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    • 专利摘要:
    Monoclonal antibodies against bambi and use for the treatment of inflammatory diseases. The present invention relates to monoclonal antibodies specific to a peptide of the bambi protein, as well as to its uses and methods comprising them. Preferably the antibodies are used for the treatment of autoimmune diseases. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2626491A1
    申请号:ES201531761
    申请日:2015-12-03
    公开日:2017-07-25
    发明作者:Jesús MERINO PÉREZ；Ramón MERINO PÉREZ
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad de Cantabria；
    IPC主号:
    专利说明:

    The present invention relates to antibodies against the BAMBI protein (BMP and Activin Membrane Bound Inhibilory) and its use for the treatment and prevention of inflammatory diseases. Therefore, the present invention can be framed in the field of Medicine.
    10 STATE OF THE TECHNIQUE
    Under the denomination of "autoimmune or chronic inflammatory" disease, there are currently more than 100 nosological entities that globally affect 10% of the world's population (Shoenfeld et al. 2008 J Autoimmun 31: 325). Autoimmune diseases are the result of the action of multiple environmental agents on a particular genetic and / or epigenetic background. The accumulation in the same individual of all these factors alters the regulation of the immune response, causing aberrant responses to external agents or the reaction of the system
    20 versus the same. The consequence is the development of autoinflammatory and / or autoimmune diseases.
    Autoimmune diseases include rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease, spondyloarthritis or lupus
    25 systemic erythematosus, share a number of etiopathogenic mechanisms and also share their response to similar or equal immunomodulatory or immunosuppressive treatments. RA is the most common autoimmune rheumatic disease.
    30 In autoimmune-based diseases, CD4 + T lymphocytes, a denomination that encompasses multiple effector sub populations (TH1, TH2, TH17, TFH) or regulatory (Treg, Tr1) lymphocytes, defined basically by the pattern of cytokines they secrete ( Zhu J el al. 2010 Annu Rev Immunol 28: 445; Zygmunt B el al. 2011 Adv Immunol 109: 159). Alterations in the control of
    35 mechanisms that regulate the differentiation and activation of different functional sub populations of CD4 + T cells have been implicated in the development of immune-based pathologies. In this sense, some severe autoimmune diseases have been associated with uncontrolled increase in the differentiation and / or functionality of TH17 lymphocytes (Rohn TA el. 2006 Eur J Immunol 36: 2857; Kebir H el al. 2007 Nat Med 13: 1173 ; multiple sclerosis or rheumatoid arthritis between
    5 others) or TFH (Tangye SG al. 2013 Nat Rev Immunol 13: 412; systemic lupus erythematosus). In contrast, the decrease in the number and / or suppressive activity of Tregs cells is critical in IPEX syndrome, observed in patients with mutations in the foxp3 gene (Bennett el al. 2001 Nat Genet 27:20) or in mice "scurfy" deficient in this gene (Khattri R al. 2003 Nat. ImmunoI4: 337).
    10 For the treatment of autoimmune diseases, low-specific immunosuppressive drugs have been used and therefore have multiple adverse effects. More recently, specific cytokine antibodies or soluble receptors of these factors (called
    15 globally as biological drugs). These compounds have their great specificity as an advantage and the results obtained with them have been very positive. However, the use of biological drugs is not exempt from serious side effects and there is also frequent resistance to them, which forces the suspension of treatments. For all these reasons, the
    20 development of highly specific therapies using monoclonal antibodies against new biological targets
    DESCRIPTION OF THE INVENTION
    The present invention demonstrates the use of monoclonal antibodies against BAMBI for the treatment and prevention of autoimmune diseases, exemplified with recognized models of arthritis, psoriasis and colitis.
    In a first aspect the present invention relates to a monoclonal antibody that
    30 specifically recognizes an amino acid sequence comprising a peptide with at least 80% identity can SEQ ID NO: 1, preferably 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, where the size of said amino acid sequence is between 15 and 30 amino acids (15, 16, 17,18,19,20, 21 , 22,23, 24 or 25 amino acids), preferably 25 amino acids.
    The term "antibody", as used herein, refers toimmunoglobulin molecules and immunologically active portions (or fragments)of immunoglobulin molecules. That is, molecules that specifically bind(immunoreact) with an antigen, such as, for example, a peptide or a5 protein (an immunogen or epitope). The term "antibody" comprises antibodiesmonoclonal and polyclonal antibodies, in the present invention the antibody ismonoclonal, and refers to an intact antibody or immunologically fragmentsassets of it, and includes human, humanized and non-human origin antibodies,recombinant, chimeric and synthetic. In the context of this invention the term
    10 antibody refers to the immunoglobulin that the animal or a hybrid cell has synthesized specifically against the sequence described in the first aspect of the present invention.
    Examples of immunologically active immunoglobulin portions or fragments 15 include F (ab) and F (ab ') 2 fragments, which can be generated by treating the antibody with an enzyme, for example pepsin.
    "Monoclonal antibodies" are homogeneous populations of identical antibodies, produced by a hybridoma, that is, a hybrid cell product of the
    20 fusion of a clan of B lymphocytes descended from a single and single stem cell and a tumor plasma cell, which are directed against a single site or antigenic determinant. The process for obtaining the monoclonal antibodies of the invention can be carried out according to conventional methods, known in the state of the art. Optionally, said antibodies can be purified by means
    Conventional chromatography, affinity chromatography, A-Sepharose protein, hydroxyapatite chromatography, dialysis gel electrophoresis.
