia will be determined by a physician taking into account the surrounding circumstances, including the condition of the mammal, the type of antagonist, the type indication and the selected route of administration. The dosage is preferably low enough so as not to cause weight gain in any substantial degree, and this level may be determined by the doctor. Glitazone approved for the treatment of type 2 diabetes in humans (rosiglitazone/Avandia and pioglitazone/Actos), cause some weight gain, however, their use, despite the side effects, as it is proved that they are useful due to its therapeutic index. The ranges of dosages, presents what's in this document, there is no intention to limit the scope of the present invention in any way. "Therapeutically effective" amount for the purposes specified in this document in respect of hypoinsulinemia and insulin resistance, determined by the above factors, but is usually from about 0.01 to 100 mg/kg body weight/day. The preferred dose is about 0.1-50 mg/kg/day, more preferably from about 0.1 to 25 mg/kg/day. Even more preferably, at introduction of the antagonist of IL-17 daily intravenous or intramuscular dose for humans is approximately from 0.3 to 10 mg/kg of body weight per day, more preferably from about 0.5 to 5 mg/kg For subcutaneous administration, the dose of, preferably, higher than therapeutically equivalent dose, administered intravenously or intramuscularly. Preferably, a daily subcutaneous dose for humans is approximately from 0.3 to 20 mg/kg, more preferably from about 0.5 to 5 mg/kg for both indications.
The present invention involves various dosing regimens. The present invention includes a continuous schedule of doses, wherein the antagonist of IL-17 is introduced on a regular basis (daily, weekly, or monthly, depending on the dose and dosage form), with no significant breaks. The preferred continuous dosing regimens include Radnevo continuous infusion, when the antagonist of IL-17 is administered by infusion every day, and continuous modes introduction shock doses, when the antagonist of IL-17 is injected at least once a day by injection loading dose of the substance or inhalation or intranasal ways. The present invention also includes intermittent dosing regimens. The exact parameters of the intermittent introduction will vary in accordance with formulation, method of delivery and the clinical needs of the mammal being treated. For example, if the antagonist of IL-17 is administered by infusion, injection can contain the first period of introduction, followed by a second period of introduction, in which the antagonist of IL-17 is not inserted, and this period is more than, equal to or less than the first period.
With the introduction by the injection of a test dose of a substance, in particular injection loading dose of the substance in the case of the formulation with delayed release, dosing regimens may also be continuous in the sense that the antagonist of IL-17 administered daily, or may be intermittent, with the first and second periods, as described above.
Continuous and discontinuous modes of introducing any way to also include dosing regimens in which the dose is changed in the first period so that, for example, in the beginning of the first period, the low dose and increasing down on the end of the first period, dose initially high and decreases during the first period, the dose is initially low, increases to a peak, and then decreases until the end of the first period, and any combination of this.
The effects from the introduction of the antagonist of IL-17 on insulin resistance can be measured using various methods known in this field. Most often the mitigation of the effects of diabetes will lead to improved glycemic control (as measured by serial analyses of glucose in the blood), reduction in insulin requirements to maintain good glycemic control, lower levels of glycosylated hemoglobin, reducing the level of improved glycosylation end products (AGE) in the blood, decrease morning hyperglycemia, reduction of ketoacidosis and improve lipid profile. Alternatively, the introduction of antagonist IL-17 can lead to stabilization of the symptoms of diabetes, as evidenced by the reduction of glucose levels in the blood, reducing the need for insulin, reduction of glycosylated hemoglobin and AGE in the blood, softening vascular, renal, neuronal and retinal complications, alleviating the complications of pregnancy and improve lipid profile.
The effect of reducing blood sugar antagonist of IL-17 can be assessed by determining kontsentratsiyami or Hb (hemoglobin) A 1cplasma venous blood from the subject before and after administration, followed by comparison of the obtained concentration before administration and after administration. HbA1cindicates glycosylated hemoglobin, which is gradually formed in response to the concentration of glucose in the blood. Therefore, it is believed, HbA1cimportant as an indicator of blood sugar control, which is less affected by rapid changes in blood sugar in patients with diabetes.
Proof treatment hypoinsulinemia demonstrate, for example, to increase circulating levels of insulin in patients.
Dosage to repair damage and regeneration of muscles is usually from about 0.01 to 100 mg/kg body weight, more preferably from 1 to 10 mg/kg, depending on the patient's condition, what specific type of repair damaged muscles necessary, etc., the dosage corresponds to the standard mode used by clinicians in this area. Proof of repair damage and regeneration of muscles demonstrated through tests to determine various parameters, are well known in this field, including analysis of proliferation and differentiation of muscle cells and testing with polymerase chain reaction (see, e.g., Best et al., J. Orthop. Res., 19:565-572 (2001), where the analysis changes the content of mRNA gene products in cultured myoblasts and fibroblasts in the treatment of skeletal muscle rabbit using a quantitative method of reverse transcription and subsequent polymerase chain reaction).
3. Products and kits
The present invention also aims at creating sets for the treatment of insulin resistance and hypoinsulinemia and to repair damage and muscle regeneration. Kits according to the invention, contain one or more containers antagonist of IL-17, preferably antibodies, in combination with a set of instructions, generally written instructions, relating to the use and dosage of the antagonist of IL-17 for the treatment of insulin resistance or hypoinsulinemia, or any other target diseases associated with insulin resistance. The instructions included with the kit generally include information on dosage, dosage and method of administration for treatment of the target disease, such as insulin resistance or hypoinsulinemia violation. Containers antagonist of IL-17 may be one-time doses, bulk packages (e.g., mnogorazovyj packaging) or partial doses.
The product includes a container and a label or leaflet insert in the package, or together with the container. Suitable containers include, for example, vials, ampoules, syringes, etc. Containers can be made of various materials, such as glass or plastic. The container holds a composition which is effective for treating the condition and may have input overstable sterile access (for example, the container may be a package solution for intravenous infusion or a bottle with a stopper that can be pierced by a needle for subcutaneous injection). At least one active agent in the composition is an antagonist of IL-17 in this invention. The label or leaflet-to the package insert indicates that the composition is used for treating a particular condition. The label or the leaflet insert in the package will contain instructions introduction the composition of the antibody to the patient. Also planned products and kits containing combination medications described in this document.
Leaflet-liner in the package refers to the instructions, usually included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, introduction, contraindications and/or warnings relating to the use of such therapeutic products.
