WO2018034597A1 - Антитело или его антигенсвязывающий фрагмент, способный связываться с рецептором интерлейкина-6 человека - Google Patents
Антитело или его антигенсвязывающий фрагмент, способный связываться с рецептором интерлейкина-6 человека Download PDFInfo
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Definitions
- the invention relates to medicine.
- the present invention relates to the creation of antibodies or fragments thereof having the ability to specifically bind to the human interleukin-6 receptor, which would be useful as drugs for the treatment or diagnosis of diseases or to alleviate the symptoms mediated by interleukin-6.
- the invention also relates to methods for producing these antibodies and to a method for treating human diseases using these antibodies.
- Interleukin-6 is one of the main mediators of inflammatory reactions, produced by a wide range of antigen-presenting cells, including B and T cells.
- IL-6 is produced by activated monocytes or macrophages, endothelial cells, fibroblasts, activated T-cells, as well as a number of cells that are not immunocytes.
- monocytes or macrophages activated monocytes or macrophages, endothelial cells, fibroblasts, activated T-cells, as well as a number of cells that are not immunocytes.
- cytokines it is involved in the processes of the immune response, angiogenesis, inflammation, and bone metabolism.
- the main effect of IL-6 is associated with its participation as a cofactor in the differentiation of B-lymphocytes, their maturation and conversion into plasma cells that secrete immunoglobulins.
- IL-6 promotes the expression of IL-6 receptor on activated immunocytes, and also induces the production of IL-2 by T cells.
- This cytokine stimulates the proliferation of T-lymphocytes and the reaction of hematopoiesis.
- IL-6 is one of the most active cytokines involved in the implementation of the immune response and inflammatory response. It has been shown that the imbalance between the pro and anti-inflammatory actions of IL-6 leads to various autoimmune diseases, chronic inflammation and osteoporosis, psoriasis, and excess production leads to various forms of cancer.
- a cytokine signal into the cell is possible in 2 ways.
- the first - to cells having a membrane-bound alpha subunit of the receptor, interleukin-6 attaches to an already assembled receptor from the alpha subunit and other 130 molecules.
- the second - to cells that have only dr130 molecules on the membrane an interleukin-6 complex with a soluble form of the alpha subunit is attached.
- the complete complex is assembled and the cascade of reactions in the cell is subsequently launched.
- Blocking the action of the cytokine and, consequently, the inflammatory reaction can be achieved by interfering with the assembly of the full complex of the receptor, consisting of the alpha subunit, dr130 and interleukin 6 molecules.
- Specific antibodies, binding to one of the molecules can interfere with the assembly of the complete complex and, accordingly, block the conduction signal inside the cell.
- Polypeptides that specifically bind to IL-6 (see RF patent N2 2550262), IL-6R or other 130 showed a significant inhibitory effect on the functioning of IL-6.
- Antibody-based drugs (tocilizumab) that binds to IL-6R and blocks the interaction with IL-6 are widely used in the treatment of rheumatoid arthritis and systemic juvenile idiopathic arthritis, both as monotherapy and in combination with methotrexate and / or other basic anti-inflammatory drugs.
- the IL-6 receptor (IL-6R, IL6R, CD126) is a complex of molecules that, when activated, causes a cascade of reactions in the cell, leading to the active synthesis of proteins involved in further inflammatory response reactions.
- the receptor is activated upon binding of IL-6 to the alpha subunit of the receptor with a mass of 80 kDa that does not transmit a signal, and two molecules of dr 130 with a mass of 130 kDa that transmit a signal into the cell (Simon A. Jones TheFASEB Journal 15 (1): 43-58).
- the soluble form is formed as a result of proteolysis of the transmembrane part or in the process of alternative splicing of mlL-6R mRNA.
- the soluble form of IL-6sR allows cytokine response to cells that do not have mlL-6R on the membrane surface.
- Blocking the IL-6 signal with receptor binding polypeptides has several advantages over blocking IL-6 binding directly. So, antibodies against IL-6 bind to both the target intra-articular ligand and to circulating IL-6 in the blood. While antibodies that bind to IL-6R act on cells containing mlL-6R on the surface, and on cells containing only dr130.
- IL-6R refers to an interleukin-6 receptor.
- slL-6R refers to the soluble form of the interleukin-6 receptor.
- mlL-6R refers to the membrane-bound receptor for interleukin-6.
- antibody and “immunoglobulin” are used interchangeably and refer to a full-sized antibody in the form in which it is synthesized by cells of the immune system or other organisms modified by genetic engineering.
- Full-sized antibodies consist of four polypeptide chains: two heavy chains (N) (approximately 50-70 kD at full length) and
- the ⁇ -terminal (amino-terminal) part of each chain includes a variable region of approximately 100-1 10 or more amino acids, which are mainly responsible for antigen recognition.
- the C-terminal (carboxy-terminal) part of each chain defines a constant region, mainly responsible for the function of the effector.
- variable regions of the heavy and light chains form an antigen-binding center (or region).
- Light chains of antibodies are classified as kappa or lambda, each of which has a specific constant region.
- Each light chain consists of a variable portion of the ⁇ -terminal portion of the light chain (“LCVR” or “VL”) and a constant portion of the light chain consisting of one CL domain.
- 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 lgG1 , logG2, logG3, logG4, logA1 and logA2.
- 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”) and a constant region of the heavy chain of CH.
- the heavy chain constant region consists of three domains (CH1, CH2 and CH3) for IgG, IgD and IgA and 4 domains (CH1, 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) interspersed with more conservative regions called framework regions (FR).
- CDRs Rf hypervariable regions
- FR framework regions
- Each HCVR and LCVR consists of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- CDRs contain most residues that form specific interactions with the antigen.
- variable regions of each of the light / heavy chain pairs form the antigen-binding sites of the antibody.
- the “antigen binding portion”, or “antigen binding site”, or “antigen binding domain” or “antigen binding center” refers, interchangeably, to that part of an antibody molecule that contains amino acid residues that interact with the antigen and confer antibody 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.
- antibody in this application does not necessarily mean a human antibody, it can be a rodent antibody, an antibody of the animal family of primates or Camelidae, preferably an antibody of a mouse, macaque, a camel or a llama antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
- antibody as used herein also encompasses artificially engineered single chain antibodies having a structure similar to that of natural antibodies.
- Antibodies referred to in this text may be glycosylated or free from polysaccharide residues.
- An antibody of the invention may be understood to mean a monoclonal antibody.
- monoclonal antibody is meant a homogeneous or substantially homogeneous antibody population (i.e., at least about 89, 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 b sequence).
- Monoclonal antibodies are also understood to mean antibodies having an identical or substantially identical amino acid sequence, although they may differ in post-translational modification, for example, pattern glycolization.
- 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 other technologies well known in the art.