    As is known to one skilled in the art, there are five isotypes or major classes of immunoglobulins: immunaglobulin M (lgM), immunoglobulin D (lgD),
    30 immunoglobulin G (lgG) (which in turn has the following subtypes in the mouse: IgG1, IgG2a, IgG2b and IgG3), immunoglobulin A (lgA) and immunoglobulin E (lgE). The monational antibodies included in the present invention are: clan B101 .37 (lgG1) and clone B143-14 (lgM).
    A preferred embodiment of the first aspect of the invention specifically recognizes the sequence SEa ID NO: 1: Murine BAMBI peptide (109-133): LHDVLSPSKSEASGQGNRYQHDSSR or SEQ ID NO: 2: Human BAMBI peptide (109-133): LHDVLSPPRGEASGQGNRYQHDGSR.
    A more preferred embodiment of the first aspect of the invention relates to the5 antibody where said antibody is expressed from the cell line(hybridoma) deposited with an international authority.
    In a particular embodiment the antibody may comprise a detectable label. An even more preferred embodiment of the first aspect of the invention relates to the
    10 antibody where said antibody is conjugated to a fluorochrome, an enzyme, a gold particle, a nanoparticle, a peptide or other protein of interest, for example a protein or ligand peptide of a receptor.
    The term "detectable label ~ or" labeling ~ in the present invention refers to
    15 a molecular tag that allows the detection, location and / or identification of the molecule to which it is attached, by procedures and equipment suitable for detection, either by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of detectable labels for compound labeling include, but are not limited to radioactive isotopes, substrates.
    20 enzymatic, co-factors, ligands, chemiluminescent agents, fluorophores, enzymes (eg peroxidase), receptors and combinations thereof. In a particular embodiment the antibody is labeled with biotin, avidin, streptoavidin, alkaline phosphatase or horseradish peroxidase (HRP). Methods for marking and guidance for choosing suitable labels for different purposes are known by the
    25 subject matter expert.
    The monoclonal antibody can be altered biochemically, by genetic manipulation or it can be synthetic, it can also lack portions.
    In a more preferred embodiment of the first aspect of the invention the antibody comprises a heavy chain comprising the sequence SEQ ID NO: 3 and / or a light chain comprising the sequence SEQ ID NO: 4.
    In another more preferred embodiment of the first aspect of the invention, antibody 35 comprises a heavy chain comprising the sequence SEQ ID NO: 5 and / or a light chain comprising the sequence SEQ ID NO: 6.
    In a preferred embodiment the antibody is the antibody referred to in the present invention as clone 8110-37 (lgG1, anti-8AM81 K) and / or clone 8143-14 (lgM, anti8AM81 K).
    In a particular embodiment the present invention also relates to a
    gene construct that is capable of generating the antibody of the first aspect of the
    present invention
    10 The term "identity", as used herein, refers to the proportion of identical amino acids between two peptides or proteins that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the BLASTP or BLASTN, ClustalW and FASTA program. We can consider, which peptides or proteins with percentages of identity
    15 of at least 80% will maintain the same properties as the SEO ID sequence NO: 1.
    "Specific recognition" means "specific binding" to the binding (reaction, interaction or specific binding) between the antibody of the invention and the sequence
    20 described in the first aspect of the invention.
    A second aspect of the present invention relates to an antiserum comprising the antibody of the first aspect of the invention.
    The term "antiserum" refers to a serum obtained after immunization of an animal with an immunogen. The antiserum comprises antibodies specific to said immunogen generated after the immune response produced in the animal. In the context of the present invention the immunogen is the peptide with at least 80% identity with the SEO ID sequence NO: 1 (preferably 80, 81, 82, 83, 84,
    30 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%), preferably SEO ID NO: 1 or SEO ID NO: 2, and the antiserum comprises specific monoclonal antibodies generated against said sequence.
    A third aspect of the present invention relates to a cell that expresses the antibody of the first aspect of the invention (hybridoma).
    A fourth aspect of the present invention relates to the use of the antibody of the first aspect of the invention or of the antiserum of the second aspect of the invention for the inhibition of BMP and Activin Membrane Bound Inhibitor (BAMBI).
    5 A fifth aspect of the present invention relates to the use of the first antibodyaspect of the invention or the antiserum of the second aspect of the invention for thePreparation of a medicine.
    A sixth aspect of the present invention relates to the use of the first antibody
    Aspect of the invention or the antiserum of the second aspect of the invention for the preparation of a medicament for the treatment or prevention of autoimmune diseases.
    It is understood by autoimmune disease ~ in the present invention that
    15 disease in which the cells of the immune system trigger a chronic inflammatory response in one or several tissues of the individual causing deterioration or even destruction. In the present invention the terms "autoimmune disease" and "chronic inflammatory disease" are used interchangeably.
    20 Autoimmune disease is preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus. In an even more preferred embodiment, the autoimmune arthritis is newly started arthritis or rheumatoid arthritis.