In addition, the product may further comprise a second container with a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-saline buffer, ringer's solution and dextrose. It may additionally contain other materials required for commercial or user standpoint, including other buffers, RA is beviteli, filters, needles and syringes.
4. Preparation of antibodies
Monoclonal antibodies can be prepared using hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or can be prepared by methods using recombinant DNA (U.S. patent No. 4816567). In the hybridoma method, a mouse or other suitable animal host, such as a hamster or macaque, subjected to immunization, as described above, to extract the lymphocytes that produce or are capable of producing antibodies that will be specific to contact the protein used for immunization. In accordance with another variant lymphocytes may be immunized in vitro. After that lymphocytes merge with myeloma cells using a suitable agent that causes cell fusion, such as polyethylene glycol, with the formation of hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
Thus prepared hybridoma cells are sown and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival nesmith, source myeloma cells. For example, if the original myeloma cells lacking the enzyme gipoksantin-guanine-phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybrid the m will typically contain gipoksantin, aminopterin and thymidine (HAT-Wednesday) - substances that prevent the growth defective in HGPRT cells.
Preferred myeloma cells are cells that effectively merge, support stable high production of antibodies selected cells producing antibodies, and sensitive to the environment, as HAT-medium. In addition, the preferred lines of myeloid cell lines are murine myeloma, such as derived from tumors of mice MOPC-21 and MPC-11, available at the Salk Institute Cell Distribution Center, San Diego, California, USA, and cells from SP-2 or X63-Ag8-653, available at the American Type Culture Collection, Rockville, Maryland, USA. Also described cell lines of human myeloma and heteromyinae mouse and human to obtain monoclonal human antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
In the culture medium in which hybridoma is grown cells, conduct quantitative determination of the production of monoclonal antibodies produced against the antigen. Preferably, the binding specificity of monoclonal antibodies produced hybridoma cells is determined by thus or analysis of binding in vitro, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
After identification of hybridoma cells, prod is ziruyuschih antibodies with desired specificity, the affinity and/or activity, the clones may be subjected to subclavian methods of serial dilutions and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable for this purpose culture medium include, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells may be grown in vivo as ascitic tumors in an animal.
Monoclonal antibodies secreted by the subclones, accordingly isolated from the culture medium, ascitic fluid or serum using standard methods of purification of immunoglobulins, such as, for example, chromatography on protein A-sepharose, hydroxiapatite, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies can be readily isolated and sequenced using standard methods (e.g., using oligonucleotide probes capable of specific contact with the genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells serve as a preferred source of such DNA. After separation of the DNA may be placed into expression vectors, which are then transferout cell host, such as E. coli cells, the cells COS monkey cells Chinese hamster ovary (CHO) or myeloma cells that would otherwise not produce immuno is globulinemia proteins, to ensure the synthesis of monoclonal antibodies in the recombinant cell host. Recombinant antibodies will be described in more detail below.
In an additional variant embodiment of the antibodies or antibody fragments can be isolated from phage libraries of antibodies obtained using the methods described in McCafferty et al., Nature, 348:552-554(1990).
Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the selection of antibodies mouse and human, respectively, using phage libraries. Subsequent publications describe the obtaining of high affinity (nm range) human antibodies by combining the variable domains of light and heavy chains (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these methods are suitable alternatives to conventional hybridoma methods of producing monoclonal antibodies for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by replacing the homologous sequences of the mouse on the coding sequence of the conservative domains of the heavy and light chain human (U.S. patent No. 4816567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or covalent joining teruya immunoglobulin sequence all or part of the sequence, coding nimmanahaeminda polypeptide.
In a typical case, such nimmanahaeminda polypeptides replace conservative domains of antibodies, or they replace the variable domains of one antigennegative site of an antibody to create a chimeric bivalent antibody containing one antigennegative site with specificity for the same antigen and other antigennegative site with specificity for a different antigen.
Humanized antibodies and human antibodies
Humanitariannet antibody contains one or more amino acid residues introduced into it from a source other than human cells. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanitarian to a significant extent can be carried out according to the method of winter and co-authors (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by replacing the CDRs or CDR sequences of rodents on the corresponding sequences of human antibodies. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), while substantially less than an intact human variable domain has been substituted posledovatelno the completion of the form, different from man. Practically, humanized antibodies typically are human antibodies in which CDR residues and possibly some FR residues substituted by residues from analogous sites in rodent antibodies.
The choice of the variable domains of a person, as in the light and heavy chains, which will be done upon receipt of humanized antibodies is very important to reduce antigenicity. In accordance with the so-called method of "most relevant" conduct screening sequence of the variable domain of a rodent antibodies against full library of known sequences of the variable domains of a human. The sequence of the person that is closest to that of the rodent is then accepted as a framework region (FR) of a person for gumanitarnogo antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). If another method is used a special frame region, derived from a consensus sequence of all human antibodies with a particular subgroup of light or heavy chains. The same frame area can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).
It is also important that antibodies were humanitarian with retention of high affinity for the antigen and other favorable bio is logicheskih properties. To achieve this goal, in accordance with the preferred method, humanized antibodies are prepared by analyzing the source sequences and various speculative humanized products using three-dimensional models of the source and humanized sequences. The spatial model of immunoglobulins public and known to a person skilled in this field. Available computer programs which illustrate and display probable spatial conformational structures of selected candidate immunoglobulin sequences. The study of these displays permits analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to contact their antigen. This way, FR residues can be selected and combined from the recipient and import sequences so that will be achieved the necessary characteristics of antibodies, such as increased affinity to the target(output) antigen(s). In General, the CDR residues are directly and most substantially involved in the effect on binding to the antigen.
In accordance with another variant embodiment, at present, possible to obtain transgenic belly is s (for example, mice) that are capable, after immunization, of producing a full set of human antibodies in the absence of endogenous production of immunoglobulins. For example, it has been described that the homozygous deletion of the gene of the connecting region of the heavy chain antibodies (J. sub.H) the chimeric and with mutations in the germline of mice results in complete inhibition of endogenous production of antibodies. The transfer set immunoglobulin genes from the cells of the germ line of such mice with mutations in the germline leads to the production of human antibodies after injection of antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Measurement., 7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human antibodies can also occur from libraries based on the phage display technique (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech 14:309 (1996)). Obtaining human antibodies from libraries of antibodies on the basis of phage display is described further below.
