WO2009150275A1 - Human anti-dectin-1 antibody, hybridoma producing said antibody and applications thereof - Google Patents
Human anti-dectin-1 antibody, hybridoma producing said antibody and applications thereof Download PDFInfo
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- WO2009150275A1 WO2009150275A1 PCT/ES2009/070206 ES2009070206W WO2009150275A1 WO 2009150275 A1 WO2009150275 A1 WO 2009150275A1 ES 2009070206 W ES2009070206 W ES 2009070206W WO 2009150275 A1 WO2009150275 A1 WO 2009150275A1
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- dectin
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/7056—Lectin superfamily, e.g. CD23, CD72
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- the innate immune response is the one in charge of the immediate recognition and containment of the microbial invasion and directs the adaptive response against the pathogens through the control of their growth, of the expression of co-stimulating molecules in the phagocytes, of the antigen presentation and the production of cytokines and chemokines. These factors regulate the leukocyte traffic by initiating the inflammatory response, activating the microbicidal abilities of the phagocytes and directing the polarization of the cooperating T cells.
- the inflammatory response serves to limit the infection, but the inflammation has to be resolved to avoid pathological processes.
- IL interleukin
- DCs dendritic cells
- regulatory T cells have an important role in the late stages of infection to prevent an excessive activation of the innate response and the establishment of commensalism and perhaps of the latency and persistence of the pathogen (Romani 2004).
- the recognition of the pathogens is carried out by the phagocytic cells through the pattern recognition receptors (PRR), which detect highly conserved microbial structures.
- PRR pattern recognition receptors
- the PRR prototype is the TLR (Toll-like receptors), but recently it has been shown that receptors of the lectin family such as Dectin-1 can function as PRRs (Akira and Takeda 2004; Gordon 2002; Brown 2005).
- the human Dectin-1 membrane receptor recognizes polysaccharides with ⁇ (1 ⁇ 3) and / or ⁇ (1 ⁇ 6) bonds, the so-called ⁇ -glucans, present in the fungal wall (Brown and Gordon 2001; Brown et al . 2003).
- Dectin-1 is involved in the response Innate immune against fungal pathogens (Brown and Gordon 2003) in both humans and mice.
- This receptor contains an extracellular region with a single carbohydrate binding domain of the CTLD (C-type lectin-like domain) (Weis et al. 1998) connected to the intracellular domain through a neck and a transmembrane region.
- Dectin-1 undergoes an alternative processing process that results in several isoforms, only two of them functional: Dectin-1a (complete) and Dectin-1 b (predominant, lacking the neck). Although they appear to work similarly in vitro, they are differentially expressed according to cell type (Willment et al. 2001, Heinsbroek et al. 2006).
- Dectin-1 is involved in phenomena of tolerance and control of the immune response since it can induce the generation of inhibitory cytokines (Interleukin (IL) -IO, IL-2). These functions can influence the resulting immune response and, in certain circumstances, lead to pathological and autoimmunity processes (Haskard and Landi 2002). Although Dectin-1 was considered to be expressed mostly in immature dendritic cells (DCs) and Langerhans cells and that maturation stimuli decreased their expression (Ariizumi et al. 2000; Hermanz-Falcon et al. 2001; Sobanov et al. 2001; Yokota et al.
- Dectin-1 is not restricted to myeloid lineage, but is also expressed in neutrophils, eosinophils, B cells and a sub-population of T cells (Brown et al. 2002; Taylor et al. 2002; Willment et al. 2005) although not in all tissues (Reid. et al. 2004).
- Dectin-1 is crucial in the immune response against living fungal pathogens such as Candida albicans (Gantner et al. 2005; Taylor et al. 2007), Coccidiodes posadasii (Viriyakosol et al. 2005), Pneumocystis carinii (Steele et al. 2003 and 2005) and Aspergillus fumigatus (Hohl et al. 2005; Steele et al. 2005; Gersuk et al. 2006).
- the relevance of this receptor has been established thanks to the generation of mice deficient in the gene coding for Dectin-1, in which it has been seen that susceptibility to C. albicans (Taylor et al. 2007) and P.
- Dectin-1 can trigger specific responses against pathogens in collaboration with or independently of TLRs.