    For this reason, in a more preferred embodiment of the sixth aspect of the invention, autoimmune diseases are selected from the list consisting of: autoimmune arthritis, spondyloarthritis, psoriasis, systemic lupus erythematosus and inflammatory bowel disease. Preferably the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
    In the present invention the term autoimmune arthritis "encompasses both the terms
    "Rheumatoid Arthritis" and "Undifferentiated Arthritis" whether it is of recent onset
    As if it is well established.
    35 "Recent Arthritis" (ARe) (or onset arthritis) is understood in the present invention, that disease consisting of inflammation of at least one
    articulation, less than one year of evolution that meets the criteria established by the American College of Rheumatology (ACR) and EULAR of "Rheumatoid arthritis or RA" (Aletaha, Neogi el. Ann Rheum Dis 2010; 69: 1580-1588) or, that without fulfilling these criteria, does not meet 5 criteria of other autoimmune, degenerative or metabolic diseases that may explain the symptoms. This last case, has been called "Arthritis Indifference da" (Al) which, in many cases, left to its free evolution, end up leading to an RA. In this invention the terms ARe, AR or Al refer to a chronic and progressive autoimmune systemic disease, which
    10 it causes chronic inflammation mainly of the joints, and given its progressive nature, it produces their destruction, with its consequent deformation and loss of functional capacity. In addition, this disease can cause extra-articular alterations in various organs.
    15 "Spondyloarthritis" is understood in the present invention as those autoimmune diseases with axial and / or peripheral involvement that meet the classification criteria of the Assessment of SpondyloArlhritis Infernational Society (ASAS or International Society for the Evaluation of Spopodyritis) (Rudwaleit et al. Ann Rheum Dis 2011; 70: 25-31).
    20 "Systemic lupus erythematosus" is understood in the present invention as a systemic autoimmune disease defined by the criteria of the American College of Rheumatology (Tan al. Arthritis Rheum '1982; 25: 1271-1277).
    The term "inflammatory bowel disease" or "IBD" in the present invention refers to chronic inflammation of the intestine in an individual, where said inflammation is due to the individual's own immune system. The two most frequent forms correspond to ulcerative colitis and Crohn's disease. So in a preferred embodiment the autoimmune disease is ulcerative colitis or disease of
    30 Crohn.
    In the present invention "psoriasis" is understood to be that skin disease which is characterized by a malfunction of the immune system, which causes excess production of skin cells. This disease results in the formation of reddish bulges covered with scaling. In addition, excess cell production also causes the infiltration of white blood cells into the skin.
    The lesions are generally located in regions with greater friction, such as, but not limited to, elbows, knees or English.
    A seventh aspect of the present invention relates to a pharmaceutical composition comprising the monoclonal antibody of the first aspect of the invention or of the antiserum of the second aspect of the invention.
    The term "pharmaceutical composition" herein refers to any substance used for diagnosis, prevention, relief, treatment or cure of a substance.
    10 disease in humans or animals. The pharmaceutical composition of the invention can be used both alone and in combination with other pharmaceutical compositions. In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
    The term "excipient" refers to a substance that aids in the absorption of the pharmaceutical composition, which comprises the antibody of the invention, stabilizes it or aids in its preparation in the sense of giving it a consistency, form, taste or any other specific functional characteristic Thus, the excipients could have the function of keeping the ingredients together such as starches,
    20 sugars or cellulose, sweetening function, coloring function, protective function such as to isolate it from air and / or moisture, filling function of a tablet, capsule or any other form of presentation such as calcium phosphate di basic, disintegrating function to facilitate the dissolution of the components and their absorption, without excluding other types of excipients not mentioned in this paragraph.
    A "pharmaceutically acceptable vehicle" (or "pharmacologically acceptable") refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The vehicle 30 can be an inert substance or of analogous action to any of the compounds of the present invention and whose function is to facilitate the incorporation of the drug as well as other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition When the presentation form is liquid, the vehicle is the diluent. The term "pharmacologically
    Acceptable "means that the compound referred to is allowed and evaluated so as not to cause damage to the organisms to which it is administered.
    The pharmaceutical composition of this invention can be provided by any route.
    for administration, for which said composition will be formulated in the form
    Pharmaceutical appropriate to the route of administration chosen.
    An eighth aspect of the present invention relates to the in vitro use of the antibody of the first aspect of the invention or of the antiserum of the second aspect of the invention for the screening of drugs, preferably drugs directed to the treatment and / or prevention of autoimmune diseases. Preferably, autoimmune diseases are selected from the list consisting of: autoimmune arthritis, spondyloarthritis, psoriasis, systemic lupus erythematosus and inflammatory bowel disease. More preferably, inflammatory bowel disease is ulcerative colitis.
    or Crohn's disease.
    The term "in vitro" refers to the method of the invention being performed outside the subject's body. That is, it is performed on a biological sample of a subject.
    The term "biological sample" in the present invention refers to any sample that allows drug screening, and includes, but is not limited to, Biological Fleets or
    20 tissues of an individual, obtained by any method known to a person skilled in the art that serves this purpose. The biological sample could be, for example, but not limited to, a sample of fluid, such as blood, plasma, serum or tissue. The biological sample in the present invention can be fresh, frozen, fixed or fixed and embedded in paraffin.
    In the present invention the terms "subject" and "individual" are used interchangeably.
