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Definitions

• the present invention relates to the field of medicine, in particular to the field of monoclonal antibodies against human IL-17.
• the invention relates to monoclonal antibodies, antagonists of IL-17A, which bind with high affinity to the epitope of IL-17, while the antibodies contain amino acid substitutions in the hypervariable regions of the heavy and light chains.
• Antibodies according to the invention can be chimeric, humanized or human antibodies, or antigen-binding fragments thereof, and can be used as a medicament for the treatment of autoimmune and inflammatory disorders and cell proliferation and cell development disorders.
• the invention also relates to methods for producing said antibodies.
• the main function of the immune system is to protect the body from infections, such as bacteria or viruses, as well as from cancer.
• This body system includes several types of lymphoid and myeloid cells, such as monocytes, macrophages, dendritic cells, eusinophils, T cells, B cells and neutrophils. Said lymphoid and myeloid cells can produce signaling proteins known as cytokines.
• the immune response includes inflammation, i.e. accumulation of immune cells systemically or in a specific
• SUBSTITUTE SHEET (RULE 26) body area.
• immune cells secrete cytokines, which in turn modulate the proliferation of immune cells, their development, differentiation or migration.
• the immune response can produce pathological processes, for example, when triggering excess inflammation, as in the case of autoimmune diseases (see, for example, Abbas et al. (Eds.) (2000) Cellular and Molecular Immunology, WB Saunders Co., Philadelphia, Pa .; Oppenheim and Feldmann (eds.) (2001) Cytokine Reference, Academic Press, San Diego, Calif .; von Andrian and Mackay (2000) New Engl. J. Med. 343: 1020-1034; Davidson and Diamond (2001) New Engl. J. Med. 345: 340-350).
• the IL-17 family of cytokines includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. All members of the IL-17 family have four highly conserved cysteine residues that are involved in the formation of interchain disulfide bonds, and have two or more cysteine residues that can be involved in interchain disulfide bonds. Members of the IL-17 family bear no resemblance to the sequences of any other known cytokines.
• Interleukin-17A also known as cytotoxic IL-17A
• T-lymphocyte-associated antigen 8 (CTLA-8), IL-17) is a 20-30 kDa homodimeric cytokine produced by memory T cells, followed by antigen recognition. Growth of such CTL-8, IL-17
• T cells are stimulated by interleukin-23 (IL-23).
• IL-23 interleukin-23
• IL-17A acts through two receptors (IL-17RA and
• IL-17RC to initiate the production of many molecules involved in processes involving neutrophils, inflammatory processes and organ destruction.
• This cytokine has a synergistic effect with factor
• SUBSTITUTE SHEET (RULE 26) tumor necrosis (TNF) or interleukin 1 beta (IL- ⁇ ) to achieve a more significant pro-inflammatory state.
• TNF tumor necrosis
• IL- ⁇ interleukin 1 beta
• RA rheumatoid arthritis
• osteoarthritis osteoporosis with rheumatoid arthritis
• inflammatory fibrosis e.g.
• These antibodies are characterized by high affinity for IL-17, with a dissociation constant K D of approximately 122 ⁇ .
• K D dissociation constant
• the use of these antibodies as inhibitors of the binding of IL-17 to the corresponding receptor is also described, in particular for the treatment of uveitis.
• the described antibody is undergoing clinical trials for the treatment of rheumatoid arthritis, ankylosing spondylitis, disease
• SUBSTITUTE SHEET (RULE 26) ozone air. This antibody has been shown to be safe to use and effective in treating rheumatoid arthritis in 46% of patients (Durez et al., Communication EULAR 2009-RA).
• humanized antibodies against IL-17 are disclosed according to the international publication of application WO 2007/070750 (LY2439821, ikzekizumab, Eli Lilly). These antibodies contain variable regions of the heavy and light chains with amino acid substitutions. Moreover, the described antibody is characterized by a strong affinity for binding (KD) of human IL-17, i.e. less than about 7.0-4.0 pM, and a koff excretion rate for human IL-17 of less than 2 ⁇ 10 "5 s " 1 . Antibodies have been clinically tested for safety, tolerability and efficacy when given intravenously to patients with rheumatoid arthritis. (Genoevse et al., Communication EULAR 2009-RA; Genoevse et al, Arthritis & Rheumatism, 62: 929-39, (2010)).
• IL-17 antibodies to IL-17 are also described in the prior art: mouse anti-human neutralizing antibodies eBio64CAP17 (eBioscience) or derivatives thereof.
• Other examples of antibodies to IL-17 are disclosed in patent applications US2009 / 0175881A1, US2008 / 0269467A1, and international publications WO2008 / 001063A1, and WO2007 / 117749A1.
• IL-17 is a new target for the treatment of inflammatory and autoimmune diseases with a potentially greater safety profile than drugs that target pro-inflammatory cytokines of the pulmonary circulation, such as TNF (tumor necrosis factor) )
• TNF tumor necrosis factor
• SUBSTITUTE SHEET (RULE 26) antagonistic effect or neutralize the activity of IL-17A for the treatment of disorders, diseases or conditions where the presence of biological activity of IL-17A causes or contributes to an undesirable pathological result, or where a decrease in the biological activity of IL-17A contributes to the desired therapeutic result, including inflammatory disorders, disorders cell proliferation and development; autoimmune disorders such as rheumatoid arthritis (RA), multiple sclerosis (PC), and inflammatory bowel disease (IBD).
• RA rheumatoid arthritis
• PC multiple sclerosis
• IBD inflammatory bowel disease
• the present invention provides antibodies against IL-17A containing amino acid substitutions, while the proposed antibodies have high affinity and improved solubility, as well as increased 1C 50 .
• FIG. 1 The foretogram of the resulting protein solution of IL-17A.
• FIG. 2 The foretogram of the resulting protein solution of IL-17A-Fc.
• FIG. 3 Gel electrophoresis in 12% SDS-PAGE under reducing conditions (A), non-reducing conditions (B).
• FIG. 4 The synthesis scheme of combinatorial Fab-llama library.
• FIG. 5. Phagmid for cloning Fab phage display libraries.
• FIG. 6 Scheme for the synthesis of combinatorial libraries of hybrid Fab antibodies.