Various methods have been developed to obtain fragments of antibodies. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). Now, however, these fragments can be obtained directly from recombinant who's host cells. For example, antibody fragments can be isolated from phage libraries of antibodies, as discussed above. In accordance with another variant, fragments, Fab'-SH can be directly extracted from E. coli and chemically connected with the formation of fragments F(ab')2(Carter et al., Bio/Technology 10:163-167 (1992)). In another embodiment, as described in the example below, F(ab')2form using protein latinboy zipper GCN4, facilitating the Assembly of the molecule F(ab')2. In accordance with another method, the fragments F(ab')2can be selected directly from the culture of the recombinant host cells. Other methods of obtaining fragments of antibodies will be apparent to a person skilled in this field. In other embodiments, the embodiment selected antibody is a single-chain Fv fragment (scFv). Cm. WO 93/16185.
Multispecific antibodies have binding specificity for at least two different epitopes, the epitopes usually belong to different antigens. Although these molecules in normal will only communicate with two different epitopes (i.e., to be bispecific antibodies, BsAb), antibodies with additional specificnosti, such as trapezitinae antibodies included in this expression when used in this document. Methods of obtaining bispecific antibodies known in this about the Asti. Traditional getting a full-sized bispecific antibodies based on simultaneous expression of two pairs of heavy and light chains of immunoglobulins, with two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to a random combination of heavy and light chains of immunoglobulins, such hybridoma (quadroma) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually carried out with the help of stages affinity chromatography, quite complex, and the product will be low. Such methods are described in WO 93/08829 and in Traunecker et al., EMBO J., 10:3655-3659 (1991). In accordance with another method of the variable domains of the antibodies with the desired specificnosti binding (the binding sites of the antibody with the antigen) sew with sequences conservative domain of immunoglobulin. The stitching is preferably carried out with a conservative domain of the heavy chain of immunoglobulin containing at least part of the hinge, CH2 and CH3 regions. Preferably, the first conserved region of the heavy chain (CH1) containing the site necessary for binding to the light chain was present at least on one of the links. DNA encoding joining the heavy chains of immunoglobulins, and, if necessary, a light chain immunoglobulin is on, implemented in a separate expression vector and co transferout they fit the host body. This provides greater flexibility in adjusting the ratio relative to each other of the three polypeptide fragments in variants of the embodiments when unequal amounts of the three polypeptide chains used in the construction provide the optimum output. It is possible, however, to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high output or when the ratio does not play a significant role.
In a preferred variant embodiment of this method bispecific antibodies are composed of a hybrid heavy chain immunoglobulin with a first binding specificity in one arm and hybrid pairs of heavy chain and light chain of immunoglobulin (providing a second binding specificity) in the other shoulder. It was found that this asymmetric structure facilitates the separation of the desired bispecific connections from unwanted combinations of immunoglobulin chains, because the presence of the light chain of immunoglobulin in only one half of bispecific molecules provides an easy way of separation. This method is described in WO 94/04690. For more podrobno the descriptions get bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
In accordance with another method described in WO96/27011, the region of interaction between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers, which is extracted from cultures of recombinant cells. The preferred interaction area contains at least a part of the CH3 domain of the conservative domain of the antibody. In this method, one or more small side chains of the amino acids on the surface of interaction of the first molecule antibodies are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar with large side chains (chain) size creates on the contact surface of the second molecule antibodies by replacing large side chains of amino acids into smaller (e.g., alanine or threonine). This provides a mechanism to increase the output of heterodimer against unwanted end-products such as homodimers.
Bispecific antibodies include cross stitched or heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate can be connected with Avidya, the other with Biotin. Such antibodies have, for example, proposed to target immune system cells to unwanted cells (patentsa No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373). Heteroconjugate antibodies may be obtained using any standard method of cross-linking. Suitable agents for cross-linking are well known in this field and are described in U.S. patent No. 4676980, together with a number of methods of cross-linking.
Methods of obtaining bispecific antibodies, fragments of antibodies have also been described in the literature. For example, bispecific antibodies can be prepared using chemical crosslinking. Brennan et al., Science 229:81 (1985) describe the way in which the intact antibody proteoliticeski cleaved to obtain fragments F(ab')2. These fragments regenerate in the presence of dayalbagh complexing agent sodium arsenite to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide bonds. The resulting fragments, Fab' after this, in turn dinitrobenzene (TNB) derivatives. One of the derivatives of Fab'-TNB then again converted into Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB for education bispecific antibodies. Received bispecific antibodies can be used as agents for the selective immobilization of enzymes.
Fragments, Fab'-SH can also be directly extracted from E. coli and chemically coupled the education bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the receiving molecule F(ab')2fully gumanitarnogo bispecific antibodies. Each fragment Fab' separately secretarials the E. coli cells and was subjected to a direct chemical blending in vitro with education bispecific antibodies.
Also described different ways of obtaining and allocating fragments bispecific antibodies directly from a culture of recombinant cells. For example, bispecific antibody was obtained with the application of latinovich fasteners. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Peptides with latinoware clasps of proteins Fos and Jun made with Fab'-part of two different antibodies by gene fusion. Homodimeric antibodies recovered in the hinge region to form monomers and then re-oxidized to education heterodimeric antibodies. This method can also be used to obtain homodimeric antibodies. The method of obtaining "diatel described in Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative method of preparation of fragments bispecific antibodies. The fragments contain the variable domain of the heavy chain (VH) connected to the variable domain of the light chain (VL) by a linker that is too short for there to be a pairing between the two domains on the same chain. Accordingly, the domains VH and VL of a single piece of stimulated is received to pair with complementary domains VL and VH another fragment, thereby forming two antigenspecific site. Also reported another method of obtaining fragments bispecific antibodies with the use of dimers of single-chain Fv (sFv). Cm. Gruber et al., J. Immunol. 152:5368 (1994).
Assumed antibodies with more than two valencies. For example, can be prepared trapezitinae antibodies. Tuft et al. J. Immunol. 147:60 (1991).
Design effector functions
It may be desirable to modify the antibody according to the invention in respect to effector function, so as to reinforce the effectiveness of antibodies. For example, the Fc region may be introduced cysteine residue (residues), which will make possible the education miaocheng disulfide bond in this area. Thus obtained homodimeric antibody may have an improved ability to internalize and/or enhanced activity in the presence of complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Cm. Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be obtained by applying heterobifunctional cross-links, as described in Wolff et al. Cancer Research 53:2560-2565 (1993). In accordance with another variant can be constructed antibody that contains two Fc region and the benefit is are this may have enhanced activity in complement lysis and ADCC. Cm. Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).