    A ninth aspect of the present invention relates to a method of obtaining a monoclonal antibody that recognizes an amino acid sequence that comprises a peptide with at least 80% identity with the SEO ID NO: 1 sequence comprising:
    to. obtaining the serum previously extracted from a non-human animal immunized with a recombinant protein comprising an amino acid sequence comprising a peptide with at least 80% of
    35 identity with the sequence SEa ID NO: 1,
    b. Obtain a hybridoma from the serum that generates specific monoclonal antibodies against an amino acid sequence comprising a sequence with at least 80% identity with the SEO ID NO: 1 sequence.
    In a more preferred embodiment of the ninth aspect of the invention the method
    further comprises a step (c) of isolation of the monoclonal antibody from
    of the hybridoma generated in stage (b).
    In another more preferred embodiment of the ninth aspect of the invention in step (a) the amino acid sequence is the SEO ID NO: 1 or SEQ ID NO: 2 sequence.
    In an even more preferred embodiment of the sixth aspect of the invention the nonhuman animal is a mammal that is selected from the list consisting of pig, chimpanzee, mouse, rat, rabbit and guinea pig.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the
    The invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    BRIEF DESCRIPTION OF THE FIGURES
    25 FIG. 1: Characterization of the murine anti-BAM81 (109-133) AcMs 8101.37 and B143-14. A) Specificity of the anti-BAMBI 8101 .37 and B143-14 AcMs evaluated by Western Blot in heart cell lysates of normal 86 and B6.BAMBI-KO mice. B) Recognition of human BAMBI by the AcM B101 .37 evaluated
    30 by Western Blot in lysates of human heart cell membranes. DC) Sequence of the CDRs of the heavy and light chains of the AcMs 8101.37 and 8143-14. The rearrangement VDJ and VJ of the heavy and light chains, respectively, of both AcMs is indicated.
    35 FIG. 2: Induction of 8AMBI expression in murine and human CD4 + T cells after activation. A) Comparative analysis by flow cytometry of the expression of
    BAMBI in CD4 + T lymphocytes of normal B6 and B6 mice.BAMBI-KO before and 48 hours after in vitro activation with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGF¡3 or IL-2 (upper panels). B101-37 staining of activated CD4 + T lymphocytes from B6 BAMBI-KO mice is compared in the lower panels.
    5 with that of an isotypic control IgG1 in activated CD4 + T lymphocytes of normal mice. B) Induction of BAMBI in human T lymphocytes stimulated in vitro for 48 hours with anti-CD3 and anti-CD28 AcMs. BAMBI expression was analyzed by Western Blol in plasma membrane lysates. As a charge control, the expression of N-Ras in the same lysates was compared.
    FIG. 3: Effect of BAMBI inhibition on in vitro differentiation of murine and human CD4 + T lymphocytes to Treg and TH17 cells. A) CD4 + CD25CD62L + CD44-na "lve cells of normal B6 and SAMSI-KO mice were stimulated for 5 days with anti-CD3 and anti-CD28 antibodies under polarizing conditions Treg 15 (upper panels) or TH17 (lower panels) in the presence of AcM B143-14 (lgM anti-BAMBI) or a polyclonal mouse IgM (Sigma) The percentages of CD4 + FoxP3 + and CD4 + IL-17 + cells in the selected regions analyzed by flow cytometry are shown. and D) Human CD4 + T lymphocytes (in Treg differentiation) or human memory (in TH17 differentiation), purified by magnetic separation, 20 were activated in vilro for 10 days with anti-CD3 and anti-CD28 antibodies conjugated to balls under conditions of differentiation THO, Treg or TH17 The effect of SAMBI inhibition with AcM B143-14 on differentiation to Tregs (e) and TH17 (D) cells is shown, as in (A) the IgM of polyclonal mouse as a negative control The percentages of each of the populations CD4 ones analyzed are
    25 determined by flow cytometry. Statistical differences are represented as: ·· p <0.01.
    FIG. 4: The AcM B101-37 inhibits the development of arthritis in the CIA model. A and B) For the induction of CIA, normal S10Rlil mice were immunized with bovine type II collagen emulsified in CFA. The different experimental groups received treatments with 2 mg / mouse / week, 0.3 mg / mouse / week of B101-37 or with 2 mg / mouse / week of an irrelevant murine IgG1 (IgG1-C) during the first 4 weeks after immunization The degree of clinical severity of each mouse (A) and of different radiological lesions (mean ± SD) associated with joint destruction at 8th week after immunization (B) is shown. As controls of the previous experiments, the development of CIA between normal B10RIII and BAMBI mice was compared
    KO The evolution of the clinical severity of arthritis in these animals expressed as mean ± SD is shown. (C) and of different radiological lesions (mean ± SD) associated with joint destruction one week after immunization (D). Statistical differences are represented as: · p <0.05. ·· p <0.01.
    FIG. 5: Effect of treatment with B101-37 on the development of psoriatic arthritis induced by injection of mannan. A) Normal B10RIII or BAMBI-KO mice treated or not since the start of the experiment with the B101-37 AcM (2 mg / mouse / week) received an Lp injection. of 10 mg of Mannan obtained from the
    10 Saccharomyces cerevisiae yeast. The evolution in the severity of arthritis and in the percentage of increase in the thickness of the ears (mean ± SD) in the different experimental groups is represented. B) Photographs representative of the macroscopic appearance of the auricular pavilion of the experimental groups described in (A). Statistical differences are represented as: · p <O, 05, ·· p <0.01, ••• p <0.001.