• FIG. 7 Scheme for the synthesis of combinatorial libraries of hybrid Fab antibodies.
• FIG. 8 The combination of pools of variable domains.
• FIG. 9 ELISA screening Fab specific against human IL-17A.
• FIG. 10 Competitive ELISA screening of Fab candidates blocking the interaction of IL-17A / IL-17R.
• FIG. 11 Results of koff screening of anti-1b-17A Fab human candidates.
• FIG. 12 Electrophoregram of the solution BCD085.
• FIG. 13 The gel-filtration profile of the purified preparation BCD085.
• FIG. 14 IL-6 Inhibitory ability of BCD085 compared with AIN
• FIG. 15 The interaction curve of BCD085 with human IL-17.
• FIG. 16 Interaction curve of BCD085 with macaque IL-17 (Tasas mulatta).
• FIG. 17 Chromatogram of antibody BCD085 obtained after thermal stress.
• FIG. 18 Pharmacokinetics of the preparation of antibodies against IL-17A BCD085 with a single subcutaneous administration to rhesus monkeys (Masas mulatta) at a dose of 40 mg / kg.
• Interleukin 17 also referred to as IL-17 or IL-17A, is a 20-30 kD glycosylated homodimeric protein.
• the human IL-17 gene encodes a 155 amino acid protein that has a 19 amino acid signal sequence and a 136 amino acid mature segment.
• a full-sized antibody as it is found in nature, is an immunoglobulin (antibody) molecule that consists of four peptide chains, two heavy (H) chains (approximately 50-70 kD at full length) and two light (L) . chains (approximately 25 kD at full length) interconnected by disulfide bridges.
• the amino-terminal portion of each chain includes a variable region of approximately 100-1 10 or more amino acids, which are mainly responsible for antigen recognition.
• the carboxy-terminal part of each chain defines a constant region, mainly responsible for the function of the effector.
• the antibody consists of several fragments: light and heavy chains.
• the variable regions of the heavy and light chains form an antigen-binding center (or region).
• the structure of the antibody is shown in the figure below:
• Light chains are classified as kappa or lambda and are characterized by a specific constant region. Each light chain consists of a variable region of the ⁇ -terminal light chain (in this application, "LCVR” or “VL”) and a constant region of the light chain, consisting of one domain, CL. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and they determine the isotype of an antibody, such as IgG, IgM, IgA, IgD and IgE, respectively, and several of them can be further divided into subclasses (isotypes), for example IgGl , IgG2, IgG3, IgG4, IgAl and IgA2.
• Each type of heavy chain is characterized by a specific constant region, Fc.
• Each heavy chain consists of a variable region of the ⁇ -terminal heavy chain (“HCVR” or “VH” in this application) and a constant region of the heavy chain, CH.
• the heavy chain constant region consists of three domains (CHI, CH2, and CH3) for IgG, IgD, and IgA, and 4 domains (CHI, CH2, CH3, and CH4) for IgM and IgE.
• the HCVR and LCVR regions can be further divided into hypervariability regions called Rf hypervariable regions (CDRs) alternating with
• Each HCVR and LCVR consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CO, FR4.
• 3 CDRs of the heavy chains are designated as “CDRH1, CDRH2 and CDRH3”
• 3 CDRs of the light chain are designated as “CDRL1, CDRL2 and CDRL3”.
• CDRs contain most residues that form specific interactions with the antigen.
• anti-1b-17 antibody “anti-IL-17 antibody”, “antibody specifically binding to enterleukin IL-17” or “anti-1b-17A antibody” are used interchangeably in the context of this application and refer to an antibody that specifically binds to IL-17A.
• antibody in connection with the anti-1L-17A monoclonal antibody of the invention (or, more simply, the “monoclonal antibody of the invention”), as used herein, refers to a monoclonal antibody.
• Monoclonal antibodies of the invention can be prepared using, for example, hybridoma techniques well known in the art, as well as recombinant technologies, phage display technologies, synthetic technologies, or combinations of such technologies, or
• the term "monoclonal antibody”, as used in this application, is not limited to antibodies obtained using hybridoma technology.
• “Monoclonal antibody” refers to an antibody obtained from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone, and not to a method for its preparation.
• a “monoclonal antibody” can be an intact antibody (containing a full or full-sized Fc region), a substantially intact antibody, a part or fragment of an antibody containing an antigen binding part, for example, a Fab fragment, a Fab'fragment, or a P (ab ') 2 fragment antibodies.
• the “Fab” fragment contains the variable and constant domain of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain.
• the "F (ab ') 2" fragments of the antibody contain a pair of Fab fragments that are mainly covalently linked near their carboxy-ends with hinge amino acids between them: Fv - variable fragment of an antibody (variable fragment), scFv (single chain Fv) - variable fragment of an antibody in which light and heavy chain fragments are linked by a polypeptide linker, (scFv) 2 - fragment consisting of two molecules scFv connected by a disulfide bond, dsFv - var anabel fragment stabilized by an additional intramolecular disulfide bond Other chemical linkages of antibody fragments are also well known in the art.
• antibody and "immunoglobulin” are used interchangeably in the context of this application.
• variable regions of each of the light / heavy chain pairs form the antigen binding sites of the antibody.
• the "antigen binding portion" or the “antigen binding site” or the “antigen binding domain” or the “antigen binding center” form the antigen binding sites of the antibody.
• SUBSTITUTE SHEET refer, interchangeably, to that part of the antibody molecule that contains amino acid residues that interact with the antigen and give the antibody its specificity and affinity for the antigen.
• This part of the antibody includes the "frame" amino acid residues necessary to maintain the proper conformation of antigen binding residues.
• the CDR of the antigen binding site or the entire antigen binding site of the antibodies of the invention is from a human donor library or essentially human from certain amino acid residues altered, for example, substituted by different amino acid residues in order to optimize specific properties of the antibody, for example K D , kof f , 1C 50 .
• the antibody fragments of the invention are of human origin or essentially human origin (at least 80, 85, 90, 95, 96, 97, 98, or 99% human origin).
• Preferred framework regions of an antibody of the invention have the following amino acid sequences corresponding to framework regions of a human antibody:
• variable domain of the heavy chain is variable domain of the heavy chain
• the antigen binding site of an IL-17 antibody of the invention may be derived from, but not limited to, other non-human species, including a rabbit, rat, or hamster. Alternatively, the antigen binding site may be derived from human species.