- the term "monoclonal antibody” is not limited to antibodies obtained using only hybridoma technology. This term may refer to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone.
- the numbering of amino acid residues in this text is carried out with reference to a reference antibody, which is understood to mean an antibody whose heavy chains have the sequence SEQIDNO: 10, and light chains the sequence SEQIDNO: 9.
- a reference antibody which is understood to mean an antibody whose heavy chains have the sequence SEQIDNO: 10, and light chains the sequence SEQIDNO: 9.
- the numbering and positioning of CDR amino acid residues within the HCVR and LCVR regions of antibodies is carried out in accordance with the nomenclature of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) unless otherwise indicated.
- Antibodies according to the invention can be obtained by various construction techniques, including using recombinant methods, including shuffling DNA obtained from various sources.
- the antibodies of the present invention may be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of the same target polypeptide or may contain antigen binding domains that are specific for one or more antigens other than IL-6R or different epitopes of IL-6R (see, for example, Tuttetal. (1991) J. Immunol. 147: 60-69).
- the relationship between the antigen binding parts of mono-, bi- and multispecific antibodies can be different.
- the bonds between the antigen binding parts can be chemical, or different antigen binding parts can be combined through a common polypeptide chain, or by non-covalent association of different chains with each other, or the antigen binding parts can be combined with each other via another antibody or antibody fragment.
- aHTH-slL-6R-aHTHTeno refers to an antibody that specifically binds to the interleukin-6 receptor.
- mll_-6R refers to a membrane-bound interleukin-6 receptor.
- slL-6R refers to the soluble form of the interleukin-6 receptor.
- antibody fragment any fragment of a natural and artificially engineered antibody containing three or less heavy CDRs and three or less light chain CDRs.
- An antibody fragment may mean, in particular, a substantially full-length antibody, with a shortened heavy chain framework, an entire or full-length Fc region, a portion or antibody fragment containing an antigen-binding portion, for example, a Fab fragment, Fab' fragment, or P ( a ') a 2-fragment of an antibody, scFv, (scFv) 2, dsFv, 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).
- antibody fragment should not be understood as indicating the origin of a full-sized antibody.
- Frament antibodies can be obtained completely independently of full-sized antibodies by any known method.
- antibody fragment is also meant an artificially engineered polypeptide or product containing peptide and non-peptide moieties if it comprises the above three or less heavy chain CDRs and three or less light chain CDRs arranged so that the polypeptide or said product retains the ability of a specific or preferred binding of its target (e.g., epitope or antigen).
- target e.g., epitope or antigen
- the term “specifically binds” as used in this application 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 antibody of the invention or its fragment specifically binds human slL-6R, while it practically does not bind to human proteins IL12, IL23, EGFR, CD3, IGFR, CTGF, FGF2, PD1, PSCK9, CD38, GCSF, interferon alpha 2b.
- 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 or sugar side chains, and possess specific three-dimensional structural characteristics, as well as specific charge characteristics.
- inhibitor 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 reduction of the biological activity of the molecule or organism that contains the molecule, invivo or invitro.
- 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 determined 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 to 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 for SIL-6R, the ability to be an antagonist of IL-6, the 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 expression protein in mammalian cells.
- ELISA analysis ELISA analysis
- competitive ELISA analysis or KINEXA surface plasmon resonance analysis
- invitro or invivo 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 any nother source.
- inhibitor 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, IL-6 activity) or a property, disease, or condition.
- Inhibition or neutralization of IL-6 activity by binding of an antibody of the invention to SIL-6R 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, mammals, farm animals, mammals, sports animals and mammals of domestic animals; 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-6, which can be improved by decreasing the bioactivity of IL-6.
- vector includes a nucleic acid molecule capable of transporting another nucleic acid with which it linked, including plasmids and viral vectors, but not limited to. 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.
- 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 isolated polynucleotide according to the invention or any recombinant vector (any recombinant vectors) that contain the sequence encoding the HCVR, LCVR or monoclonal antibody of the invention.
- Host cells include the offspring of an individual host cell, and the offspring need not be completely identical (in morphology or complete DNA complement) to the original parent cell due to natural, accidental or intentional mutations and / or changes.
- 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 the 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.
- affinity refers to measuring the attraction between an antigen and a binding molecule, for example, an antibody.
- the intrinsic ability to attract a binding molecule to an antigen is typically expressed as the equilibrium constant of binding affinity (KD) of a particular interaction of the binding molecule and antigen. It is believed that the binding molecule specifically binds to the antigen when the KD is ⁇ 1 mM, preferably ⁇ 100 nM.
- KD affinity binding constant can be measured, for example, using surface plasmon resonance (BIAcoreTM) or biolayer interferometry, for example, using the ProteOnTM XPR36 SPR system from Bio-Rad or the OctetTM system.
- Ka refers to the rate of association of a particular antibody-antigen interaction
- Kd refers to the dissociation rate of a specific antibody-antigen interaction
- KD refers to the affinity constant, which is obtained from the ratio of Kd to Ka (ie Kd / Ka), and it is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art.
- a preferred method for determining KD antibodies is a method using resonance of surface plasmons, preferably using a biosensor system such as the Biacore® system.
- the term “high affinity” for an IgG antibody refers to an antibody having KD - E-10 - E-08 M, more preferably E-10 - E-09 M or less, and even more preferably E-10 M or less in relation to the target antigen.
- the “high affinity” of binding may vary for other antigen isotypes.
- IgM refers to an antibody having a KD E-10 - E-07 M or less, more preferably E-10 - E-08 M or less, even more preferably E-10 - E-09 M or less.
- K 0 ff refers to the dissociation rate constant of a particular interaction of a binding molecule and antigen.
- the koff + dissociation rate constant can be measured by bi-layer interferometry, for example using the Octet TM system.
- epitope refers to a part (determinant) of an antigen that specifically binds to a binding molecule (eg, an antibody or a related molecule, such as a bispecific binding molecule).
- a binding molecule eg, an antibody or a related molecule, such as a bispecific binding molecule.
- Epitope determinants usually consist of molecules, such as amino acids or carbohydrates, or Sugar side chains, and, as a rule, have specific three-dimensional structural characteristics, as well as specific characteristics of charges.
- the epitope can be “linear” or “conformational”. In a linear epitope, all points of interaction between a protein (eg, an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein.
- a conformational epitope interaction points occur through amino acid residues on a protein that are separated from each other in the primary amino acid sequence.
- antibodies to this epitope can be generated using techniques well known in the art.
- the generation and characterization of antibodies or other binding molecules can shed light on information about the desired epitopes. Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example, by conducting competition studies to find binding molecules that compete for binding to antigen.
- epitope refers to the part of the 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 means of a conventional immunoassay.
- test antibody if the test antibody is unable to bind to the target antigen at the same time, then the test antibody binds to the same epitope that overlaps the epitope or epitope that is in close proximity to the epitope associated with the binding molecule.