Chimera antibody epitope binding to receptor recycling
In some embodiments, embodiments of the present invention may be desirable to use a fragment antibodies, but not of intact antibodies. In this case, it may be desirable to hold a modification of the antibody fragment to increase its half-life in serum. This can be achieved, for example, by introducing the epitope of the receptor binding recycling in a fragment antibodies (e.g., by mutation of the appropriate region in the antibody fragment or the implementation of the epitope into a peptide tag that is then stitched to the antibody fragment at either end or in the middle, for example, using the synthesis of DNA or peptide synthesis).
The epitope binding to receptor recycling preferably contains an area where any one or more amino acid residues from one or two loops of the Fc domain transferred to a similar position of the fragment of the antibody. Even more preferably, transferred three or more residues from one or two loops of the Fc domain. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (e.g., IgG) and transferred to the area CH1, CH3 or V. sub.H, or more than one such region of the antibody. In accordance with another variant of the epitope is taken from the CH2 domain of the oblast is ti Fc and transferred to the area of CL or VL region, or in both regions of the antibody fragment.
Other covalent modifications of the antibody
Covalent modifications of the antibody are within the scope of the present invention. They can be made by chemical synthesis or by enzymatic or chemical cleavage of the antibodies, if applicable. Other types of covalent modifications of the antibody is introduced into the molecule by reaction of the target amino acid residues of the antibody with an organic agent to obtain the derivative, which is capable of reacting with selected side chains or the N - or C-terminal residues. Examples of covalent modifications described in U.S. patent No. 5534615 specifically included in this document as a reference. The preferred type of covalent modification of the antibody comprises linking the antibody to one of the many non-protein polymers, such as polyethylene glycol, polypropyleneglycol or polyoxyalkylene, the method described in the U.S. patents№№ 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337.
Obtaining antibodies from a synthetic phage libraries of antibodies
In a preferred variant embodiment of the present invention is directed to a method of obtaining and selection of new antibodies using a unique method of phage display. The method includes obtaining a synthetic phage libraries of antibodies OS is ove single matrix frame region, the design is sufficient diversity within the variable domains, obtaining the display of polypeptides having different variable domains, the selection of candidate antibodies with high affinity to the target antigen and the selected antibodies.
A detailed description of the methods of phage display can be found, for example, WO03/102157, published December 11, 2003, the complete disclosure of which is specifically incorporated in this document by reference.
In one variation of the embodiment of the present invention, the library of antibodies used in the invention may be obtained by mutations in the solvent-accessible and/or vysokopribylnyj positions in at least one CDR of the variable domain of the antibody. Some or all of the CDR can be subjected to mutations, using the method described in this document. In some embodiments, the embodiments may be preferred for various libraries of antibodies by mutation engine positions in CDRH1, CDRH2 and CDRH3 with the formation of a single library, or by mutation engine positions in CDRL3 and CDRH3 with the formation of a single library, or by mutation engine positions in CDRL3 and CDRH1, CDRH2 and CDRH3 with the formation of a single library.
For example, can be obtained library variable domains of antibodies having mutations in the solvent-accessible and/or vysokopribylnyj positions CDRH1, CDRH2 THE CDRH3. Can be obtained from another library with mutations in CDRLl, CDRL2 and CDRL3. These libraries can also be used in conjunction with each other to obtain binding molecules with desired affinity. For example, after one or more rounds of screening libraries of the heavy chains binding to the target antigen library of light chains can be moved in the population of binding molecules on the basis of the heavy chains for further rounds of selection to increase the affinity binding molecules.
Preferably, the library is generated by replacement of the initial amino acids of variant amino acids in the CDRH3 region of the variable domain (region, region) sequences of the heavy chain. The resulting library can contain many sequences of antibodies and the sequence diversity observed mainly in the field of CDRH3 sequences of the heavy chain.
In one variation of the embodiment of the present invention, the library is created in the context of sequence gumanitarnogo antibody 4D5 or amino acid sequence frame sequences gumanitarnogo antibody 4D5. Preferably, the library is created by substitution of at least residues 95-100a in the heavy chain amino acids encoded by the set of codonsDVKwhile the set of codonsDVKremaneat to set encoding variant amino acids for each of these positions. An example set of oligonucleotides, useful for performing these substitutions comprises the sequence(DVK)7.In some embodiments, embodiments of the present invention the library create by replacing residues 95-100a amino acids encoded by sets of codons asDVK,andNNK. An example set of oligonucleotides, useful for performing these substitutions comprises the sequence(DVK)6(NNK).In another variant embodiment of the present invention, the library is generated by replacing at least residues 95-100a amino acids encoded by sets of codons asDVKandNNK. An example set of oligonucleotides, useful for performing these substitutions comprises the sequence(DVK)5(NNK).Another example of a set of oligonucleotides, useful for performing these substitutions comprises the sequence(NNK)6. Other examples of suitable oligonucleotide sequences can be determined by the person skilled in the art in accordance with the criteria described in this document.
In another variant embodiment of the present invention various designs CDRH3 used for selecting high-affinity binding molecules and the selection of binding molecules for different epitopes. The range of CDRH3 lengths obtained in this library, sostavlayut 11 to 13 amino acids, although there may be obtained, and fragments of other lengths. The diversity of H3 can be extended with sets of codonsNNK, DVK and NVKand more limited diversity at the N - and/or C-end.
Diversity can also be obtained in CDRH1 and CDRH2. When creating diversity in CDR-H1 and H2 follow the strategy of the desire to recreate the similarity with the set of natural antibodies, as described in the case of modifications, which addresses diversity, more closely corresponding to the natural variety than the previous design.
For diversity in CDRH3 can be constructed separately multiple libraries with different lengths of H3, which then unite for the selection of binding molecules for the target antigen. Many libraries can be merged and sorted using selection techniques on a solid substrate and sorting in solution, as described previously and below in this document. Can be applied different strategies of sorting. For example, one alternative embodiment includes sorting by the target immobilized on a solid substrate, followed by the sorting on the label that may be present in the chimeric polypeptide (for example, the label anti-gD), followed by another sorting by the target immobilized on a solid substrate. In accordance with another variant library can nachlassgericht by the target, immobilized on a solid substrate, and erwerbende binding molecules are then sorted using bind soluble phase with decreasing concentrations of the target antigen. The use of combinations of different ways of sorting minimizes selection only sequences with a high level of expression and focus on the selection of the number of different clones with high affinity.