• the "monoclonal antibody” as used in this application may be a single chain Fv fragment that can be obtained by linking DNA encoding LCVR and HCVR to a linker sequence (see Pluckthun, The Pharmacology of Monoclonal Antibodies , vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, p. 269-315, 1994). It is understood that, regardless of whether fragments or parts are indicated, the term “antibody” as used herein includes such fragments or parts as well as single chain forms. As long as the protein retains the ability to specifically or preferentially bind its target (eg, epitope or antigen), it refers to the term “antibody”. Antibodies may or may not be glycosylated, and are within the scope of the invention.
• a “monoclonal antibody” population refers to a homogeneous or substantially homogeneous antibody population (ie at least about 85, 90, 91, 92, 93, 94, 95, 96%, more preferably at least about 97 or 98% or even more preferably at least 99% of the antibodies in the population will compete in the ELISA for the same antigen or epitope, or more preferably the antibodies are identical in amino acid sequence).
• Antibodies may or may not be glycolized and still fall within the scope of the invention.
• Monoclonal antibodies can be homogeneous if they have the same amino acid sequence, although they
• SUBSTITUTE SHEET (RULE 26) may differ in post-translational modification, such as pattern glycolization.
• an “embodiment” of an antibody refers in this application to a molecule whose amino acid sequence differs from the amino acid sequence of the “parent” antibody by adding, deleting, and / or replacing one or more amino acid residues in the sequence of the parent antibody.
• the variant antibody contains at least one amino acid (e.g., from one to about ten and preferably 2, 3, 4, 5, 6, 7, or 8) additive, deletion, and / or substitution in the CDR regions of the parent antibody .
• Identity or homology with respect to the sequence of the variant antibody is defined in this application as the percentage of amino acid residues in the sequence of the variant antibody identical to the residues of the parent antibody, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity.
• a variant antibody retains the ability to bind an antigen, or preferably an epitope, to which a parent antibody binds, or preferably has at least one property or biological activity that exceeds that of a parent antibody.
• the antibody preferably has a stronger binding affinity, a longer half-life, lower 1C 50, or an increased ability to inhibit the biological activity of the antigen than the parent antibody.
• a variant antibody of particular interest in this application is an antibody exhibiting at least about 2-fold, preferably at least about 5-fold, 10-fold or 20-fold increase in biological activity compared to the parent antibody.
• a "parent” antibody in this application is an antibody encoded by the amino acid sequence that is used to produce the variant.
• the parent antibody may have a frame sequence of origin from Lama glama, but preferably the frame sequence is fully or essentially human in origin.
• the parent antibody may be from a llama, a chimeric, humanized, or human antibody.
• Antibodies according to the invention can be obtained by various construction techniques, including using recombinant methods, including shuffling DNA obtained from various sources.
• the term “specifically binds,” as used herein, refers to a situation in which one member of a specific binding pair does not significantly bind molecules other than its specific binding partner (s).
• the term is also applicable when, for example, the antigen binding domain of an antibody of the invention is specific for a particular epitope that is carried by a number of antigens, in which case a specific antibody having an antigen binding domain will be capable of specific binding of various antigens bearing the epitope.
• the monoclonal antibody of the invention specifically binds human IL-17 (i.e., IL-17A), while it does not specifically bind human IL-17B, IL-17C, IL-17D, IL-17E, IL-17F .
• epitope refers to that part of a molecule that is capable of being recognized and bound to an antibody in one or more antigen-binding regions of an antibody. Epitopes often consist of chemically active surface groups of molecules such as amino acids
• inhibitory epitope and / or “neutralizing epitope” is meant an epitope which, in the context of an intact antigenic molecule and when bound by an antibody specific for the epitope, results in the loss or decrease in the biological activity of the molecule or organism that contains the molecule, in vivo or in vitro.
• epitope also refers to a portion of a polypeptide that has antigenic and / or immunogenic activity in an animal, preferably a mammal, such as a mouse or human.
• antigenic epitope is defined as the portion of a polypeptide to which an antibody can specifically bind, as determined by any method well known in the art, for example, by conventional immunoassay. Antigenic epitopes need not be immunogenic, but may also be immunogenic.
• An “immunogenic epitope”, as used herein, is defined as part of a polypeptide that elicits an antibody response in an animal, as established by any method known in the art.
• a “non-linear epitope” or “conformational epitope” contains non-contiguous polypeptides (or amino acids) within the antigenic protein with which the antibody specific for the epitope binds.
• biological property or “biological characteristic” or the terms “activity” or “bioactivity” with respect to an antibody of the invention are used interchangeably herein and include, but are not limited to, epitope / antigenic affinity and specificity, ability neutralize or antagonize IL-17 activity in vivo or in vitro,
• SUBSTITUTE SHEET (RULE 26) IC 50 , stability of antibodies and immunogenic properties of antibodies in vivo.
• Other biological properties or characteristics of the antibody identified by the prior art include, for example, cross-reactivity (i.e., with non-human homologs of the target peptide or with other proteins or targets, in general), and the ability to maintain high levels of protein expression in mammalian cells.
• the above properties or characteristics may be observed, measured or evaluated using methods recognized in the art, including, but not limited to, ELISA analysis, competitive ELISA analysis, BIACORE or KINEXA surface plasmon resonance analysis, in vitro or in vivo neutralization assays, without limitation receptor binding, production and / or secretion of a cytokine or growth factor, signal transduction and immunohistochemistry of tissue sections obtained from various sources, including human, primate or L. Any other source.
• inhibit or “neutralize,” as used herein, with respect to the activity of an antibody of the invention, means the ability to significantly counteract, inhibit, prevent, limit, slow down, interrupt, destroy, terminate, reduce or reverse for example, the development or severity of what is inhibited, including but not limited to the above, biological activity (eg, activity of IL-17) or a property, disease or condition.
• Inhibition or neutralization of the activity of IL-17 by binding of an antibody of the invention to IL-17 is preferably at least about 20, 30, 40, 50.60, 70, 80, 90, 95% or higher.