- This experiment can be performed using ELISA, RIA, analysis of intermolecular interactions on BIACORETM, biolayer interferometry or flow cytometry.
- the two-way competitive assay described above can be used, i.e., determine whether a known binding molecule blocks the test binding molecule, and vice versa.
- Such cross-competition experiments can be performed, for example, using an IBIS MX96 SPR instrument or an OctetTM system.
- the binder the molecule proposed in this invention is a monoclonal antibody.
- TAB used herein refers to a monoclonal antibody, that is, an antibody that is synthesized and isolated by a separate clonal cell population.
- a clonal population may be a clonal population of immortalized cells.
- immortalized cells in a clonal population are hybrid cells, hybridomas that are usually obtained by fusing individual B lymphocytes from immunized animals with individual lymphocyte tumor cells. Hybridomas are a type of engineered cell and are not found in nature.
- the class (isotype) and subclass of antibodies can be determined by any method known in the art.
- the class and subclass of an antibody can be determined using antibodies specific to a particular class and subclass of antibodies. Such antibodies are commercially available.
- the class and subclass can be determined using ELISA, Western blot analysis, as well as other methods.
- the class and subclass can be determined by sequencing all or part of the constant domains of the heavy and / or light chains of antibodies, comparing their amino acid sequences with known amino acid sequences of different classes and subclasses of immunoglobulins, and determining the class and subclass of antibodies.
- sequence identity refers to residues in two sequences that are the same when aligned for maximum match.
- a sequence identity comparison can take place over a length of at least about nine nucleotides, typically at least about 18 nucleotides, more often at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides.
- polynucleotide sequences can be compared using FASTA, Gap, or BESTFIT, which are programs in the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin.
- FASTA which includes, for example, the FASTA2 and FASTA3 programs, provides alignment and percent sequence identity in the areas of best coverage between the requested and the desired sequences (Pearson, Methods Enzymol. 183: 63 98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71 -84 (1998)).
- the default parameters for a particular program or algorithm are used.
- the percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (word size 6 and NOPAM factor for the score matrix) or using Gap with default parameters, as provided in GCG version 6.1.
- nucleic acid sequence should be interpreted as a nucleotide sequence exhibiting at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity with respect to the nucleic acid sequence.
- bispecific antibodies includes an antibody capable of selectively binding two or more epitopes.
- Bispecific antibodies can include two different antigen-binding parts, where these antigen-binding parts specifically bind different epitopes either on different molecules (e.g., antigens), or on the same molecule (e.g., on the same antigen ) If a bispecific antibody is capable of selectively binding two different epitopes (first epitope and second epitope), the affinity of the first antigen-binding part for the first epitope will typically be at least one to two, three, or four orders of magnitude lower than the affinity of the first antigen -binding parts for the second epitope, and vice versa.
- Epitopes recognized by a bispecific antibody can be the same or different target (for example, on the same or different protein). Bespecifically antibodies can be obtained, for example, by combining heavy chains that recognize different epitopes on the same antigen. For example, nucleic acid sequences encoding variable sequences of heavy chains that recognize different epitopes can be fused to nucleic acid sequences encoding different constant regions of the heavy chain, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
- a typical bispecific antibody has two heavy chains, where each has three heavy chain CDRs, followed by the (from the ⁇ -end to the C-end) CH1 domain, hinge region, CH2 domain and CH3 domain, and immunoglobulin light chain that is either not provided with antigen binding specificity, but can combine with each of the heavy chains, or can combine with each of the heavy chains and can bind one or more epitopes bounded by antigen-binding regions of the heavy chain, or can combine with each of the heavy chains and It binds either one or both heavy chains to one or both epitopes.
- the present invention provides an antibody or antigen-binding fragment thereof, in which the ability to bind to the human interleukin-6 (IL-6) receptor is ensured by the fact that it contains an amino acid sequence of at least 75% homology the sequence of SEQ ID NO: 3.
- IL-6 human interleukin-6
- the present invention relates to an antibody or fragment thereof which comprise the amino acid sequence of SEQ ID NO: 3.
- the implementation of the present invention relates to an antibody or its fragment, which contain:
- the present invention relates to an antibody or fragment thereof that contains the amino acid sequences of SEQ ID NO: 1-3.
- the binding fragment competes for binding or binds to the same epitope as the binding domain containing the amino acid sequence of SEQ ID NO: 7.
- the binder the fragment is at least 90% homologous to the amino acid sequence of SEQ ID NO: 7.
- the binding domain comprises the amino acid sequence of SEQ ID NO: 7.
- the antibody or antigen binding fragment thereof is characterized in that it is of the human IgG1, IgG2, IgG3, IgG4 isotype.
- the antibody or fragment thereof has a heavy chain sequence homologous to at least 90% of the sequence of SEQ ID NO 9.
- the antibody or fragment thereof has a light chain sequence homologous to at least 90% of the sequence of SEQ ID NO 10.
- the Fc constant portion of the IgG1 isotype comprises mutations E233P, L234A, L235A, E236P, L237V and / or L238A.
- the Fc constant portion of the IgG1 isotype contains mutations that increase the pharmacokinetic parameters in an animal or human, such as ⁇ / 2
- the Fc constant portion of the IgG1 isotype comprises M255Y, S257T, and / or T259E mutations that increase the pharmacokinetic parameters in an animal or human, such ⁇ 8 ⁇ ⁇ 1 / 2 ⁇ (hour) or Stax ( ⁇ g / ml).
- the antibody or antigen binding fragment thereof has at least one of the following properties:
- b) has such aggregation stability that when concentrations of more than 10 mg / ml and with increasing temperature to 37 ° C for more than 2 weeks, the content of aggregates does not increase by more than 5% of the initial content in the solution.
- c) has such aggregation stability that, at concentrations of more than 10 mg / ml and increasing the temperature to 50 ° C for more than 24 hours, the content of aggregates does not increase by more than 5% of the initial content in the solution;
- d) has a KD affinity constant with the human IL-6 receptor of not more than E-10 - E-09 M;
- e has a kinetic constant of association kon (1 / Ms) with the human IL-6 receptor of at least E + 05 (1 / Ms);
- f has a kinetic dissociation constant dis (1 / s) with the human IL-6 receptor of not more than E-04 (1 / s).
- e demonstrates antiproliferative activity in a culture of interleukin-6 dependent DS1 cells with a calculated IC50 value of not more than 10 "8 M.
- h shows the blocking of STAT-3 signaling in a culture of interleukin-6 dependent cells with a calculated IC50 value of not more than 10 "8 M.
- the present invention also provides a bispecific antibody comprising an antigen binding fragment of the above antibody.
- An example of such an antibody may be a bispecific antibody containing antigen-binding parts whose CDRs are different from each other and have a total homology higher than 95% of each other.