High-affinity binding molecule for the target antigen can be isolated from libraries. Limiting the diversity in the field H1/H2 reduces the degeneracy of about 104-105time and a resolution of greater diversity in H3 provides reception of binding molecules with a higher affinity. Application libraries with different types of diversity in CDRH3 (for example, using DVK or NVT) provides the selection of binding molecules that can communicate with different epitopes on the target antigen.
For binding molecules isolated from the joint, as described above, libraries, it was found that the affinity can be further improved by providing a limited diversity in the light chain. A variety of light chain get in this embodiment as follows: in CDRL1: the amino acid position 28 is encoded RDT; amino acid position 29 is encoded RKT; position aminoxy is lots 30 is encoded RVW; the amino acid position 31 is encoded ANW; amino acid position 32 is encoded THT; optional position of 33 amino acids is encoded by CTG; CDRL2: position 50 amino acids encoded KBG; amino acid position 53 is encoded AVC; and, optionally, the amino acid position 55 is encoded GMA; CDRL3: the amino acid position 91 is encoded TMT or SRT, or both; position 92 amino acids encoded DMC; amino acid position 93 is encoded RVT; amino acid position 94 is encoded NHT; and the amino acid position 96 is encoded TWT or YKG, or both.
In another variant embodiment of the present invention receive a library or libraries with diversity in regions CDRH1, CDRH2 and CDRH3. In this embodiment, the diversity in CDRH3 get with the use of a variety of lengths, areas H3 and using mostly sets of codonsXYZ and NNK or NNS.Libraries can be generated using a single oligonucleotide and the joint, or oligonucleotides can be combined to form subtypes libraries. Libraries in this embodiment can be sorted with respect to the target immobilized on a solid substrate. Clones isolated from a variety of types that can be thrown screening specificity and affinity by using ELISA. To determine the specificity of the clones can be subjected to screening for the desired target antigen, as well as others, untargeted, antigens. After that, the binding molecules that bind to the target antigen, can be subjected to screening for affinity with the use of ELISA for competitive binding in solution or analysis of competitive binding in spots. High-affinity binding molecules can be isolated from a library that uses sets of codonsXYZprepared as described above. These binding molecules can be easily obtained in the form of antibodies or antigenspecific fragments with high yield in cell culture.
In some embodiments, embodiments of the present invention can be preferably obtaining a library with a large variety of lengths CDRH3 region. For example, it may be desirable receipt of libraries with CDRH3 regions in the range of from about 7 to 19 amino acids.
High-affinity binding molecules isolated from libraries in these embodiments, easy to get into the culture of a bacterial or eukaryotic cells with high yield. Can be constructed vectors, which can easily be removed sequences, such as labels gD, sequence component of the viral envelope protein, and/or insertion in the sequence of conserved regions to ensure the production of full-size antibodies or antigenspecific fragments with high you who Odom.
Library with mutations in CDRH3 can be combined with the library containing the variant versions of other CDR, such as CDRL1, CDRL2, CDRL3, CDRH1 and/or CDRH2. Thus, for example, in one embodiment, the CDRH3 library combined with the library CDRL3 created in the context of sequence gumanitarnogo antibody 4D5 with variant amino acids at positions 28, 29, 30, 31 and/or 32 using pre-defined sets of codons. In another embodiment, a library with mutations in CDRH3 can be combined with a library containing variant CDRH1 and/or CDRH2 in the variable domains of the heavy chain. In one embodiment, the library CDRH1 created on the basis of sequence gumanitarnogo antibody 4D5 with variant amino acids at positions 28, 30, 31, 32 and 33. Library CDRH2 can be created on the basis of sequence gumanitarnogo antibody 4D5 with variant amino acids at positions 50, 52, 53, 54, 56 and 58 using pre-defined sets of codons.
It is assumed that the above written description is sufficient for a person skilled in the art could practice the present invention. The following examples are given only for purposes of illustration and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of izobreteny is, in addition to those shown and described in this document will be obvious to the person skilled in the art from the foregoing description and fall within the scope of the attached claims.
Commercially available reagents referred to in the examples were used according to manufacturer's instructions, unless otherwise indicated. Source of cells identified in the following examples and in the description of the invention the access numbers ATCC is the American Type Culture Collection, Manassas, VA. Unless otherwise stated, the present invention apply standard methods of recombinant DNA technology, such as described above in this document and in the following manuals: Sambrooket al., above;Ausubelet al.,Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N. Y., 1989); Inniset al.,PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N. Y., 1990); Harlowet al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coliganet al.,Current Protocols in Immunology, 1991.
Additional detailed features of the invention are given in the following non-limiting examples.
All references cited in the disclosure of the invention specifically included here in its entirety as a reference.
The role of members of the family of IL-17 in diabetes and insulin resistance
Mouse IL-17Rc KO and study of mo who ate diets high in fat
8-week-old males of IL-17Rc (UNQ6118.KO.lex) knock-out and odnopolnyh wild-type (WT) control mice received either normal food diet, or 60% diet high fat diet (HFD).
GROUP 1: IL-17Rc knockout (KO) mice on a diet high in fat (5 animals)
GROUP 2: IL-17Rc wild-type odnomomentnye control animals on a diet high in fat (5 animals)
GROUP 3: IL-17Rc KO mice on a normal diet (3 animals)
GROUP 4: IL-17Rc wild-type odnomomentnye control animals on a normal diet (3 animals).
The experimental design is shown in Fig.7.
Mice were test glucose tolerance (GTT) to assess their status of insulin resistance.
GTT was performed using the following method.
Measurement of glucose and insulin in the blood: blood samples were obtained by taking blood from the saphenous vein and immediately analyzed for the concentration of glucose using glucometer (OneTouch glucometer production Lifescan, USA). The content of insulin in serum were determined using ELISA method.
Test for glucose tolerance (GTT): after an overnight fast (14 hours) were tested on animals in the morning, at 9:00. The level of glucose in the blood was measured in samples obtained by drawing blood from the saphenous vein before intraperitoneally injection of glucose, 1.5 mg/gram t the La of each animal, and after 30, 60, 120 and 150 minutes after administration of glucose. Values were calculated as the concentration of glucose in mg/DL.
GTT was performed for the initial level (before switching to a diet high in fat), and at week 8, week 10, week 12 and week 14 after the start of participation in the group with a diet high in fat. Mice that were fed normal food diet was used as control groups. Other conditions were similar to knock-out and wild-type (WT) odnopolnyh control mice.
In addition to the GTT weekly measured total body weight of the animal, as well as serum insulin and glucose on an empty stomach.