• patient in this application refers to a mammal, including, but not limited to, mice, monkeys, humans,
• SUBSTITUTE SHEET mammals, farm animals, mammals, sports animals, and mammals, pets; preferably, the term refers to people.
• the patient preferably a mammal, preferably a human, is further characterized by a disease or disorder, or condition, mediated by IL-17A, which can be improved by decreasing the bioactivity of IL-17.
• vector includes a nucleic acid molecule capable of transporting another nucleic acid to which it is bound, including but not limited to plasmids and viral vectors. Certain vectors are capable of autonomous replication in the host cell into which they are introduced, while the remaining vectors can be integrated into the genome of the host cell and, thus, replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are referred to in this application as “recombinant expression vectors" (or, simply, “expression vectors”), and illustrative vectors are well known in the art.
• cell As used herein, the expressions "cell”, “host cell”, “cell line” and “cell culture”, “cell line as producer” are used interchangeably and include an individual cell or cell culture that is a recipient of any an isolated polynucleotide according to the invention or any recombinant vector (any recombinant vectors) that contain a sequence encoding an HCVR, LCVR or a monoclonal antibody of the invention.
• Host cells include the offspring of an individual host cell, and the offspring may not
• a host cell includes cells transformed, transduced or infected with a recombinant vector, or a monoclonal antibody that expresses the polynucleotide of the invention or its light or heavy chain.
• a host cell that contains a recombinant vector of the invention may also be referred to as a “recombinant host cell.”
• Preferred host cells for use in the invention are CHO cells (e.g., ATCC CRL-9096), NS0 cells, SP2 / 0 cells, COS cells (ATCC, e.g. CRL-1650, CRL-1651) and HeLa (ATCC CCL-2 )
• Additional host cells for use in the invention include plant cells, yeast cells, other mammalian cells, and prokaryotic cells.
• IL-17A mediated disease or disorder includes a disease or disorder associated with abnormal levels of IL-17 in the body or tissues.
• This disease or disorder can be either infectious or inflammatory in nature, or autoimmune, and can be selected from rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondylarthropathy, systemic lupus erythematosus , ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis, scleroderma, reactions " ransplantat versus host ", organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular
• SUBSTITUTE SHEET (RULE 26) coagulation, Kawasaki disease, Graves disease, nephrotic syndrome, chronic fatigue syndrome, Wegener granulomatosis, Shenlein-Genoch purple, microscopic renal vasculitis, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, infectious diseases, parasites , acquired immunodeficiency syndrome, acute transverse myelitis, Huntington’s chorea, Parkinson’s disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemo of anemic anemia, malignant tumors, heart failure, myocardial infarction, Addison’s disease, type I sporadic polyglandular deficiency and type I polyglandular deficiency, Schmidt’s syndrome, adult (acute) respiratory distress syndrome, alopecia, focal alopecia, seronegative arthropathy, aronegative arthropathy, psoriatic arthr
• SUBSTITUTE SHEET (RULE 26) lung diseases, cryptogenic fibrotic alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis associated with connective tissue disease interstitial lung disease associated with mixed connective tissue disease lung disease associated with systemic scleroderma lung disease associated with rheumatoid arthritis of interstitial lung disease, systemic lupus erythematosus pulmonary disease associated with dermatomyositis / polymyosis om lung disease associated with Sjogren's disease lung disease associated with ankylosing spondylitis lung disease, vasculitis diffuse lung disease associated with hemosiderosis lung disease, drug-induced interstitial lung disease, fibrosis associated with radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lung diseases with lymphocyte infiltration, post-infectious interstitial lung disease, gouty art ita, autoimmune hepatitis, autoimmune hepatitis type
• SUBSTITUTE SHEET polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic scleroderma, Schengen syndrome, Takayasu disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune hypothyroidism, hyperthyroidismosis, hypothyroidismosis, hypothyroidismosis, myxedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, alcohol alcohol-induced irrosis, liver damage, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergies and asthma, group B streptococcal infection (GBS), mental disorders (including depression and schizophrenia) mediated by type Th2 and type YO acute and chronic pain (GBS), mental
• SUBSTITUTE SHEET (RULE 26) arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (permanent or paroxysmal), atrial flutter, atrioventricular block, B-cell lymphoma, bone graft rejection, bone marrow transplant rejection (BMT), blockade of the bundle of His, heart lymphoma, Burkita, Berkitt's lymphoma, stunned heart syndrome, heart tumors, cardiomyopathy, inflammatory responses to cardiopulmonary bypass, cartilage graft rejection, cerebral cortical degeneration, cerebellar disturbances, chaotic or multifocal atrial tachycardia associated with chemotherapy disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease, chronic intoxication with salicylates, colorectal carcinoma, colon carcinoma, colon and rectal carcinoma, colon carcinoma , contact dermatitis,
• SUBSTITUTE SHEET gas gangrene, stomach ulcers, glomerulonephritis, transplant rejection of any organ or tissue, gram-negative sepsis, gram-positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Gallervorden-Spatz disease, Hashimoto thyroiditis, hemorrhagic fever, hemorrhoid fever, heart disease, syndrome / thrombolytic thrombocytopenic purpura, blood loss, hepatitis (A), arrhythmias of the bundle of His, HIV infection / HIV neuropathy, disease kin, hyperkinetic motor disorders, hypersensitivity reactions associated with hypersensitivity to pneumonitis, hypertension, hypokinetic motor disorders, examination of the hypothalamic-pituitary-adrenal system, idiopathic Addison’s disease, idiopathic pulmonary fibrosis, mediated by antibodies of cytotoxicity, asthenia, inflammation, infantile influenza A virus, exposure
• SUBSTITUTE SHEET (RULE 26) nephritis, nephrosis, neurodegenerative diseases, neurogenic muscle atrophy I, neutropenic fever, non-Hodgkin’s lymphomas, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, OKTZ® therapy, orchitis / epididymitis, orchitis / recurrent procedures after vasectomy, otorrhea, organ pancreatic transplant, pancreatic carcinoma, paraneoplastic syndrome / hypercalcemia with a malignant tumor, parathyroid transplant rejection, inflammatory pelvic diseases, perennial rhinitis, pericardial disease, peripheral arteriosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumonia Pneumocystis carinii, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy syndrome, and monoclonal gamm
• SUBSTITUTE SHEET (RULE 26) thromboangitis obliterans, thrombocytopenia, toxicity, transplantation, trauma / blood loss, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, heart valve diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, venous thrombosis, and fungal infections, encephalitis with a high risk of death / aseptic meningitis, hemophagocytic syndrome with a high risk of death, Wernicke-Korsakov syndrome, more Wilson’s illness, xenograft rejection of any organ or tissue, acute coronary syndrome, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, Still's disease, focal alopecia, anaphylaxis, atherosclerosis syndrome, anthrophosphoplipitis, anemia, art
• SUBSTITUTE SHEET (RULE 26) fever, Hughes syndrome, idiopathic Parkinson’s disease, idiopathic interstitial pneumonia, mediated IgE allergy, immune hemolytic anemia, inclusion bodies, infectious inflammatory eye disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, idiopathic pulmonary fibrosis and pulmonary fibrosis ulceris, keratitis, dry keratoconjunctivitis, Kussmaul disease or Kussmaul-Meyer disease, Land paralysis ri, histiocytosis of Langerhans cells, marbled skin syndrome, macular degeneration, microscopic polyangiitis, ankylosing spondylitis, impaired motor neurons, pemphigoid mucous membranes, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-neuropathy, non hepatitis, optic neuritis,
• SUBSTITUTE SHEET (RULE 26) Sneddon-Wilkinson dermatosis, ankylosing spondylitis, Stevens-Johnson syndrome, systemic inflammatory response syndrome, temporal arteritis, toxoplasma retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (periodic syndrome associated with tumor necrosis factor receptor), type I allergic reaction II, urticaria, common interstitial pneumonia, vasculitis, spring conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet yellow heel degeneration to, wound healing and related arthropathies Yersinia and salmonella.