    FIG. 6: Effect of treatment with B101-37 on the development of DSS colitis. Normal B6 or BAMBI-KO mice treated or not since the start of the experiment with the AcM 810137 (2 mg / mouse / week) received 3% dissolved DSS in the bottle water for 5 days. The severity of colitis was assessed by quantifying the ICD (A) or
    20 analyzing the shortening of the colon (B). C) Mortality in the different experimental groups. Statistical differences are represented as: · p <0.05, ·· p <O, 01.
    EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    MATERIAL AND METHODS.
    30 Obtaining and characterizing murine anti-BAMBI monoclonal antibodies:
    B6.8AMBI-KO mice were immunized with the BAMBI peptide (109-133) murine keyhole conjugate limpet hemocyanin (KLH) and emulsified in Freund's complete adjuvant 35 (CFA). The mice were immunized twice more (one month apart between each immunization) with the same peptide emulsified in
    Freund's incomplete adjuvant (IFA). The murine BAMBI (109-133) peptide is located in the extra cellular region of BAMBI and differs with its human counterpart in 4 amino acids (positions 8, 9, 10 and 23 of SEQ ID NO: 1). The presence of circulating murine anti-BAMBI antibodies (109-133) in the immunized mice was evaluated 15 days after each immunization by ELlSA. Mice with higher titers of these antibodies were used to obtain murine anti-BAMBI monoclonal antibodies (AcM). For this, spleen cell suspensions were fused with the non-secreting myeloma line SP2 / 0-Ag14 as described previously (24). Hybridomas producing human anti-BAMBI AcM were selected by enzymatic immunoassay (ELlSA). In the present invention two of the obtained mAbs have been characterized; clone B110-37 (lgG1, K anti-BAMBI) and clan B143-14 (lgM, K anti-BAMBI). The specificity of the MMAs was subsequently evaluated by Western blotting in lisa two of cell membranes of hearts from normal B6 and B6.BAMBI-KO mice and samples of 15 human myocardium obtained from surgical biopsies.
    Ribonucleic acid (RNA) from AcM 8110-37 and 8143-14 was isolated by kit
    commercial RNeasy Mini Kit (Oiagen). To define the coding sequences for the complementarity determining regions (CDRs) in the heavy and light chain of both AcMs, RT-PCRs were performed from the purified RNAs, as previously described (25). The following amplimers were used to define the CDR of the heavy chain of 8110-37: degenerate 5 'amplifier of the FR1 region of the heavy chain: 5'-CTI CCG GAA TIC SAR GTN MAG CTG SAG SAG TC-3 (SEO ID NO: 7) '; 3 'amplifier of the constant region of IgG1: 5'-GGA AGA TCT ATA GAC 25 AGA TGG GGG TGT CGT TTI GGC-3' (SEO ID NO: 8). To define the CDR of the heavy chain of B143-14, the degenerate amplifier of the FR1 region of the heavy chain mentioned above and the 3 'amplifier of the constant region of IgM were used: 5'-GGA AGA TCT GAC ATI TGG GAA GGA CTG ACT CTC-3 '(SEO ID NO: 9) .To define the CDR of the light chains of B110-37 and B143-14 30 used the following amplimers: degenerate amplifier of the FR1 region of the
    light chain K: 5'-GG GAG CTC GAT ATI GTG MTS ACM CAR WCT MCA-3 '(SEO ID NO: 10); 3 'amplifier of the constant region of the light chain K: 5'-GGT GCA
    TGC GGA TAC AGT TGG TGC AGC ATC-3 '(SEO ID NO: 11). PCR products
    were subsequently sequenced (STABVida, Caparica, Portugal) and the sequences analyzed by the IgBLAST program.
    Study of the expression of BAMBI in murine and human CD4 T lymphocytes.
    The expression of BAMBI was studied by flow cytometry in CD4 + T lymphocytes of normal B6 mice after stimulation using biotinylated B101 .37 in AcM. The 5 isolated CD4 + T lymphocytes from the spleen of normal B6 mice were stimulated in vitro for 48 hours with anti-CD3 (1 g / well) and anti-CD28 (0.5 g / well) antibodies attached to the plate in the presence or absence of 2 ng / ml of recombinant murine TGF¡3 and / or 1 ng / ml of recombinant murine IL-2 (PeproTech, London). Spleen cells from B6 BAMBI-KO mice stimulated in the same manner were used as negative controls
    10 forms and colored with biotinylated B101 .37 and of normal B6 colored mice an isotypic control biotinylated IgG1. The colored cells were analyzed in a FACSCanto 11 cytometer equipped with the FACSDiva software (BD Biosciences).
    BAMBI expression in human CD4 + T cells after in vitro stimulation is
    15 analyzed by Westem Blol. These lymphocytes were purified from 50 ml of buffy-coats from healthy donors of the Blood and Tissue Bank of Canlabria (Marqués de Valdecilla University Hospital, Santander). The mononucleated cells obtained after Ficoll gradient were subjected to a positive selection after labeling with microparticle-conjugated CD4-specific AcM.