- ligand binding causes an intracellular signaling cascade: the phosphorylation of the proximal tyrosine of the motive for ITAM activation by Src tyrosine kinase, allows the spleen tyrosine kinase to be recruited (Syk, Spleen Tyrosine Kinase).
- Syk is necessary for signaling through the specific T and B cell antigen receptor, and in myeloid cells it has been described as crucial to stimulate the production of reactive oxygen species through Dectin-1 (Underhill et al. 2005; Rogers et al. 2005). After the activation of Syk, a phosphorylation cascade is produced that involves, among others, the transcription factor NF- ⁇ B (Dennehy. And Gordon 2007; Dennehy et al. 2008). Recently, it has been shown that signaling via Dectin-1 (induced by particulate ⁇ -glucan (zymosan) or with C.
- albicans spores can directly modulate gene expression through the activation of the NFAT transcription factor, both in macrophages as in DCs, in turn inducing the transcription factors Egr2, Egr3 and COX-2 (Goodridge. et al. 2007).
- Dectin-1 participates in a new signaling route independent of TLRs in DCs, directly mediating Ia NF- ⁇ B activation via the CARD9 signaling adapter molecule (Gross et al. 2006).
- the Dectin-1-Syk-CARD9 route combines the immune and adaptive response, being CARD9 necessary for the development of a cooperative T-lymphocyte response (T H -17) against pathogens such as C. albicans (LeibundGut-Landmann et al . 2007).
- beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus. PLoS Pathog. 1 (4): e42 (2005).
- Coccidioides posadasii is dependent on Toll-like receptor 2 and dectin-1. I infected. Immun 73, 1553-1560 (2005).
- An object of the present invention constitutes the anti-antibody
- ID NO2 either a monoclonal or polyclonal antibody, hereinafter the antibody of the invention.
- a more particular object of the invention is the antibody of the invention characterized in that it is monoclonal that is produced by the MGD3 hybridoma (deposited in the ECACC culture collection -
- Another object of the present invention is the nucleotide sequence characterized by being coding for domain 71-247 of the human protein Dectin-1 (SEQ ID NO1), and for being: a) a nucleotide sequence analogous to SEQ ID NO1 b ) a fragment of the sequences of a, and c) a nucleotide sequence comprising any sequence belonging to a) and b).
- Another object of the invention constitutes the isolated amino acid sequence of the human protein Dectin-1 characterized in that it corresponds to domain 71-247 thereof (SEQ ID NO2), and for being: a) an amino acid sequence analogous to Ia SEQ ID NO2 b) a fragment of the sequence of a, and c) an amino acid sequence comprising any sequence belonging to a) and b).
- a particular object of the invention is the use of the nucleotide sequence (SEQ ID NO1) for the production of human anti-Dectin-1 antibodies, whether monoclonal or polyclonal.
- a particular object of the invention is the use of the amino acid sequence (SEQ ID NO2) for the production of human anti-Dectin-1 antibodies, whether monoclonal or polyclonal.
- An object of the present invention is the hybridoma characterized in that it produces an antibody specific for domain 71-247 of the human protein Dectin-1 (SEQ ID NO2).
- a particular object of the invention is the hybridoma of the invention characterized in that it is the hybridoma MGD3 producing the antibody of the invention (deposited in the ECACC-European Collection of Animal CeII Culture-e ⁇ 6 February 2008 crop collection with reference
- Another more particular object of the invention is the use of hybridoma of the invention for the production of the human anti-Dectin-1 monoclonal antibody, preferably the monoclonal antibody of the invention.
- An object of the present invention is a biotechnological, pharmaceutical, diagnostic or therapeutic composition characterized in that it contains the antibody of the invention.
- Another object of the invention is the biotechnological, pharmaceutical or diagnostic pharmaceutical composition characterized in that it comprises the antibody of the invention.
- Another particular object of the invention is the use of the composition of the invention and the antibody of the invention in an ex vivo method of detection, identification and / or quantification of the human Dectin-1 protein.
- Another particular object of the invention is the use of the composition of the invention and the antibody of the invention in an ex vivo procedure for the diagnosis of an autoimmune, infectious and / or inflammatory disease.