• TRAPS peripheral syndrome associated with tumor necrosis factor receptor
• type I allergic reaction II urticaria
• urticaria common interstitial pneumonia
• vasculitis spring conjunctivitis
• viral retinitis Vogt-Koyanagi
• V H antibody heavy chain variable domain
• the hypervariable region of HCDR1 comprises the sequence of SEQ NO: l;
• the hypervariable region of HCDR2 includes SEQ ID: NO 2, and
• the hypervariable region of HCDR3 includes SEQ ID NO: 3, and
• the hypervariable region LCDR1 includes SEQ ID NO: 4,
• the hypervariable region LCDR2 includes SEQ ID NO: 5 and
• the hypervariable region of LCDR3 includes SEQ ID NO: 6 in the variable regions of light chains (VL).
• the antibody is an isolated monoclonal antibody or fragment thereof
• SUBSTITUTE SHEET (RULE 26) containing the variable domains of the heavy chain VH and light chain VL, where the specified antibody or fragment contains:
• HCDR1 comprising the sequence F-T-F-S-N-Y-A-M-S (SEQ ID NO: 7);
• HCDR2 comprising the sequence R-I-E-G-G-I-S-S-T-Y (SEQ ID NO: 8);
• HCDR3 comprising the sequence C-A-V-N-Y-Y-G-M-Y-Y (SEQ ID NO.- 9);
• variable region of the light chain VL contains:
• LCDR1 comprising the sequence T-G-T-S-E-D-V-G-F-G-N-Y (SEQ ID NO: 10);
• LCDR2 comprising the sequence R-V-N-T-R-P-S (SEQ ID NO:
• LCDR3 comprising the sequence C-S-S-Y-K-A-G-G-T-Y
• the antibody is an isolated monoclonal antibody that specifically binds to human IL-17A, or a fragment thereof comprising the variable domains of the heavy chain (VH) and light chain (VL), where the specified antibody or its active the fragment contains at least one variable domain, including:
• variable domain of the heavy chain (VH) of the antibody which includes the amino acid sequence of SEQ ID NO: 13.
• variable domain of the light chain (VL) of the antibody which includes the amino acid sequence of SEQ ID NO: 14.
• the antibody may contain a heavy chain amino acid sequence identical to SEQ ID NO: 15.
• the antibody comprises a light chain with an amino acid sequence identical to SEQ ID NO: 16.
• antibodies of the present invention may contain additional mutations M252Y / S254T / T256E, N434W, N434A, N434F, H433K / N434F / Y436H, H433K / N434F / Y436H + M252Y / S254T / T256E, T307A / E34A / E303A / E330 M428L. These substitutions do not lead to a loss of the ability of an antibody to bind IL-17A, but can lead to a decrease in ADCC (antibody-dependent cell-mediated cytotoxicity) or an increase in the affinity or other biological properties of antibodies.
• ADCC antibody-dependent cell-mediated cytotoxicity
• the antibodies of the present invention have improved biological properties, in particular, better inhibitory ability (IC50) against IL-17A and increased aggregation stability.
• the invention also provides a DNA construct encoding the antibodies described, as well as an expression vector containing the DNA construct for producing antibodies of the invention.
• a cell line that contains in the 'cells an expression vector with the DNA described above, which can be used as a producer of antibodies according to this invention.
• An antibody of the invention may be included in a pharmaceutical composition suitable for administration to a patient (see Example 14).
• Antibodies of the invention may be administered alone or in combination with a pharmaceutically acceptable carrier, diluent and / or excipient in single or multiple doses.
• the pharmaceutical compositions for administration are designed to suit the chosen mode of administration, and pharmaceutically acceptable diluents, carriers and / or excipients, such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonic agents, stabilizers, and the like. used properly (see example 14).
• compositions have been developed in accordance with traditional methods described in, for example, Remington, The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995, which describes various methods for preparing the compositions, generally known to specialists.
• a pharmaceutical composition containing an anti-1L-17 monoclonal antibody of the invention can be administered to a patient at risk of development or having a pathology, as described herein, using standard methods of administration, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or with the help of suppositories.
• composition of the invention preferably contains or is a "therapeutically effective amount" of
• a “therapeutically effective amount” refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result.
• a therapeutically effective amount of an antibody can vary depending on factors such as the condition of the disease, the age, gender and weight of the individual, and the ability of the antibody or part of the antibody to elicit the desired response in the individual.