    20 magnetic (MACS) using a magnetic separator (AutoMACS, Miltenyi Biotec). CD4 + T lymphocytes were subsequently stimulated in vitro for 48 hours with anti-CD3 (1 g / well) and anti-CD28 (0.5 g / well) antibodies bound to the culture plate. Lysates of the cell membranes of activated lymphocytes were obtained as described above.
    25 In vitro differentiation cultures of murine and human CD4 + T lymphocytes to Treg and TH17 cells.
    The inhibitory capacity of anti-BAMBI mAbs directed against the BAMBI peptide (10930 133) was explored in vitro in cultures of murine and human CD4 + T lymphocytes differentiated to Treg and TH17 cells. In these experiments the AcM 8143 was used
    14. In the murine lymphocyte experiments, CD4 + naiVe cells (CD4 + CD25-CD62L + CD44-) were purified from the spleens of normal B6 mice by cell sorting (FACSAria, BD Biosciences). 5 x 105 CD4 + naiVe cells were stimulated
    35 for 5 days with anti-CD3 and yanti-CD28 antibodies bound to the plaque and culture plastic, under Treg polarizing conditions (2 ng / ml of murine TGF¡3) or TH1 7 (1 ng / ml of murine TGFj3 and 10 ng / ml of murine IL-6 IL-6) in the presence of 20! -Ig / ml B143-14 or 20! -Ig / ml of murine IgM (Sigma, St Louis, Missouri) as an isotypic control. The percentages of TCD4 + FoxP3 + (Treg) and CD4 + IL-17 + (TH17) lymphocytes at the end of the culture were analyzed by flow cytometry, as described above.
    5 (21).
    NaiVe human CD4 + T lymphocytes (in Treg differentiation) or CD45RO + memory (in TH17 differentiation), purified by magnetic separation, were activated in vitro for 10 days with anti-CD3 and anti-CD28 antibodies (Acs) conjugated to 10 balls in Treg differentiation conditions (5 ng / ml of TGF ~) or TH17 (20 ng / ml of IL-1 ~, 30 ng / ml of IL-6, 30 ng / ml of IL-23, 3 ng / ml of TGF ~ 1, 1 ~ g / ml of anti-IFNy and 2.5! -Ig / ml of anti-IL-4), in the presence of 20! -Ig / ml B143-14 or 20! -Ig / ml of IgM murine The percentages of TCD4 + FoxP3 + (Treg) and CD4 + IL-17 + (TH17) lymphocytes at the end of
    Culture were analyzed by flow cytometry.
    15 Experimental model of arthritis after immunization with bovine type 11 collagen emulsified in CFA (CIA).
    Groups of 10 B10RIII mice (MHC H-2r) deficient or not in BAMBI were
    20 immunized before the 12th week of age intradermally at the base of the tail with 150! -Ig of bovine type 11 collagen (MD Biosciences, Zurich) emulsified (vol. 1/1) in CFA containing a concentration of Mycobacterium 4 mglml tuberculosis (MD Biosciences), as described previously (21). To study the effect of 8AM81 inhibition on the development of CIA normal 810RIII mice
    25 immunized patients were treated intraperitoneally (j.p.) during the first 4 weeks after immunization with 2 or 0.3 mg / week of 8101.37 or with 2 mg / week of an anti-TNP IgG1 used as an isotypic control (lgG1-C). The evolution of arthritis was monitored weekly from the 3rd (date of onset of arthritis) to the 8th week after immunization. The severity of arthritis was assessed in each
    30 of the four limbs by the method described by Wooley et al (26). Likewise, at 8 weeks after immunization, joint lesions in the front and rear legs were evaluated by radiology, as we have described previously (21).
    35 Experimental model of dextran sulfate colitis (DSS colitis).
    For the induction of colitis, the DSS (MPbio.com) was dissolved in 3% water. This solution (with daily water changes) was supplied in the bottle to the mice of the different experimental groups for a variable period of time [until the mice of the control group reach a disease activity index (DAI) 5 of between 1.5-2 (approximately 4-5 days)]. Daily the water consumed by each experimental group was measured and DA1 was assessed by calculating the clinical score of the following parameters: A) weight loss: 0 = no weight loss, 1 = 1-5% weight loss, 2 =: loss of 5-10% of weight, 3 = loss of 10-20% of weight and 4 = loss of more than 20% of weight; B) stool consistency (the same is granted
    10 value to all animals that are in the same box): 0 = feces of normal consistency, 2 = soft stools and 4 = diarrhea; C) rectal bleeding (the same value is given to all animals that are in the same box): 0 = no bleeding, 2 = mild bleeding and 4 = severe bleeding. The final DAI value was calculated by adding the values of the different parameters and dividing it by 3.
    Induction of psoriatic arthritis with mannan.
    For the induction of psoriatic arthritis groups of B10RIII mice deficient or not in BAMBI received an i.p. of 10 mg of mannan obtained from the yeast S. 20 cerevisiae (Sigma-Aldrich) dissolved in 200 1-11 of PBS (27). To study the effect of BAMBI inhibition on the development of psoriatic arthritis the mice were treated from the time of administration of mannan with 2 mg / week of B101 .37 or with 2 mg / week of an anti-TNP IgG1 used as IgG1. -C. Psoriasis lesions were assessed at the level of the ear pavilions by quantifying the thickness of the same with the help of a digital caliper. The severity of the arthritis will be evaluated on each of the four legs, on a scale of 0-10 (total range of 0-40) as follows: severe inflammation of the carpus / tarsus = 5 points adding 1 additional point for each swollen finger If the inflammation is mild = 3 points per carpus / tarsus
    + 0.5 points for each swollen finger.