- the present invention also provides DNA for the preparation of the above antibody or antigen binding fragment thereof, comprising sequences corresponding to the sequences of said CDRs.
- Expression vector also provides an expression vector comprising one or more of the above DNAs.
- the present invention also provides a cell line containing the above vector or the above DNA in cells.
- the present invention provides a method for producing the above antibody or antigen binding fragment thereof, comprising culturing the above cell line in a culture medium under conditions sufficient to produce said antibody or antigen binding fragment, followed by isolation and purification of the resulting antibody or antigen binding fragment thereof.
- the present invention provides a pharmaceutical composition for treating a disease or condition associated with the action of interleukin-6 (IL-6), or to eliminate or alleviate a symptom associated with the undesirable effects of IL-6, containing an effective amount of an antibody or antigen-binding fragment thereof paragraph 1, or according to any one of the paragraphs of paragraph 1, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.
- IL-6 interleukin-6
- composition according to paragraph is a solution for parenteral administration.
- composition according to paragraph may be lyophilized powder.
- composition according to paragraph may be intended for use in therapy and / or in the diagnosis of a disease selected from the following group of diseases and / or disorders: rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondylarthropathy, systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, asthma, allergic disease, psoriasis, atopic dermatitis, with Leroderma, graft versus host reaction, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, Kawasaki disease, Graves disease, nephrotic syndrome, chronic fatigue syndrome, Wegener granulomatosis, Shenlein-Genoch purple, microscopic vasculitis , chronic active hepatitis, uvenitis, septic shock, toxic shock syndrome,
- 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.
- Pharmaceutical compositions for administration are designed to correspond to 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, cryoprotectants are selected taking into account composition of the necessary dosage form.
- compositions are developed in accordance with conventional methods described, for example, in Remington, TheScienceandPracticeof Pharmacy, 19th Edition, Gennaro, Ed., MackPublishingCo., Easton, PA 1995, which describes various methods for preparing compositions generally known to those skilled in the art.
- a pharmaceutical composition containing an antibody or fragment thereof of the invention can be administered to a patient using standard methods of administration, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository.
- the pharmaceutical composition of the invention contains an effective amount of an antibody of the invention.
- effective amount is meant an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result.
- a therapeutically effective amount of an antibody may 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.
- An effective amount is also an amount in which the therapeutically beneficial effect of the antibody prevails over the toxic or harmful effect. If an antibody is used for prophylactic purposes, then “effective amount” is understood to mean the amount effective at dosages and over the periods of time necessary to achieve desired preventive 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.
- an effective amount is at least the minimum dose, but less than the toxic dose of the active agent, necessary to provide a therapeutic or prophylactic effect in a patient.
- an effective amount of an antibody of the invention is an amount that, in mammals, preferably humans, reduces the biological activity of IL-6.
- 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 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. Dose may vary depending on the type and severity of the disease.
- the 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 -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.
- 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.
- Antibodies or fragments thereof may be frozen either 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 the treatment or prophylaxis of 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-6, and may have sterile access (for example, the container may be an intravenous solution bag or a vial having a plug that is permeable to the hypodermal injection needle) .
- the active agent in the composition is an anti-L6HH 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. 10. Application
- 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. 10.
- the present invention provides the use of the above antibody or fragment thereof for the manufacture of a medicament.
- FIG. 1 Gel electrophoresis under denaturing conditions of the accumulated and purified human SIL-6R in 12% PAGE.
- Lanes a-d Fermentas unstained marker, IL6R-H6F culture fluid before application to the column 10 ⁇ l, IL6R-H6F culture fluid after application to the column 10 ⁇ l, IL6R-H6F elution 5 ⁇ l.
- FIG. 2 Western blot analysis of accumulated and purified human SIL-6R. After gel electrophoresis under denaturing conditions of human SIL-6R in 12% PAGE and transfer to the membrane, binding was performed with tocilizumab, and then anti-GoatahlgGlgG HRP. Coloring was carried out using DBA. Lanes a-b: Prestained marker, IL6R-f lag-his 5 ⁇ l.
- FIG. 3 Gel electrophoresis under denaturing conditions of the accumulated and purified human IL6-H6-EPEA (with 6 histidines and EPA sequence at the C-terminus of the protein) in 12% PAGE.
- Lanes a-d Fermentas unstained PW marker, IL6-H6-EPEA ql before applying to a column of 5 ⁇ l, IL6-H6-EPEA ql after applying to a column of 5 ⁇ l, IL6-H6-EPEA elution from a column of 5 ⁇ l.
- FIG. 4 Fagmida for cloning Fab phage display libraries.
- FIG. 5 Expression plasmid for cloning and production
- FIG. 6 Kinetics of association and dissociation of Fab fragments with the alpha subunit of human IL-6R on ForteBio.
- FIG. 7 Gel electrophoresis under denaturing conditions of the developed and purified full-length human antibodies aHTH-IL-6sR in 12% PAGE in the presence of beta-mercaptoethanol.
- Lanes a-f a - Fermentas unstained marker, b-f - aHTH-IL-6sR antibody.
- FIG. 8 Denaturing gel electrophoresis of the developed and purified full-length human aHTH-IL-6sR antibodies in 12% PAGE in the presence of beta-mercaptoethanol.
- Lanes a-f a - Fermentas unstained marker, b-f - aHTH-IL-6sR antibody.
- FIG. 9 Cellular antibody proliferation inhibition test for DS-1 aHTH-IL-6R antibodies.
- FIG. 10 Cell test of STAT3 pathway inhibition assay in HEK-BlueTM IL6 cell culture with BCD089 antibody (aHTH-IL-6sR) compared to Tocilizumab.
- FIG. 1 1 - Enzyme-linked immunosorbent assay for the interaction of BCD089 antibody with human IL-6R, cynomolgus monkey, rat and dog
- FIG. 12 Enzyme-linked immunosorbent assay for the interaction of BCD089 antibodies with human, rabbit and guinea pig IL-6R receptors.
- FIG. 13 Kinetics of the association and dissociation of BCD089 with the alpha subunit of human IL-6R on ForteBio.
- FIG. 14 Kinetics of the association and dissociation of BCD089 with the alpha subunit of the cynomolgus IL-6R on ForteBio.
- FIG. 15 Kinetics of the association and dissociation of BCD089 antibody BCD 089 with human and mouse IL-6R (mouse IL-6R 250 nM-black curve, human IL-6R 50 nM-blue curve)
- FIG. 16 Kinetics of the association and dissociation of BCD089 with the alpha subunit of guinea pig IL-6R on ForteBio.
- FIG. 17 Gel-filtration profiles of the BCD089 molecule before (red) and after incubation for 12 hours of incubation at 50 ° C (blue).