• a therapeutically effective amount is also an amount in which the therapeutically beneficial effect of the antibody prevails over the toxic or harmful effect.
• a “prophylactically effective amount” refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Since a prophylactic dose is used for individuals before or at an early stage of the disease, typically a prophylactically effective amount may be less than a therapeutically effective amount.
• a therapeutically effective or prophylactically effective amount is at least the minimum dose, but less than the toxic dose of the active agent, necessary to provide therapeutic benefit to the patient.
• a therapeutically effective amount of an antibody of the invention is an amount that, in mammals, preferably humans, reduces the biological activity of IL-17, for example, IL17R binding, where the presence of IL-17 causes or contributes to undesirable pathological effects, or a decrease in IL-17 levels causes a beneficial therapeutic effect in a mammal, preferably a human.
• the route of administration of the antibodies of the invention may be oral, parenteral, by inhalation, or topical.
• the antibodies of the invention may be included in a pharmaceutical composition suitable for parenteral administration.
• parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration.
• Advantageous is the introduction by intravenous or intraperitoneal, or subcutaneous injection. Suitable carriers for such injections are directly known in the art.
• compositions must be sterile and stable under the conditions of manufacture and storage in a container that is provided, including, for example, a hermetically sealed vial (ampoule) or syringe. Therefore, pharmaceutical compositions can be sterile filtered after preparation, or otherwise made microbiologically suitable.
• a typical intravenous infusion composition may have a volume of 250-1000 ml of liquid, such as sterile Ringer's solution, physiological saline, dextrose solution, and Hank's solution, and a therapeutically effective dose (e.g., 1-100 mg / ml or more) of antibody concentration. The dose may vary depending on the type and severity of the disease.
• dosages for any of the patients depend on many factors, including patient size, body surface area, age, specific compound to be administered, gender, time and route of administration, general health and other medications that are administered simultaneously.
• a typical dose may be, for example, in the range of 0.001 to 1000 ⁇ g; however, doses below or above this illustrative range are anticipated, especially considering the above factors.
• the dosage regimen of daily parenteral administration may be from 0.1 ⁇ g / kg to 100 mg / kg of the total body weight, preferably from 0.3 ⁇ g / kg to 10 mg / kg and more preferably from 1 ⁇ g / kg to 1 mg / kg , even more preferably 0.5 to 10 mg / kg body weight per day.
• the treatment process can be monitored by periodically evaluating the patient's condition. For repeated administration over several days or longer, depending on the condition of the patient, the treatment is repeated until the desired response is achieved or the symptoms of the disease are suppressed. However, other dosage regimens that are not described in this application may also be used.
• the desired dosage may be administered by single-pole administration, multiple bolus administrations, or by continuous infusion of an antibody, depending on the pharmacokinetic decay sample that the practitioner wants to achieve.
• the therapeutic agents of the invention can be frozen or lyophilized and reconstituted before use in a suitable sterile vehicle. Lyophilization and recovery can lead to varying degrees of loss of antibody activity. Dosages may be adjusted to compensate for this loss. In general, pH values of the pharmaceutical composition of 6 to 8 are preferred.
• the invention provides a finished product containing materials useful for treating or preventing the disorders or conditions described above.
• the finished product contains a container containing a pharmaceutical composition with an antibody, with a designation and possibly instructions.
• Suitable containers include, for example, vials, ampoules, syringes, and assay tubes. Containers may be made of a variety of materials, such as glass or polymeric material.
• the container contains a composition according to the invention, which is effective for treating a disease or disorder mediated by IL-17A, and may have a sterile access (for example, the container may be an intravenous solution bag or a vial having a plug that is permeable for hypodermal injection needles) .
• the active agent in the composition is an anti-Lb-17 antibody of the invention.
• the designation on the container and the instructions attached to it indicate that the composition is used to treat the selected disease.
• the finished product may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's saline and dextran solution. It may further include other materials desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
• Antibodies and antigens were produced in cells of a constant cell line derived from Chinese hamster ovary cells (CHO-K1 line) according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91 (6): 670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun; 28 (l 1): 843-848; Biotechnol Bioeng. 2003 Nov 5; 84 (3): 332-342].
• a recombinant IL-17A protein containing six His amino acids at the C-terminus of the protein was isolated and purified from the culture fluid using Profmity IMAC Ni-charged resin (from Bio-Rad). Prior to purification, NiCl 2 was added to the culture liquid to a concentration of 1 mM. Next, 5 ml of Profmity IMAC Ni-charged resin was added to the culture fluid and stirred for 1 hour at room temperature.
• SUBSTITUTE SHEET (RULE 26) The sorbent was transferred to a 5 ml Thermo scientific Polypropylene columns column, washed with 5 column volumes of FSB to wash the non-specific binding components. The bound antigen was eluted using 0.3 M imidazole, pH 8, 150 mM NaCl. Then the protein was transferred to PBS (pH 7.4) using dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 ⁇ m), transferred to test tubes and stored at - 70 ° ⁇ . The purity of the resulting protein solution was evaluated using SDS gel electrophoresis (Figure 1).
• Recombinant protein IL-17A-Fc from the culture fluid was isolated and purified using a Protein A affinity chromatography column.
• the clarified culture fluid was passed through a 5 ml HiTrap rProtein A Sepharose FF column (GE Healthcare) that was equilibrated with phosphate salt buffer (FSB, pH 7.4). Then the column was washed with 5 column volumes of FSB to wash the non-specific binding components.
• the bound antigen was eluted using 0.1 M glycine buffer pH 3.
• the main protein peak of elution was collected and adjusted to neutrality with 1 M Tris buffer (pH 8). All stages were carried out at a flow rate of PO cm / h.
• the protein was transferred to PBS (pH 7.4) using dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 ⁇ m), transferred to tubes and stored at -70 ° C.
• the purity of the resulting protein solution was evaluated using SDS gel electrophoresis ( Figure 2).
• IgGl antibodies were purified on a 1 ml Hi Trap rProteinA FF column (GE Healthcare) according to the procedure described above for IL-17A-Fc antigen. The purity of the resulting protein solution was evaluated using SDS-gel electrophoresis (Fig.Z).
• Lama Glama animal was sequentially immunized 5 times by subcutaneous administration of antigenic material mixed with an equal volume of Freund's complete (in the first injection) or incomplete (in the remaining injections).