The results are presented in Fig.8-11.
While odnomomentnye control mice with IL-17Rc wild type showed a significant increase in weight and development of the phenotype with insulin resistance, knock-out by IL-17Rc mice were significantly thinner and withdrew the glucose much better than their wild-type odnomomentna control group. Even after receiving diets with high fat diet for more than 12 weeks knockout mice did not gain weight. Both groups demonstrated similar levels of circulating insulin on an empty stomach. There were no significant differences between knockout and wild type mice in the groups receiving the control diet.
Besides the op is sliding above experiment with IL-17Rc KO mice were performed TWO additional lines of research to explore the role of proinflammatory cytokines IL-17A and IL-17F in diabetes and insulin resistance.
Effect of monoclonal antibodies (mAb) against IL-17 and against IL-17F in mice with a model of insulin resistance caused by a diet high in fat
The aim of this study was to examine the effectiveness of mAb against IL-17 and against IL-17F in preventive and established model of insulin resistance and comparison with therapeutic effect muTNFRII-Fc.
The planning of the experiment and groups:
Group 1: Agglutinin ragweed, 6 mg/kg in 100 μl solution of sodium chloride IP 3 times per week for 10 weeks (n=10).
Group 2: MuTNFRII-IgG2a, 4 mg/kg in 100 μl solution of sodium chloride 3 times a week for 10 weeks (n=10).
Group 3: MuAnti-IL-17, 6 mg/kg in 100 μl solution of sodium chloride IP 3 times per week for 10 weeks (n=10).
Group 4: MuAnti-IL-17+MuAnti-IL-17F mAb, 6 mg/kg in 100 μl solution of sodium chloride IP 3 times per week for 10 weeks (n=10).
Group 5: MuTNFRII-Fc 4 mg/kg = MuAnti-IL-17 6 mg/kg+MuAnti-IL17F mAb 6 mg/kg for 18 weeks and 24 weeks (10 animals).
All groups received a diet high in fat. To assess the status of insulin resistance mice were test glucose tolerance (GTT) every 2 weeks HFD (diets high in fat) and doses of antibodies.
The Protocol is illustrated in Fig.12. Effect of monoclonal antibodies against IL-17A and anti IL-17F on glucose tolerance after 9 weeks introduction the Oia doses shown in Fig.13.
The effect of overexpression of IL-17 on the status of insulin resistance, assessed using GTT
The study is based on hydrodynamic injection into the tail vein (HTV) plasmid DNA for expression of native proteins of mouse IL-17A and IL-17F in normal and treated with a diet high in fat mice to high expression levels of Pro-inflammatory cytokines mouse IL-17A and IL-17F in mice to study their role in insulin resistance.
Group 1: without plasmids
Group 2: only vector pRK
Group 3: pRK-IL-17A
Group 4: pRK-IL-17F
Within each group were injected with 5 subgroups of mice for screening blood at different time points (0 h, 2 h, 6 h, 24 h and 72 h after injection of DNA) to measure levels of circulating cytokines in serum. When this parameter has been set, has been overexpression of IL-17A and IL-17F in mice with diet high fat diet (HFD) for estimating change of status of insulin resistance.
Experiments on injection into the tail vein of:
1) Structure of DNA (vector pRK or pRK-IL-17A and pRK-IL-17F) were diluted in sodium chloride solution (preferably, ringer's solution) to a concentration that will give a final dose of 50 μg/mouse/injection.
2) of Each mouse was done by intravenous injection into the tail vein of approximately 1.6 ml of a solution containing DNA in solution of sodium chloride sludge is the ringer's solution.
3) Dose was administered as intravenous loading dose (tail vein) over a period of 4-5 seconds (maximum of 8 seconds) for maximum absorption of DNA.
The results are presented in Fig.14. A) the Eight-week c57BL/6 mice were injected with 50 μg of plasmid DNA (pRK-lL-17A) or only vector pRK. After 48 hours in both groups were collected serum and measured the level of IL-17 in serum using ELISA. B) Three groups of mice after an overnight fast subjected intraperitoneally GTT, and the results are presented in the form of a graph based on time elapsed after the injection of glucose (*p>0,05).
Although the present invention has been described with reference to specific examples of embodiments, it is understood that the present invention is not limited to such embodiments. On the contrary, it is understood that the present invention covers various modifications and equivalents included in the nature and scope of the attached claims.
1. A method of treating insulin resistance in a mammal, which includes an introduction to the needy in the mammal an effective amount of an antagonist of IL-17A and/or IL-17F, where specified, the antagonist is an antibody or antigennegative fragment.
2. The method according to p. 1, wherein the mammal is a violation selected from the group consisting of insulinnezawisimy the CSOs diabetes (NSSD), obesity, giperandrogenii ovarian cancer and hypertension.
3. The method according to p. 2, characterized in that the violation is NSSD or obese.
4. The method according to p. 1, wherein the mammal is a human, and the introduction of a system.
5. The method according to p. 1, wherein the antibody is an antibody selected from the group consisting of antibodies against IL-17A, against IL-17F, against the IL-17A/F, against IL-17Rc and against IL-17RA.
6. The method according to p. 5, characterized in that the antibody is a monoclonal antibody.
7. The method according to p. 6, characterized in that the antibody is chimeric, humanized antibody or human antibody.
8. The method according to p. 7, characterized in that the antibody is bispecific, multispecific or have cross-reactivity with the antibody.
9. The method according to p. 8, characterized in that it further includes introducing an effective amount of an agent for the treatment of insulin resistance.
10. The method according to p. 9, characterized in that the agent for the treatment of insulin resistance is insulin, IGF-1 or sulfonylurea.
11. The method according to p. 9, characterized in that it further includes introducing an effective amount of an additional agent suitable for the treatment specified disorders associated with insulin resistance.
12. The method according to p. 11, characterized in that the more the agent is Dickkopf-5 (Dkk-5).
13. The method according to p. 1, characterized in that the introduction of the antagonist reduces the level of glucose in the blood at the specified mammal.
14. Pharmaceutical composition comprising an antagonist of IL-17A and/or IL-17F with the addition of pharmaceutically acceptable excipients, for the treatment of insulin resistance, where the antagonist is an antibody or antigennegative fragment.
15. The pharmaceutical composition according to p. 14, characterized in that the antibody is an antibody selected from the group consisting of antibodies against IL-17A, against IL-17F, against the IL-17A/F, against IL-17RC and against IL-17RA.