- FIG. 18 Evaluation of anti-inflammatory activity of the drug BCD-089 in a model of collagen-induced arthritis of primates DETAILED DESCRIPTION OF THE INVENTION
- polypeptides were selected that specifically bind to the soluble form of the human interleukin-6 receptor (slL-6R), i.e., its alpha subunit.
- phage libraries constructed on the basis of the amino acid sequences of the heavy and light chains of human immunoglobulins were used.
- RNA molecules were isolated and purified from blood cells of more than 1000 donors, of which the genes of the heavy and light chains of donor immunoglobulins were synthesized in several steps by reverse transcription and PCR.
- Various combinations of genes of the binding sites of the heavy and light chains were inserted into bacteriophage vectors in the correct orientation and used to create hybrid phage libraries (Fig. 4).
- the selection of the obtained phage libraries was carried out on the human protein SIL-6R, which was produced in the transient system of CHO-T cells and purified from the culture fluid on the IMAC BIORAD sorbent.
- Cells producing SIL-6R were obtained by reclining the alpha subunit of the human interleukin-6 receptor gene into a vector for transient production of pEE with subsequent transfection of CHO-T.
- Producer cells were grown in an incubator for 7-10 days.
- the protein was purified in a single step and verified that it was targeted using the Westernblot method using specific antibodies (Human IL-6sR Detection antibody PartNo. 840244 from Human IL-6sR DuoSet ELISA Development Kit Catalog Number: DY227 R&D System).
- the selection of the obtained phage libraries was carried out in two or more stages. Phages that specifically bind to SIL-6R immobilized on plastic were selected. As a result, we received many phages that bind to slL-6R from which bacteriophage vectors were isolated. Of these vectors, the genes of polypeptides that specifically bind to the protein were amplified and transferred to expression vectors for the synthesis of protein molecules in prokaryotic E. coli cells. After transformation of the cells with the obtained plasmids and synthesis of individual polypeptides, they were screened and selected molecules that specifically bind to IL-6sR and block the binding of the ligand to the receptor.
- the affinity constants of the obtained proteins were compared with each other and the most successful genes were inserted into the expression peF vectors for the expression of already full-sized antibodies.
- affinity was evaluated and they were confirmed to block the signal of interleukin 6 in various functional cell tests.
- one antibody molecule was selected that specifically binds to SIL-6R and blocks the effect of IL-6 in cell tests. Its heavy and light chain nucleotide sequences were recloned into the psX vector, using which cell lines stably producing the antibody were obtained. Subsequently, using the obtained cell line, the selected antibody was developed for which a purification scheme and storage conditions were selected. Preclinical studies of the resulting preparation were planned and started.
- Antibodies and antigens were produced in cells of a transient cell line obtained from Chinese hamster ovary cells (CHO-K1 line), according to the protocols Suspension cultivation was carried out in flasks on an orbital shaker using serum-free media for suspension cultures manufactured by Life Technologies Corporation, according to the manufacturer's instructions.
- cells were transfected using linear polyethyleneimine (PEI "MAX", the company "Polysciences”). The ratio of DNA / PEI was 1: 3-1: 10. 7-10 days after transfection, the culture medium was centrifuged at 2000 g for 20 minutes and filtered through a filter with a pore size of 0.22 ⁇ m.
- Target proteins were isolated from the culture fluid by affinity chromatography.
- a recombinant SIL-6R protein containing six His amino acids at the C-terminus of the protein was isolated and purified from the culture fluid using a Profinity IMAC Ni-charged resin (Bio-Rad). Prior to purification, N 1CI2 was added to the culture fluid to a concentration of 1 mM. Next, 1 / 200-1 / 500 volumes of Profinity IMAC Ni-charged sorbent were added to the culture fluid and stirred for 1 hour at room temperature. The sorbent was transferred to a 5 or 10 ml Thermoscientific Polypropylene columns column, washed with 5 column volumes of 10 mM pH8 phosphate buffer with 5 mM imidazole and 0.3 M sodium chloride to wash the non-specific binding components.
- the bound antigen was eluted using 0.3 M imidazole, pH 8, 0.3 M NaCl.
- the protein was transferred to PBS (pH 7.4) using dialysis using Snake Skin Dialysis Tubing technology, filtered (0.22 ⁇ m) and transferred to tubes.
- the tubes were stored at -70 ° C.
- the purity of the resulting protein solution was evaluated by SDS gel electrophoresis, and identified using the Western blot method using a specific anti-11_-6Ph antibody and conjugated goat anti-human antibody antibodies ( Figures 1 and 2).
- the anti-L6HH antibodies tested were purified on a 1 ml HiTrap rProteinA FF column (GE Healthcare).
- the clarified culture fluid was passed through 1 ml HiTrap rProtein A FF (GE Healthcare), which was equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to wash the non-specific binding components. Bound protein was eluted using 0.1 M glycine pH 3 buffer. The main protein elution peak was collected and adjusted to neutral pH using 1 M Tris buffer (pH 8). All stages were carried out at a flow rate of 1 ml / min. Next, the protein was transferred to PBS (pH 7.4) by dialysis using Snake Skin 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 (Fig.7 and 8).
- the recombinant ligand IL6-H6-EPEA (interleukin 6 with a peptide tail of 6 histidines and an EPA sequence at the C-terminus) was purified on a GE Healthcare C16 / 20 column with 5 ml Capture Select C-tag Affinity Matrix. All stages were carried out at a flow rate of 5 ml / min.
- the clarified culture fluid was passed through a column that was equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with PBS until the signal of the spectrophotometer of the chromatographic system reached a plateau in order to wash the non-specific binding components.
- PBS phosphate-buffered saline
- Bound protein was eluted using 20mM Tris 2M MgCl2 pH7.1. The major elution protein peak was collected and transferred to PBS (pH 7.4) by dialysis using SnakeSkinDialysisTubing technology. Then it was filtered through a filter (0.22 ⁇ m) and transferred to tubes and stored at -70 ° C. The purity of the resulting protein solution was evaluated using SDS gel electrophoresis (Fig.Z).
- SIL-6R-binding phage particles with Fab- human fragments were isolated from the MeganLib TM combinatorial phage Fab display library (BIOCAD) containing more than 10 1 1 independent clones and synthesized based on the human immunoglobulin variable domain genes ( Figure 4), according to a modified protocol (J BiolChem. 1999 Jun 25; 274 (26): 18218-30). The lymphocytes of more than a thousand donors were used to create the library. Selection was performed on recombinant human SIL-6R-H6F under conditions as previously described (NatBiotechnol. 1996 Mar; 14 (3): 309-14; J Mol Biol. 1991 Dec 5; 222 (3): 581 -97).
- human SIL-6R-H6 or slL-6R-Fc in 50 mM carbonate buffer (pH 9.5) was adsorbed overnight at 4 ° C on the surface of EIA / RIA high binding (Greineer bio- one).