• a mixture of recombinant proteins (0.2 mg of each of the proteins per 1 injection) was used as antigen, one of which was human IL-17A (kit from R&D Systems).
• the second injection (stage of immunization) was carried out 3 weeks after the first, then three more immunizations were carried out with an interval of two weeks. Blood sampling (50 ml) was performed 5 days after each injection, starting with the third.
• Blood was diluted 2 times with a solution of PBS containing 1 mm EDTA.
• 35 ml of the diluted blood solution was layered on Histopaque®-1077 medium (from Sigma) with a density of 1.077 g / ml, volume of 15 ml and centrifugation was carried out for 20 min at 800 g.
• Mononuclear cells (lymphocytes and monocytes) were selected from the interphase plasma / Histopaque medium, and then washed with a FSB solution containing 1 mM EDTA.
• RNA concentration was determined using Nanovue (GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis on a 1.5% agarose gel.
• SUBSTITUTE SHEET (RULE 26) using MMuLV reverse transcriptase and random hexamer oligonucleotides as a seed.
• variable domain genes flanked by restriction sites using a set of oligonucleotides and the protocols of the authors [J Biol Chem. 1999 Jun 25; 274 (26): 18218-30; WO 2010/001251].
• connection of the genes of the variable domains of light and heavy chains into one fragment was carried out by sequential restriction, ligation and amplification reactions, according to the scheme shown in Figure 1.
• the heavy chain genes were connected to the kappa genes and separately to the light chain lambda genes.
• the calculated number of matrix molecules in all reactions was at least 10 p .
• the resulting DNA preparation VL-CK-VH was digested with Nhel / Eco911 restriction enzymes and ligated into the original pH 5 phagemid.
• the structure of the phagemid is shown in FIG. 2.
• Ligation products were transformed into electrocompetent SS320 strain cells prepared according to protocols [Methods Enzymol. 2000; 328: 333-63].
• the repertoire of the Kappa derivative Fab library was 5.1 * 10 and lambda 3.7 * 10, respectively.
• a phage library preparation was prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5; 222 (3): 581-97].
• Unbound phages were removed during a series of washing cycles using a PBS solution (pH 7.4) -Tween 20 (0.1% v / v). The number of washes increased from the first round to the third round 20-30-40 times, respectively. The remaining phage particles bound were eluted with 100 mM Gly-HCl solution (pH 2.5) for 15 minutes with stirring, then neutralized with 1 M TRIS-HC1 (pH 7.6). Bacteria of E. coli TG1 strain were infected with the obtained phages, then the phages were isolated and used in the next selection cycle.
• RNA of B lymphocytes from the blood of five hundred and fifty human donors was isolated using the RNeasy Mini Kit according to the proposed protocol (from QIAGEN). The concentration of RNA was determined using the Nanovue kit (from GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis on a 1.5% agarose gel.
• the reverse transcription reaction was performed using the MMLV RT kit (from Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as a seed.
• variable domain gene of the light chain of the clone A1 antibody of the llama with the genes of the variable domains of the human donor heavy chains with the gene of the variable domain of the heavy chain of the antibody of clone A 1 llama in one fragment was carried out by sequential restriction, ligation and amplification reactions according to the scheme shown in FIG. . 6B.
• the calculated number of molecules of the matrix of genes of human variable domains in all reactions was at least 10 12 .
• the obtained DNA preparation VL-CK-VH was treated with restriction enzymes Nhel Eco9P and led into the original phagemid pH5. Ligation products were transformed into electrocompetent cells of strain SS320 prepared according to protocols [Methods Enzymol.
• ELISA ELISA
• Fab with the published sequence AIN457 (from Novartis) was used as a positive control.
• wells of ELISA plates (from Nunc ImmunoMaxisorp) were coated with 50 ⁇ l (0.5 ⁇ g / ml in IX coating carbonate buffer) IL-17A-Fc, sealed and incubated overnight at 4 ° C. All subsequent steps were carried out according to the standard ELISA protocol using a high-performance automated platform based on GenetixQ-pix2xt robotic systems (from Molecular Device) and Tesap Freedom EVO 200 (from Tesap).
• blocking buffer BB 200 ⁇ l of 0.5% nonfat milk in the FSB. The plates were incubated on a shaker for one hour at room temperature. After washing with FSB-Tween, 50 ⁇ l was added
• the colorimetric signal was developed by adding TMB (100 ⁇ l / well) to saturation (average 3-5 min), then further development was stopped by adding a stop solution (100 ⁇ l / well, 10% sulfuric acid).
• the color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise tablet reader (from Tesap). The degree of antibody binding was proportional to the production of a color signal.
• test cell supernatant containing the test Fab was mixed in a non-binding 96-well plate with 50 ⁇ l of 0.4 ⁇ g / ml IL-17A-His6-Flag in 1% milk in PBS-Tween. Incubated for 1 hour at 37 ° C and shaking at 500 rpm.
• SUBSTITUTE SHEET (RULE 26) stopped by the addition of stop solution (100 mcd / well, 10% sulfuric acid).
• the color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise tablet reader (from Tesap). The degree of antibody binding was proportional to the production of a color signal.
• Koff is a screening of anti-1b-17A Fab candidates. Koff - screening was performed using a Pall Forte Bio Octet Red 96 instrument. Anti FABCH1 biosensors were rehydrated for 30 minutes in a working buffer containing 10 mM FSB, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA. In the test samples of E coli supernatants, a 10x working buffer was added to a final concentration of 1x. Then, anti FABCH1 biosensors were immersed in E. coli supernatants containing Fab fragments of antibody candidates for 12 hours at 4 ° C.
• Sensors with Fab fragments immobilized on the surface were transferred to wells with a working buffer, where a baseline (60 s) was recorded.
• the sensors were then transferred to wells with analyte solution (IL-17A, 30 ⁇ g / ml) for association of the antigen-antibody complex (300 s). Then the sensors were returned to the wells containing the working buffer for the subsequent stage of dissociation (300 s).