- Another particular object of the invention is the use of the composition of the invention and the antibody of the invention in a therapeutic procedure of a human disease where the human Dectin-1 protein has an etiopathogenic role.
- Another more particular object of the invention is the use of the composition of the invention and the antibody of the invention where the disease to which the therapeutic procedure is applied is an inflammatory, infectious and / or autoimmune disease.
- the present invention is based on the fact that the inventors have observed that a specific antibody, specifically a monoclonal antibody and more specifically the monoclonal antibody MGD3, generated by the inventors by immunizing mice with a fusion protein comprising the region between amino acids 71-247 (Dectin-1 / 71-247 domain) of the human Dectin-1 protein (SEQ ID NO2), presents a series of characteristics that confer a series of technical advantages with respect to the antibodies described in the state of the art, by Therefore, the MGD3 antibody represents a new biotechnological tool useful in the field of biomedical research and in the medical care field, both in the area of diagnosis and in the therapeutic field.
- the technical advantages of the MGD3 antibody of the invention are the following: it is capable of inhibiting the binding of the natural ligand to its receptor - in flow cytometry tests ( Figure 6) - analogously to soluble ⁇ -glucans (laminarin ). It is also capable of significantly inhibiting the production of pro and anti-inflammatory cytokines such as TNF- ⁇ and interleukin (IL) -IO, respectively, in dendritic cells derived from monocytes in response to the interaction with spores of C. albicans ( Figures 7A and 7B).
- pro and anti-inflammatory cytokines such as TNF- ⁇ and interleukin (IL) -IO
- Dectin-1 receptor expression levels in human leukocytes can be used to detect changes in Dectin-1 receptor expression levels in human leukocytes, which could indicate the ability of these cells to detect the presence of fungal pathogens such as Candida albicans, Coccidiodes posadasii, Pneumocystis car ⁇ nii and / or Aspergillus fumigatus and therefore, the susceptibility to these.
- MGD3 antibodies of the invention can be used as indicators of the therapeutic effect of different anti-inflammatory and immunosuppressive agents.
- the MGD3 antibody of the invention :
- the invention also relates to a cloned hybridoma (MGD3) (deposited in the ECACC-European Collection of Animal CeII Culture crop collection on February 6, 2008 with reference 08020601) that produces a monoclonal antibody, hereinafter referred to as MGD3 , which specifically recognizes the human Dectin-1 membrane receptor.
- MGD3 cloned hybridoma
- Said clone was obtained by standard procedures of cell fusion between a B lymphocyte of the spleen of an immunized BALB / c mouse and myeloma cells (X63.Ag.653 NP3) (Example 1).
- one aspect of the present invention is a human anti-Dectin-1 antibody, hereinafter antibody of the invention, which specifically binds and recognizes domain 71-247 of the human protein Dectin-1 (SEQ ID NO2) , either a monoclonal or polyclonal antibody.
- a more particular aspect of the invention constitutes the monoclonal antibody of the invention and produced by the hybridoma MGD3 (deposited in the ECACC culture collection - European Collection of Animal CeII Culture - on February 6, 2008 with reference 08020601), in below monoclonal antibody MGD3 of the invention.
- the term "antibody” refers to a recombinant antibody or mini-antibody that maintains its antigen binding capacity.
- mini-antibody is defined as fragments derived from antibodies constructed by recombinant DNA technology, which, despite their smaller size, retain the antigen binding capacity since they maintain at least one variable immunoglobulin domain where the binding areas reside to antigens, and includes, by way of illustration and without limiting the scope of the invention, the following group: Fab, F (ab ') 2, scFv, and recombinant monodomain antibodies (dAbs).
- recombinant monodomain antibodies and / or immunoglobulin-like domains with independent binding and recognition capacity both to the domains heavy chain variables (VH), to light chain variable domains (VL), to recombinant camelid antibodies (VHH), recombinant antibodies to humanized camelids, recombinant antibodies to other camelized species, fish monodomain antibodies IgNAR cartilaginous; that is, that both domains that are naturally monodomain (case of VHH and IgNAR) are included, as well as engineering antibodies that have been altered so that by themselves they are able to interact with the antigen and improve its stability and solubility properties .
- Any modification of the recombinant antibodies such as their multimerization or fusion to any molecule (eg toxins, enzymes, antigens, other antibody fragments, etc.) is included in this definition.