16. The pharmaceutical composition according to p. 15, wherein the antibody is a monoclonal antibody.
17. The pharmaceutical composition according to p. 16, wherein the antibody is a chimeric, humanized antibody or human antibody.
18. The pharmaceutical composition under item 17, characterized in that the antibody is bispecific, multispecific or have cross-reactivity with the antibody.
19. The use of an antagonist of IL-17A and/or IL-17F for the manufacture of a medicinal product for the treatment of insulin resistance, where the antagonist is an antibody or antigennegative fragment.
20. Set for the treatment of insulin resistance, comprising: (a) a container containing the antagonist is East of IL-17A and/or IL-17F; and (b) the label or instructions for the introduction of this antagonist for the treatment of the indicated insulin resistance, where the antagonist is an antibody or antigennegative fragment.
SUBSTANCE: humanised antibody against human IFN-α is proposed, obtained based on the murine antibodies ASO-1. Also the therapeutic composition and method of prevention, treatment or diminution of intensity of the symptoms of an autoimmune or inflammatory disease or disorder is considered.
EFFECT: invention may find further application in treatment of IFN-α-related diseases.
13 cl, 13 dwg, 18 tbl, 16 ex
SUBSTANCE: invention refers to immunology and biotechnology. What is presented is a single-domain antibody (VHH) able to bind the cytokine human necrosis factor (TNF), a DNA fragment coding the antibody according to the invention, as well as a method for identifying the human TNF and measuring it in a biological sample.
EFFECT: invention provides the specific block of macrophague-monocytic TNF cells and can find further application in therapy of the diseases associated with an undesired TNF-dependent inflammatory response.
4 cl, 5 ex, 7 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to immunology. Claimed are versions of applying versions of sets of genes (pharmacodynamic (PD) markers) with increased regulation of expression or action, induced by alpha interferon (αIFN). Described are versions of method of identifying patients for obtaining MEDI-545 by identification of such profiles of expression of αIFN-induced FD markers.
EFFECT: application of invention ensures identification of patients, for which treatment with neutralising antibody MEDI-545 can be really effective, which can be applied in medicine.
10 cl, 90 dwg, 33 tbl, 22 ex
SUBSTANCE: present invention refers to immunology and biotechnology. What is presented is an IL-1β-binding antibody or its IL-1β-binding fragment containing V heavy and light chain regions. The above antibody binds to human IL-1β with dissociation constant less than 1pM. Versions of the antibody are described. There are disclosed corresponding coding nucleic acids (NA), as well as: a NA passage vector to a host cell, the host cell producing a coded polypeptide. What is described is using the antibody for preparing the other format of the above antibody: "camel-like", VHH antibody, nanobody. What is disclosed is a pharmaceutical composition for treating or preventing an IL-1β-related disease in a mammal on the basis of the antibody, as well as a method of treating or preventing the IL-1β-related disease in a mammal.
EFFECT: using the invention provides the novel IL-1β-specific antibodies with high IL-1β affinity that can find application in medicine for preventing, treating the diseases mediated by IL-1β activity.
39 cl, 20 dwg, 6 tbl, 14 ex
SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed are versions of an isolated monoclonal antibody, specific to hGM-CSF, where each version is characterised by a heavy and light chain. Each of the versions is characterised by the fact that it contains six appropriate CDR. Described are: a pharmaceutical composition, and a set, representing medication, based on the antibody application. Disclosed are: a coding isolated nucleic acid, an expression vector, containing it, and a vector-carrying host cell, used for the antibody obtaining. Described is a method of obtaining the antibody with the cell application.
EFFECT: claimed inventions can be applied for treating disease or disorder, associated with superexpression of hGM-CSF.
25 cl, 9 dwg, 14 tbl, 15 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions relates to methods of obtaining preparation of antibody against IL-18 or its antigen-binding part with reduced content of host cell protein (HCP) from sample of mixture, containing antibody against IL-18 or its antigen-binding part, and, at least, one HCP. Method includes reduction of mixture sample pH from 3.0 to approximately 4.0, bringing pH from 4.5 to approximately 5.5 and conductivity to 9±0.5 mS/cm, application of sample on cation-exchange resin, carrying out chromatography of hydrophobic interaction and sample collection. Version of method includes realisation of anion-exchange chromatography before the stage of cation-exchange chromatography. Version of method also includes sample filtration before anion-exchange chromatography.
EFFECT: group of inventions makes it possible to obtain preparation of antibody with increased output and purity Output constitutes 96±4%, purity 99,51±0,26%.
28 cl, 2 dwg, 9 tbl, 2 ex
SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.
EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.
2 cl, 4 dwg, 3 ex
SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.
EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.
22 cl, 14 dwg, 16 ex
SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.
EFFECT: invention allows obtaining antibodies to IL-21 of a human being.
48 cl, 4 dwg, 16 tbl, 23 ex
SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.
EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.
3 cl, 5 tbl, 7 ex
SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.
EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.
18 cl, 124 dwg, 10 ex
SUBSTANCE: therapeutic agent containing activated-potentiated forms of anti-histamine, anti-tumour necrosis factor alpha (anti-TNF - α) and anti-S-100 brain specific antibodies is administered.
EFFECT: treating functional bowel disorders by ensuring the spasmolytic action and normalising the motor-evacuation function of the intestine.
11 cl, 4 ex, 4 tbl
SUBSTANCE: method includes carrying out drug treatment, diet therapy, physical exercise, balneotherapy. Drug treatment includes intake of an ACE inhibitor or a beta-blocker 30-40 minutes before the first breakfast. 2-2.5 hours after supper methmorphine in a dose of 500-1000 mg/day is taken in. Diet therapy includes fractional feeding with the calorie content of 1200 kilocalories from Monday to Friday. On Saturday the calorie content is reduced to 800-900 kilocalories, and on Sunday - to 600-700 kilocalories. The physical exercise is carried out in the form of walking in a slow tempo, with stops, with the total duration of 90-120 minutes. Balneotherapy is started with carrying out in the morning of a rain fresh water shower. Then, manual superficial massage of the neck zone is carried out. Baths take place in the period from 12 to 17 o'clock. For their carrying out hydrocarbonate-sulfate-sodium mineral water from Belokurikha resort with an increased content of silicic acid and fluorine, and mineralisation of 0.4 g/l is applied. Radon concentration for bath realisation constitutes 3.9-4.6 nCi/dm3, water temperature is 36-37°C. The duration of the bath time is five minutes on the first day, on the second day - eight minutes, on the third day - ten minutes, on the fourth day - rest, on the fifth and sixth days - twelve minutes, on the seventh day - fifteen minutes, on the eighth day - rest, on the following three days the baths are being carried out for 15 minutes.