    Example 1: Molecular characterization of the anti-BAMBI AcM.
    The specificity of the anti-BAMBI B110-37 and B 143-14 mAbs was initially determined by ELlSA (during the hybridoma screening process) and subsequently by Western Blot. Both AcM recognize a band of approximately 2729 kDa and another of approximately 54 kDa, compatible with BAMBI monomers and
    dimers resistant to sodium dodecyl sulfate (SDS) and reducing conditions, such and
    as previously described (Xavier S et al. 2010 PLoS Qne 5: e12995), in the
    lysates of B6 mice but not those of B6 mice. BAMBI-KO (Figure 1A).
    5 Although the murine BAMBI peptide (109-133) used for the development of MCAsanti-BAMBI differs from its human counterpart in 4 amino acids, Western studiesBlot in myocardial plasma membrane lysates indicate that B110-37 alsorecognize human BAMBI (Figure 1 B).
    10 Subsequently, we characterized the CDRs of the heavy and light chains of both anti-BAMBI mAbs by deoxyribonucleic acid (DNA) sequencing (SEQ ID NO: 3, SEO ID NO: 4, SEO ID NO: 5, SEO ID NO; 6 ). The comparison of the DNA sequences with the IgBLAST database indicates that the CDRs of the heavy and light chains of the AcM B11 0-37 are the result of the rearrangement of the
    15 segments V9-31D1-3 / J1 and V15-103 / J5, respectively, while the CDRs of the heavy and light chains of the AcM B143-14 are the result of the rearrangement of segments V1-55 / D4-lJJ2 and V5 -43 / J1, respectively.
    Example 2: Expression of BAMBI in CD4 + T cells.
    20 CD4 + T lymphocytes play a fundamental role in the development of inflammatory and autoimmune pathologies. For this reason, the regulation of BAMBI expression in this lymphocyte population in mice was studied by flow cytometry and later in humans by Western blotting, using in both cases
    25 AcM 8101.37. In murine CD4 + T lymphocytes, the expression of 8AM81 was not detected but was induced 48 hours after in vitro stimulation with anti-CD3 and anti-CD28 antibodies (Figure 2A). This expression was increased after the addition of TGF¡3 , but not IL-2, to activated CD4 + T cells (Figure 2A). As controls for specificity of AcM 8101.37, no labeling was detected in flow cytometry in both T lymphocytes
    30 activated CD4 + mice 8 AM81-KO colored 8101 .37 as in those of normal 86 mice colored with an isotypic IgG1 control, respectively (Figure 2A, lower panels).
    As in the mouse, the expression of 8AM81 was very low in human CD4 + 35 lymphocytes, inducing after stimulation with anti-CD3 and antiCD28 antibodies (Figure 28).
    BAMBI inhibition with the murine B143-14 anti-BAMBI mAb (109-133) alters in vitro differentiation of mouse and human CD4 + T lymphocytes to Treg subtypes
    yTH17.5
    In previous studies we demonstrated that the absence of BAMBI in the CD4 + T lymphocytes of the BAMBI-KO mice enhanced and inhibited their differentiation in vitro to the Treg and TH17 populations, respectively (Postigo J et al. PhD thesis defended on April 19, 2013 , University of Cantabria. ”To assess the inhibitory effect of 10 anti-BAMB I mAbs directed against the murine BAMBI epitope (109-133), we analyzed the ability of B143-14 to alter the functional differentiation of CD4 + T lymphocytes from mice normal in the same sense as observed in BAMBI-KO mice. To do this, CD4 + nai T lymphocytes from 86 normal mice and BAM81-KO were activated in vitro under conditions of polarization to Treg or TH17 cells, in the presence
    15 of AcM B143-14 or a murine IgM used as an isotypic control. As observed with the lymphocytes of the 8 AMBI-KO mice, the inhibition of 8AMBI after the addition to the culture of 8143-14, but not of the 19M control, increased and reduced the differentiation Treg and TH17 in vitra, respectively, of CD4 + T lymphocytes from normal B6 mice (Figure 3A).
    20 Finally, we observe that in the presence of AcM 8143-14, but not of the control IgM, the in vitro differentiation of CD4 + na lymphocytes (lve (to Treg) or human memory (to Th17), activated under polarizing conditions towards Treg (Figure 38) or TH17 (Figure 3C) is altered in the same direction as in the mouse: increase in Treg and
    25 decrease in TH17.
    Example 3: Therapeutic effect of AcM B101.37 on the development of CIA, psoriatic arthritis and DSS colitis.
    30 The preceding results indicate that: 1) we have AcMs capable of recognizing BAMBI in the mouse and in humans, 2) BAMBI expression is induced in CD4 + T lymphocytes after activation in both the mouse and humans; and 3) in both species the inhibition of BAMBI in vitro with a murine anti-BAMBI AcM (109-133) alters the functional differentiation of CD4 + T cells in the same sense as
    35 described in B6.BAMBI-KO mice (increase in Treg cells and decrease in TH17). These findings raise the possibility that the inhibition of 8AMBI in vivo has a therapeutic effect on inflammatory / autoimmune pathologies.