- the tubes were washed several times with PBST (PBS pH 7.4 and 0.1% Tween 20 v / V), and then the vacant protein binding sites on the surface of the tubes were blocked with a skim milk solution (0.5% m / V in PBST pH 7.4 ) The skim milk solution was incubated in tubes for 1 h. The tubes were then washed with PBST solution.
- the flasks were rearranged on a rocking chair and the phage developed for 3-5 hours at AIA.
- the cell culture was centrifuged for 20-30 minutes 10 thousand rpm and the supernatant was collected in test tubes. After that, 1/6 of the volume of the solution containing 20% polyethylene glycol and 2.5 M sodium chloride was added, and stirred vigorously. The solution was incubated in ice for at least 3 hours. Then, the solution was centrifuged for 10 minutes at 8000 d; a precipitate formed containing phages was dissolved in 1 ml of TBS buffer.
- E. coli TG1 cells were isolated phage DNA (phagemid) derived phage M13. From the phagemid, PCR genes and terminal specific primers, the genes of Fab fragments with various variable regions were amplified and transferred into the expression plasmid pI4. Replaced genes on sites Nhel, Notl. The insert was checked by sequencing of 4 random colonies.
- Fab fragments based on the obtained genes were synthesized in E. coli BL21Gold cells.
- Fab-fragment of Actemra (tocilizumab) was used as a positive control.
- 96-well ELISA plates (from NunclmmunoMaxisorp) were used.
- IL-6sR-H6 antigen was bound on the surface of the wells; for this, 50 ⁇ l of a 0.5 ⁇ g / ml solution of IL-6sR-H6 in binding carbonate buffer was added to each well. The lid was closed and incubated overnight at 4 ° C.
- the plates with solutions of Fab fragments were incubated on a shaker for one hour at room temperature. After an hour, the wells of the plates were washed five times with PBST buffer. Then a solution of conjugated secondary goat antibody was added to the wells of the plate with peroxidase (from Pierce-ThermoScientific) in a ratio of 1: 5000 in PBST at 50 ⁇ l / well. The plates were incubated on a rotary shaker for 50 minutes at room temperature, and then washed five times with PBST buffer as described above.
- peroxidase from Pierce-ThermoScientific
- a colorimetric signal was obtained by adding to the wells a solution of substrate (TMB) in acetate buffer pH5.5 and ⁇ 2 ⁇ 2 0.02% (50 ⁇ l / well), incubated until the signal was saturated (on average 3-5 min), and then the reaction stopped by adding a solution of 10% sulfuric acid at 30 ⁇ l / well.
- the signal was recorded at a wavelength of 450 nm using a Tesap-Sunrise plate reader (from Tesap). The number of Fab fragments bound to the antigen is proportional to the recorded signals. Therefore, clones were selected whose signal exceeded the background by more than 5 times.
- the ELISA method was used to test the antagonistic ability of previously selected Fab fragments specifically binding to human IL-6R.
- An anti-L6BPb fragment was used as a positive antagonist control.
- the ligand IL-6-EPA was immobilized on ELISA plates (Maxisorp), adding 50 ⁇ l of a solution with a protein concentration of 1 ⁇ g / ml in carbonate buffer per well. The solution was incubated in the plate overnight at 4 ° C. All subsequent steps were carried out according to the standard ELISA with minor modifications and using a high-performance automated platform based on GenetixQ-pix2xt robotic systems (from MolecularDevice) and TecanFreedomEVO 200 (from Tesap). To block nonspecific binding, 200 ⁇ l blocking buffer per well (0.5% skim milk in PBST) was added. The plates were incubated on a shaker for one hour at room temperature.
- a colorimetric signal was obtained using a TMB solution (50 ⁇ l / well) to saturation (average 3-5 min), the reaction was stopped by adding a solution of 10% sulfuric acid (30 ⁇ l / well).
- the color signal was measured at a wavelength of 450 nm using a Tecan-Sunrise plate reader (manufactured by Tesap). The degree of binding of the secondary conjugated antibody was proportional to the color signal.
- Fab fragments that specifically bind to the IL-6R receptor and block the interaction of the receptor with its ligand were compared with each other by receptor affinity.
- Comparative dissociation constant screening (k ⁇ jg) for aHTH-IL-6R candidates was performed using an OctetRed 96 instrument and anti-FABCHI biosensors (Pall-ForteBio) (Fig. 6). The biosensors were rehydrated for 30 minutes in a working buffer containing 10 mM PBS pH7.2-7.4, 0.1% Tween-20 and 0.1% BSA. 1/10 volume of a 10 ⁇ working buffer was added to the studied samples of E. Co // cell growth medium containing aHTH-IL-6R Fab fragments and mixed.
- anti FABCH1 biosensors were immersed in solutions containing Fab fragments for 12 hours at 4 ° C. After 12 hours, sensors with Fab fragments immobilized on the surface were transferred to wells with a working buffer, where the baseline was recorded (60 s). The sensors were then transferred to wells with analyte solution (IL-6sR-H6F, 30 ⁇ g / ml) for association of the antigen-a-fragment fragment complex (300 s). Then the sensors were returned to the wells containing the working buffer for the subsequent stage of dissociation (300 s). After each experiment, the sensors used were regenerated by placing them three times in the regeneration buffer (Gly-HCI, pH 1, 7), after which they were used in the following experiments. The analysis of the obtained curves was performed using OctetDataAnalysis software (version 7.0) according to the standard procedure using a 1: 1 interaction model.
- lgG1 antibodies The construction of full-length lgG1 antibodies was carried out by means of the reclamation of the genes of variable domains from expression plasmids, previously used to generate Fab fragments.
- an In-Fusion® HD EcoDry TM CloningKit kit from Clontech was used for cloning.
- Variable domain gene insertions were obtained by amplification in PCR with specific primers, matrix residues in PCR were removed by treatment with restriction enzyme Orp !.
- the vector is a pEE plasmid, linearized by restriction Sail and BsiWI for the light chain and Sall / Nhel for heavy.
- the insert gene and linearized vector were mixed in 10 ⁇ l of water and transferred to separate In-Fusion vialkistrips. Mix by pipetting.
- Strips were incubated for 15 minutes at 37 ° C, then 15 minutes at 50 ° C, then transferred to ice. Part of the reaction volume with the resulting constructs was used in cell transformation. Plasmids were isolated from the obtained clones and the insert was checked by sequencing.
- the growth of DS-1 cells depends on the presence of extracellular external IL6 ligand and blocking its binding to the receptor on the surface of the cells leads to inhibition of their growth.
- the DS-1 cell culture was grown on RPMI-1640 nutrient medium, 10% inactivated fetal serum, 1 mM sodium pyruvate and 7.5 ng / ml IL6. The day before the experiment, the cells were washed 2 times in PBS from IL6 residues and plated on a 96-well plate at a rate of 10,000 cells per well in growth medium without the addition of IL6.