EFFECT: method increases remission duration due to an increase of the non-specific resistance of the organism protection factors, normalisation of oxygenation and metabolic processes in tissues.
4 cl, 2 tbl, 2 ex
SUBSTANCE: present invention refers to fumaryldiketopiperazine (FDKP) microparticles applicable for pulmonary delivery and having a specific surface area within the range of 15 m2/g to 67 m2/g and a diameter within the range of 0.5 mcm to 10 mcm. The invention also refers to a dry powder comprising said microparticles that is also applicable for pulmonary delivery, to an inhalation system comprising a powder inhalation apparatus and the above dry powder, as well as to a method for delivering an active agent being an ingredient of the dry powder by inhalation of the above dry powder. There are also disclosed methods for forming the FDKP microparticles, first of which comprises dissolving FDKP in aqueous ammonia, adding an acidic solution to aqueous ammonia at temperature up to 12°C to 26°C and collecting a precipitate containing the FDKP microparticles after rinsing with deionised water. A second method for forming the FDKP microparticles involves supplying equal portions of 10.5 wt % of an acetic acid solution and 2.5 wt % of a FDKP solution at temperature 14-18°C through a high-shear mixer.
EFFECT: invention aims at preparing the FDKP microparticles applicable for pulmonary delivery that have the good aerodynamic performance and provide the enhanced drug absorption.
18 cl, 4 tbl, 9 dwg, 2 ex
SUBSTANCE: invention refers to a pharmaceutical tablet for oral administration containing ulipristal acetate 3-18 wt %; a diluent 60 - 95 wt % specified in lactose monohydrate, microcrystalline cellulose, cellulose, mannitol and combinations thereof; a binding agent 0 - 10 wt % specified in hydroxypropyl methyl cellulose, povidone and combinations thereof; sodium croscarmellose 1 - 10 wt % and magnesium stearate 0.5 - 4 wt %.The invention also refers to a method for preparing the above tablet which involves mixing the ingredients and forming the tablet by wet granulation or direct compression.
EFFECT: invention provides the new formulation of the tablet with improved disintegration.
13 cl, 2 dwg, 5 tbl, 6 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: microparticle contains an agglomerate of particles containing a hydrophilic active substance, wherein the particle contains an amphiphilic polymer composed of a hydrophobic segment of polyhydroxy acid and a hydrophilic segment of polysaccharide or polyethylene glycol, and a hydrophilic active substance. What is also disclosed is a method of producing the agglomerated microparticles, which involves (a) a stage of preparing a reverse phase emulsion, (b) a stage of preparing a solid residue containing the hydrophilic active substance, and (c) a stage of introducing the solid residue into a liquid phase containing a surface modifier.
EFFECT: agglomerated microparticles provide the effective encapsulation of the hydrophilic active substance and the release of the hydrophilic active substance at an appropriate speed.
14 cl, 22 dwg, 4 tbl, 31 ex
SUBSTANCE: medicinal agent for inhibition of MASP-2-dependent complement is an agent containing an antibody or its fragment, bound with a full-size polypeptide MASP-2, but not bound with a MASP-2 N-terminal fragment containing CUBI-EGF-CUBII domains and not bound with a MASP-2 C-terminal fragment, containing of CCPII-SP domains.
EFFECT: invention makes it possible to selectively inhibit MASP-2-dependent activation of complement, at the same time leaving Clq-dependent classic path of complement activation as functionally unaffected.
9 cl, 39 dwg, 7 tbl, 31 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to combinations of peptides in each case with the same sequence length (SEQL) which can be prepared in a stable reproducible quality and quantity of a mixture (A) containing a number of x amino acid with protected acid groups or a number of z peptides with the acid groups protected by the protective groups and the activated amino groups, with the amino acids in the mixture (A) found in a specific molar ratio, and a mixture (B), containing a number of y amino acids with the amino groups protected by the protective groups, with a molar ratio of the amino acids of the mixture (B) being the same as the molar ratio of the amino acids of the mixture (A), and the number x=y, and x is a figure from 11 to 18.
EFFECT: new combinations of the peptides are presented.
13 cl, 2 dwg, 1 ex
SUBSTANCE: invention refers to medicine, namely gynaecology, and may be used for treating chronic inflammatory genital diseases in females. That is ensured by the use of Promed balm which is introduced into vagina once a day, as well as taken orally 3 times a day. Promed balm may be introduced into vagina taking into account biological rhythms from 19 to 21 o'clock, and taking into account biological rhythms 9 to 11 o'clock, from 13 to 15 o'clock and from 19 to 21 o'clock. The method provides the improved clinical effectiveness ensured by providing the local and systemic anti-inflammatory effect due to the delayed growth of pathogens and toxin purification of the female body as a result of a diuretic and choleretic effect, immunomodulatory action, as well as due to activation of biological centres and their rhythm functioning, and regulation of metabolism and genital glands. In addition, it has a regenerating effect on skin and mucous membranes and recovers the nervous system.
EFFECT: achieved diet fortification and prevented recurrence of the disease.
3 cl, 2 ex
SUBSTANCE: claimed invention relates to medicine, namely to therapy and can be used for treatment of patient with thermoregulation disorder, in particular suffering from high temperature attacks and hot flushes, night sweats and their combinations, as well as conditions, selected from female and male hormonal changes, connected with menopause, hormonal changes, caused by chemical way, disease states, causing hormonal irregularities and any combinations of said conditions. For this purpose paroxetine dosed in terms of paroxetine component, 7.5 mg/day is introduced to said patient.
EFFECT: method ensures efficient treatment of such conditions due to optimal dose.
9 cl, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions refers to medicine and concerns a method for preparing a spray-dried stable powder composition containing an antibody or its antigen-binding fragment; a pharmaceutical preparation containing an effective amount of the spray-dried stable powder composition containing the antibody or its antigen-binding fragment and prepared by the method described above; the spray-dried stable powder composition prepared by the method described above, containing the antibody or its antigen-binding fragment, and an excipient, wherein the composition contains residual moisture of 6% or less and the antibody or its antigen-binding fragment is specified in a group consisting of MAK 195F, ABT-325, ABT-308 or ABT-147.
EFFECT: group of inventions provides preparing the spray-dried high-stability powder composition.
44 cl, 11 dwg, 14 tbl