    In the present invention we have characterized the therapeutic potential of 8101.37 in5 the development of CIA (the experimental model of rheumatoid arthritis most used bythe scientific community), psoriatic arthritis and colitis-DSS.
    Both from a clinical and radiological point of view, the treatment of miceNormal B10RIII with 2 mg / week of B101 .37 during the first 4 weeks after
    10 bovine type 1I collagen immunization inhibited the development of CIA in these animals, unlike that observed in mice treated with 0.3 mg / week of B101.37 or with 2 mg / week of IgG1-C (Figures 4A and 4B). CIA inhibition after treatment with the high dose of 8101 .37 was similar to that observed in immunized B10RIII.BAMBI-KO mice (Figures 4C and 40).
    15 As in the CIA model, treatment with 2 mg / week of 8101.37 from the time of administration of mannan to normal 810RlII mice significantly reduced the severity of joint and skin lesions in this experimental model of psoriatic arthritis, similar to what was observed in
    20 B10RIII.BAMBI-KO (Figure 5).
    Finally, we assess the therapeutic effect of 8101 .37 in the experimental model of colitis-DSS. As in 86.BAM81-KO mice, normal 86 mice treated with 2 mg / week of 8101.37 from the time of administration of 25 DSS developed significantly less severe colitis than untreated controls (Figure 6A). However, the protective effect of treatment with 8101.37 in B6 mice was not as important as that observed in animals 8 AMBI-KO, especially with regard to the degree of shortening of the colon (Figure 6A, right panel). It should be noted that both in animals 86 treated with B101 .37 and in
    30 BAM81-KO mice were completely prevented mortality associated with the induction of colitis-DSS (Figure 68).
    Therefore, the use of antibodies against BAM81 for the treatment and prevention of autoimmune diseases is demonstrated in the present invention.
    权利要求:
    Claims (17)
    [1]
    one. Monoclonal antibody that specifically recognizes an amino acid sequence comprising a peptide with at least 80% identity with SEO ID NO: 1, where the size of said amino acid sequence is between 15 and 30 amino acids.
    [2]
    2. Antibody according to claim 1 wherein said antibody specifically recognizes the SEO ID NO: 1 or SEQ ID NO: 2 sequence.
    [3]
    3. Antibody according to any one of claims 1 or 2 wherein the antibody comprises a heavy chain comprising the sequence SEQ ID NO: 3 and / or a light chain comprising the sequence SEO ID NO: 4.
    [4]
    Four. Antibody according to any one of claims 1 or 2 wherein the antibody comprises a heavy chain comprising the sequence SEQ ID NO: 5 and / or a light chain comprising the sequence SEQ ID NO: 6.
    [5]
    5. Antiserum comprising the antibody according to any of claims 1
    to 4.
    [6]
    6. Cell expressing the antibody according to any of claims 1 to 4.
    [7]
    7. Use of the antibody according to any of claims 1 to 4 or of the antiserum according to claim 5 for the inhibition of MBP and Activin Membrane Bound Inhibi / or (BAMBI).
    [8]
    8. Use of the antibody according to any of claims 1 to 4 or of the antiserum according to claim 5 for the preparation of a medicament.
    [9]
    9. Use of the antibody according to any of claims 1 to 4 or of the antiserum according to claim 5 for the preparation of a medicament for the treatment or prevention of autoimmune diseases.
    [10]
    10. Use according to claim 9 wherein the autoimmune diseases are selected from the list consisting of: autoimmune arthritis, spondyloarthritis, psoriasis, systemic lupus erythematosus and inflammatory bowel disease.
    [11 ]
    eleven . Use according to claim 10 wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
    [12]
    12. Pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 4 or the antiserum according to claim 5.
    [13]
    13. In vitro use of the antibody according to any of claims 1 to 4 or of the antiserum according to claim 5 for drug screening.
    [14]
    14. Method of obtaining a monoclonal antibody that recognizes an amino acid sequence comprising a sequence with at least 80% identity with the SEO ID NO: 1 sequence comprising:
    to. obtaining the serum previously extracted from a non-human animal immunized with a recombinant protein comprising an amino acid sequence comprising a peptide with at least 80% identity with the SEO ID NO: 1 sequence,
    b. Obtain a hybridoma from the serum that generates specific monoclonal antibodies against an amino acid sequence comprising a sequence with at least 80% identity with the SEO ID NO: 1 sequence.
    [15]
    fifteen. Method according to claim 14, further comprising a step (c) of isolation of the monoclonal antibody from the hybridoma generated in step (b).
    [16]
    16. Method according to any of claims 14 or 15 wherein in step (a) the amino acid sequence is the SEO ID NO: 1 or SEO ID NO: 2 sequence.
    [17]
    17. Method according to any of claims 14 to 16, wherein the nonhuman animal is a mammal that is selected from the list consisting of pig, chimpanzee, mouse, rat, rabbit and guinea pig.
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    同族专利:
    公开号 | 公开日
    EP3385282A1|2018-10-10|
    WO2017093589A1|2017-06-08|
    EP3385282A4|2019-08-21|
    US20190338019A1|2019-11-07|
    ES2626491B1|2018-06-25|
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