- a HEK-BluelL6 cell suspension was prepared with a concentration of 1-10 ⁇ clones / ml in DM EM growth medium with 10% inactivated serum (cell growth medium). Sown in a 96-well plate at the rate of 100 ⁇ l / well (5-10 ⁇ clones / well).
- a series of dilutions of antibodies was prepared in a cell growth medium from 200 ⁇ g / ml in increments of 3 ten points, the last control point without antibody.
- a solution of human IL6 in a cell growth medium with a concentration of 4 ng / ml was prepared.
- 50 ⁇ l of diluted antibodies were added to the cells and incubated for 45 minutes in a CO2 incubator.
- 50 ⁇ l of IL6 solution was added to the antibody cells and the antibody cells and IL6 were left to incubate overnight in a CO2 incubator.
- the absorption level was measured on a spectrophotometer at a wavelength of 630 nm (Fig. 10).
- the analysis was performed at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as the working buffer. Titration of human, cynomolgus monkey, guinea pig, dog, rabbit and mouse IL-6R was performed using a working buffer from a concentration of 126 nM to 2 nM in steps of 2.
- BCD89 binds to the recombinant preparation of human IL-6R and macaque IL-6sR with high affinity (Fig. 13, Fig. 14).
- the candidate interacts with guinea pig IL-6R with a constant lower by 3 orders of magnitude than with a human (FIG. 16). No antibodies were detected with the mouse receptor (FIG. 15).
- Test samples were concentrated to 5 mg / ml using ultrafiltration in centrifuge filters AmiconUltra 10 kDa per 0.5 ml (Millipore). Protein content was determined by UV spectrophotometry at a wavelength of 280 nm. Each obtained sample was divided into several parts of 150 ⁇ l and placed in separate tubes: one tube for each composition was stored in a refrigerator at + 4 ° C, the rest were installed in a tube thermostat and thermostated at 50 ° C for a set time.
- the flow rate was 0.7 ml / min, and the sample volume was 10 ⁇ l with a sample concentration of 5 mg / ml.
- the detector wavelength is 220 and 280 nm, the elution time is 25 minutes. (Fig.17).
- S1 is the peak area of the monomer
- XS is the sum of the areas of all the peaks.
- the introduction of the drug and placebo substances began after preliminary sensitization with collagen.
- the articular surface was measured in animals of all groups to calculate the PPV; at the time the study was completed, metacarpophalangeal and metatarsophalangeal joints were taken to assess the severity of destructive changes. Animals were divided into 5 groups. The names of the groups are given in table 3.
- bovine collagen type II (Sigma) three times.
- the first introduction of collagen The total amount of collagen administered to one experimental animal was 2 mg. For this, 2 mg of collagen was dissolved in 0.7 ml of 0.1 M acetic acid. To the resulting solution was added 0.7 incomplete Freund's adjuvant.
- the animals were kept for 28 days after the first administration of collagen.
- the second introduction of collagen The total amount of collagen administered to one experimental animal was 3 mg. For this 3 mg collagen was dissolved in 1.0 ml of 0.1 M acetic acid. To the resulting emulsion, 1.0 ml of complete Freund's adjuvant was added.
- the animals were kept 21 days after the second injection of collagen.
- the third introduction of collagen The total amount of collagen administered to one experimental animal was 3 mg. For this, 3 mg of collagen was dissolved in 1.0 ml of 0.1 M acetic acid. To the resulting emulsion, 1.0 ml of Freund's incomplete adjuvant was added.
- PS value for the longitudinal axis ⁇ value for the transverse axis x 3.14 x 0.25.
- PPV (value for PS on the day of the experiment ⁇ 100) / (average value for PS before the induction of arthritis) (Fig. 18).
- - a general blood test according to indicators: the number of red blood cells, the number of leukocytes, the concentration of hemoglobin, the number of lymphocytes, the number of monocytes, the number of neutrophils, the number of eosinophils, the number of basophils, the number of platelets (before administration, then once a week, starting from the first week of the experiment);
- the BCD89 antibody was transferred to the required buffer and the concentration was adjusted to 180 mg / ml, the resulting solution was filtered (arrow filtering) and the syringes were filled.
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Abstract
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Priority Applications (14)
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MX2019001970A MX2019001970A (es) | 2016-08-17 | 2017-08-03 | Anticuerpo o un fragmento de union a antigeno del mismo, capaz de unirse a un receptor humano de interleucina-6. |
NZ750451A NZ750451A (en) | 2016-08-17 | 2017-08-03 | Antibody or an antigen-binding fragment thereof capable of binding to a human receptor of interleukin-6 |
KR1020197007632A KR102602564B1 (ko) | 2016-08-17 | 2017-08-03 | 인터루킨-6의 인간 수용체에 결합가능한 항체 또는 항원 결합성 단편 |
AU2017313632A AU2017313632A1 (en) | 2016-08-17 | 2017-08-03 | Antibody or an antigen-binding fragment thereof capable of binding to a human receptor of interleukin-6 |
BR112019003307A BR112019003307A2 (pt) | 2016-08-17 | 2017-08-03 | anticorpo ou seu fragmento de ligação a antígeno, capaz de se conectar a um receptor humano de interleucina-6 |
JP2019509472A JP7158376B2 (ja) | 2016-08-17 | 2017-08-03 | インターロイキン-6のヒト受容体に結合することができる抗体またはその抗原結合フラグメント |
MA44917A MA44917B1 (fr) | 2016-08-17 | 2017-08-03 | Anticorps ou son fragment de liaison d’antigènes apte a se lier au récepteur d'interleukine-6 de l'humain. |
CA3033063A CA3033063A1 (en) | 2016-08-17 | 2017-08-03 | Antibody or an antigen-binding fragment thereof capable of binding to a human receptor of interleukin-6 |
EP17841757.2A EP3502135A4 (en) | 2016-08-17 | 2017-08-03 | ANTIBODIES OR ITS ANTIGEN-BINDING FRAGMENT CAPABLE OF BINDING TO THE HUMAN INTERLEUKIN-6 RECEPTOR |