• the NK codon randomization technique [Appl Environ Microbiol. 2006 Feb; 72 (2): l 141-7; MAbs.2012 May-Jun; 4 (3): 341 -8] using the Q5® Site-Directed Mutagenesis Kit (from NEB) according to the protocol, using the Al-Fab-pLL plasmid as a template. PCR products were then fractionated on low-melting agarose and purified on columns. After the ligation reaction, DNA was transformed into E. coli expressing strain BL21gold (from Stratagene). The resulting individual clones were expanded under Fab-expression conditions in 96 well plates as previously described.
• SUBSTITUTE SHEET (RULE 26) Goat anti-Human IgG (Fab ') 2 (HRP) (from Pierce) and TMB + H202 / H2S04 paint with subsequent measurement of optical density at a wavelength of 450 nm.
• amino acid positions of the CDR regions of antibody regions BCD085 tolerant of substitutions for other amino acids were screened. As a result, it was found that this set of amino acid substitutions did not significantly change the binding strength of the antibody to human I-17. A set of such positions can be used to optimize various properties of a candidate.
• a stable cell line producing the monoclonal antibody BCO085 was obtained by transfection of the parent
• SUBSTITUTE SHEET (RULE 26) suspension cell line CHO-S vector constructs containing light and heavy chains of antibodies in an optimized ratio.
• High level clonal lines (greater than 100 mg / L) were obtained using the ClonePix robotic platform (Molecular Devices).
• the productivity analysis of the selected clones was carried out on the basis of the Biomek FX robotics automated system (Beckman Coulter) and the Octet RED96 analytical system (Pall Life Sciences).
• the preparation BCD085 for preclinical studies was run in a 200-liter HyClone single-use bioreactor (Thermoscientific) fermenter.
• the resulting eluate was kept at acidic pH for 30 minutes for viral inactivation, and then neutralized with a 1M Tris-Osn solution. to a pH value of 6.8.
• the protein solution was filtered using STERICUP filter systems with a MilliporeExpressPlus® membrane (from PVDF) with a pore diameter of 0.22 ⁇ m.
• the resulting protein solution was passed through the prepared Q Sepharose FF sorbent (from GE HealthCare), pH 7.0, at a low conductivity value ( ⁇ 2 mS / cm). The purified protein was subjected to antiviral
• the concentration of the obtained protein was 50 mg / ml or more.
• the purification result of the obtained protein solution was evaluated using SDS gel electrophoresis ( Figure 1 1).
• Antibody samples were diluted with FSB buffer, pH 7.5, to a concentration of ⁇ 1 mg / ml.
• the volume of the injected sample is 10 ⁇ l.
• the calibration mixture Gel filtration standard (from Bio-Rad), KaT, was previously chromatographed. jYo 151-1901.
• IL-17 The ability of IL-17 to induce the production of IL-6 by human HT1080 cell line (ATCC: CCL-121) was used to analyze the neutralizing activity of BCD085 against human recombinant IL-17.
• Cells were grown in DMEM culture medium supplemented with 10% inactivated fetal serum, gentamicin and glutamine. 5 * 10 4 cells per well were seeded in 96-well flat-bottomed cell culture plates. The cells were left to attach for 5 hours.
• IL-6 production in HT1080 cell culture was proportional to the amount of IL-17 added.
• the amount of IL-6 released in cell supernatants was determined by ELISA using the DuoSet ELISA developmentsystemhuman IL6 system (from RD System, Cat. N ° DY206).
• the results of the analysis of the antagonistic properties of candidate BCD085 are shown in FIG. 13 in comparison with AIN457 (anti-1L-17A antibody from Novartis).
• the average value of 1 ⁇ 5 schreib for this candidate for a series of experiments is 40 ⁇ 15 pM.
• SUBSTITUTE SHEET (RULE 26) second-generation sensors (from AR2G) according to the standard protocol according to the manufacturer's instructions for the preparation and immobilization of AR2G sensors. The analysis was performed at 30 ° C using an FSB containing 0.1% Tween-20 and 0.1% BSA as a working buffer.
• BCD085 binds to a recombinant human IL-17A preparation with picomolar affinity, which is a thousand times stronger than the macaque affinity for macaque IL-17A, which has a nanomolar value.
• the candidate does not interact with rat IL-17A (curves not shown).
• SUBSTITUTE SHEET (RULE 26) with a particle diameter of 7 ⁇ m, KaT.j 08543. Elution was carried out in the isocratic mode with a mobile phase: 50 mM Na ⁇ E> 6, 0.3 M NaCl, pH 7.0 at a flow rate of 0.5 ml / min. Detection was carried out at wavelengths of 214 and 280 nm. Antibody samples were diluted with FSB buffer, pH 7.5, to a concentration of ⁇ 1 mg / ml. The volume of the injected sample is 10 ⁇ l. The calibration mixture Gel filtration standard (from Bio-Rad), cat. N ° 151-1901.
• Monoclonal antibody BCD085 against IL-17A was administered once subcutaneously to monkey rhesus monkeys (Masasa mulatta) at a dose of 40 mg / kg. At certain intervals after administration (after 0.5, 1, 3, 6, 12, 24, 36, 48, 60, 72, 96, 120, 168, 264, 336, 432, 504, 672, 840 and 1008 hours ) blood was taken from animals and serum was obtained by the standard method.
• Serum BCD085 antibody levels were assessed by enzyme-linked immunosorbent assay (ELISA) on immobilized human IL-17A (from R&D Systems), where the bound antibody was detected using goat polyclonal antibodies against the Fc fragment of human IgG antibodies (from Pierce-ThermoScientific )
• BCD085 has a significant prolonged effect or a long circulation time in the blood.
• the maximum content of monoclonal anti-IL-17A antibody in the blood of animals was observed after 72-168 hours and this level remained for 400 hours and only then a very slow decrease occurred, so that even after 1000 hours the concentration of BCD085 was significantly above the control level.
• the calculated pharmacokinetic parameters for BCD085 for single subcutaneous administration to monkeys are given in Table 2.
• Buffer citrate HUMM up to pH 6.0-7.0
• kits with a dosage form containing an anti-IL-17 antibody composition the pharmaceutical composition prepared according to Example 14 is sealed in 1 ml ampoules or syringes under sterile conditions, labeled and packaged in containers made of plastic or cardboard material.
• instructions for use are included in the container with the ampoule.

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