CR20190086A CR20190086A (es) | 2016-08-17 | 2017-08-03 | Anticuerpo o un fragmento de union a antígeno del mismo, capaz de unirsea un receptor humano de interleucina-6 |
CN201780064176.5A CN110114370B (zh) | 2016-08-17 | 2017-08-03 | 能够与人白细胞介素-6受体结合的抗体或其抗原结合片段 |
US16/325,528 US11993647B2 (en) | 2016-08-17 | 2017-08-03 | Antibody or an antigen-binding fragment thereof capable of binding to a human receptor of interleukin-6 |
ZA201901042A ZA201901042B (en) | 2016-08-17 | 2019-02-18 | Antibody or an antigen-binding fragment thereof capable of binding to a human receptor of interleukin-6 |
PH12019500342A PH12019500342A1 (en) | 2016-08-17 | 2019-02-18 | Antibody or an antigen-binding fragment thereof capable of binding to a human receptor of interleukin-6 |
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RU2016133720A RU2656160C2 (ru) | 2016-08-17 | 2016-08-17 | Антитело или его антигенсвязывающий фрагмент, способный связываться с рецептором интерлейкина-6 человека |
RU2016133720 | 2016-08-17 |
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WO2018034597A1 true WO2018034597A1 (ru) | 2018-02-22 |
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US (1) | US11993647B2 (ru) |
EP (1) | EP3502135A4 (ru) |
JP (1) | JP7158376B2 (ru) |
KR (1) | KR102602564B1 (ru) |
CN (1) | CN110114370B (ru) |
AU (1) | AU2017313632A1 (ru) |
BR (1) | BR112019003307A2 (ru) |
CA (1) | CA3033063A1 (ru) |
CL (1) | CL2019000426A1 (ru) |
CR (1) | CR20190086A (ru) |
MA (1) | MA44917B1 (ru) |
MX (1) | MX2019001970A (ru) |
NI (1) | NI201900016A (ru) |
NZ (1) | NZ750451A (ru) |
PH (1) | PH12019500342A1 (ru) |
RU (1) | RU2656160C2 (ru) |
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RU2656160C2 (ru) | 2016-08-17 | 2018-05-31 | Закрытое Акционерное Общество "Биокад" | Антитело или его антигенсвязывающий фрагмент, способный связываться с рецептором интерлейкина-6 человека |
JP7525762B2 (ja) * | 2020-05-18 | 2024-07-31 | ビオシオン インコーポレイテッド | Il6rに結合する抗体及びその使用 |
RU2745814C1 (ru) * | 2020-06-05 | 2021-04-01 | Закрытое Акционерное Общество "Биокад" | Водная фармацевтическая композиция левилимаба и ее применение |
WO2024079310A1 (en) * | 2022-10-14 | 2024-04-18 | Ebbil, Ltd. | Sil-6r and ctgf binding proteins and methods of use thereof |
Citations (4)
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US20070280945A1 (en) * | 2006-06-02 | 2007-12-06 | Sean Stevens | High affinity antibodies to human IL-6 receptor |
US20110177074A1 (en) * | 2008-03-27 | 2011-07-21 | Sivakumar Pallavur V | Compositions and methods for inhibiting pdgfrbeta and vegf-a |
US8673306B2 (en) * | 2006-01-13 | 2014-03-18 | Novartis Ag | Compositions and methods of use for antibodies of dickkopf-1 |
WO2015065987A1 (en) * | 2013-11-01 | 2015-05-07 | Ibc Pharmaceuticals, Inc. | Bispecific antibodies that neutralize both tnf-alpha and il-6: novel therapeutic agent for autoimmune disease |
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CN1162922A (zh) * | 1994-10-07 | 1997-10-22 | 中外制药株式会社 | 以il-6拮抗剂作为有效成分治疗慢性类风湿性关节炎 |
GB2401040A (en) * | 2003-04-28 | 2004-11-03 | Chugai Pharmaceutical Co Ltd | Method for treating interleukin-6 related diseases |
US8080248B2 (en) * | 2006-06-02 | 2011-12-20 | Regeneron Pharmaceuticals, Inc. | Method of treating rheumatoid arthritis with an IL-6R antibody |
MX2010003329A (es) * | 2007-09-26 | 2010-04-27 | Chugai Pharmaceutical Co Ltd | Anticuerpo anti-receptor de il-6. |
NZ596837A (en) | 2009-06-17 | 2014-02-28 | Abbvie Biotherapeutics Inc | Anti-vegf antibodies and their uses |
AU2012223449A1 (en) * | 2011-03-03 | 2013-05-02 | Apexigen, Inc. | Anti-IL-6 receptor antibodies and methods of use |
RU2550262C1 (ru) | 2014-02-28 | 2015-05-10 | Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт особо чистых биопрепаратов" Федерального медико-биологического агентства | Моноклональное антитело против интерлейкина-6 человека и гибридома, продуцирующая данное моноклональное антитело |
CN105037548B (zh) * | 2014-04-21 | 2018-08-03 | 上海市免疫学研究所 | 抗人白介素-6受体β链单克隆抗体、其制备方法和用途 |
RU2656160C2 (ru) | 2016-08-17 | 2018-05-31 | Закрытое Акционерное Общество "Биокад" | Антитело или его антигенсвязывающий фрагмент, способный связываться с рецептором интерлейкина-6 человека |
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Patent Citations (4)
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US8673306B2 (en) * | 2006-01-13 | 2014-03-18 | Novartis Ag | Compositions and methods of use for antibodies of dickkopf-1 |
US20070280945A1 (en) * | 2006-06-02 | 2007-12-06 | Sean Stevens | High affinity antibodies to human IL-6 receptor |
US20110177074A1 (en) * | 2008-03-27 | 2011-07-21 | Sivakumar Pallavur V | Compositions and methods for inhibiting pdgfrbeta and vegf-a |
WO2015065987A1 (en) * | 2013-11-01 | 2015-05-07 | Ibc Pharmaceuticals, Inc. | Bispecific antibodies that neutralize both tnf-alpha and il-6: novel therapeutic agent for autoimmune disease |
Also Published As
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NZ750451A (en) | 2023-05-26 |
CA3033063A1 (en) | 2018-02-22 |
MA44917A1 (fr) | 2020-02-28 |
US20190194313A1 (en) | 2019-06-27 |
AU2017313632A1 (en) | 2019-03-07 |
CR20190086A (es) | 2019-07-04 |
CN110114370A (zh) | 2019-08-09 |
JP2019531063A (ja) | 2019-10-31 |
PH12019500342A1 (en) | 2020-01-20 |
EP3502135A1 (en) | 2019-06-26 |
KR20190067771A (ko) | 2019-06-17 |
CL2019000426A1 (es) | 2019-07-12 |
EP3502135A4 (en) | 2020-04-22 |
KR102602564B1 (ko) | 2023-11-16 |
ZA201901042B (en) | 2019-10-30 |
AU2017313632A2 (en) | 2019-03-21 |
CN110114370B (zh) | 2023-11-03 |
BR112019003307A2 (pt) | 2019-10-15 |
MX2019001970A (es) | 2019-08-14 |
RU2656160C2 (ru) | 2018-05-31 |
NI201900016A (es) | 2019-10-31 |
JP7158376B2 (ja) | 2022-10-21 |
MA44917B1 (fr) | 2020-06-30 |
US11993647B2 (en) | 2024-05-28 |
RU2016133720A (ru) | 2018-02-22 |
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