- the antibody can be obtained from a human being or an animal (eg camels, llamas, vicunas, mice, rats, rabbits, horses, nurse sharks, etc.) or by recombinant DNA techniques or chemical gene synthesis, or either from or in vitro or in vivo selection of antibody libraries.
- a human being or an animal eg camels, llamas, vicunas, mice, rats, rabbits, horses, nurse sharks, etc.
- recombinant DNA techniques or chemical gene synthesis or either from or in vitro or in vivo selection of antibody libraries.
- nucleotide sequence characterized by being coding for domain 71-247 of the human protein Dectin-1 (SEQ ID NO1), as well as any nucleotide sequence analogous to it.
- analogous is intended to include any nucleotide sequence that can be isolated or constructed based on the nucleotide sequence shown herein, for example, by introducing conservative nucleotide substitutions or non-conservative, including the insertion of one or more nucleotides, the addition of one or more nucleotides at any of the ends of the molecule or the deletion of one or more nucleotides, at any end or inside the sequence, and constituting a coding sequence or peptide with activity similar to the sequence of the human protein Dectin-1.
- an analogous nucleotide sequence is substantially homologous to the amino acid sequence discussed above.
- the expression “substantially homologous” means that the nucleotide sequences in question have a degree of identity of at least 40%, preferably of at least 85%, or more preferably of At least 95%.
- This nucleotide sequence can be used either in its entirety or partially by means of fragments thereof for the expression of the corresponding peptide / s in different cells for subsequent biotechnological uses.
- Another aspect of the present invention is the amino acid sequence isolated from the human protein Dectin-1 corresponding to domain 71-247 thereof (SEQ ID NO2), as well as any amino acid sequence analogous to it.
- the term "analogous” is intended to include any amino acid sequence that can be isolated or constructed based on the amino acid sequence shown herein, for example, by introducing conservative amino acid substitutions or non-conservative, including the insertion of one or more amino acids, the addition of one or more amino acids at any of the ends of the molecule or the deletion of one or more amino acids, at any end or inside the sequence, and constituting a peptide with activity similar to the sequence of the human protein Dectin-1.
- an analogous amino acid sequence is substantially homologous to the amino acid sequence discussed above.
- the expression "substantially homologous” means that the amino acid sequences in question have a degree of identity of at least 40%, preferably of at least 85%, or more preferably of At least 95%.
- Another aspect of the invention is the use of the nucleotide and amino acid sequence - SEQ ID NO1 and SEQ ID NO2, respectively - to produce human anti-Dectin-1 antibodies, both monoclonal and polyclonal.
- Another aspect of the invention is a hybridoma, hereinafter hybridoma of the invention, which produces an antibody specific for domain 71-247 of the human protein Dectin-1 (SEQ ID NO2).
- Another more particular aspect of the invention constitutes the hybridoma of the invention called the MGD3 hybridoma and producer of the MGD3 antibody (deposited in the ECACC culture collection -
- Another aspect of the invention constitutes the use of the antibody of the invention in the elaboration of a biotechnological composition or a diagnostic or therapeutic pharmaceutical composition useful for performing tests to identify the human Dectin-1 protein or for the treatment and / or diagnosis of autoimmune and / or inflammatory diseases.
- Another aspect of the invention constitutes a biotechnological composition, diagnostic or therapeutic pharmaceutical composition comprising the anti-Dectin-1 antibody of the invention.
- Another aspect of the invention constitutes the use of the antibody or
- composition of the invention in an ex vivo method of detection, identification and / or quantification of the human anti-Dectin-1 protein, either for research purposes or for diagnostic purposes of a human disease.
- Another more particular aspect of the present invention is the use of the human anti-Dectin-1 antibody or of the biotechnological composition of the invention, in an ex vivo biotechnological procedure where the identification of the human Dectin-1 protein is required, as for example and without limiting the scope of the invention, in: - immunofluorescence and immunohistochemical tests
- Dectin-1 for example TNF-a, IL-6, IL-10, IL-2, etc.
- Another more particular aspect of the present invention is the use of the antibody anti-human Dectin-1 or the diagnostic pharmaceutical composition of the invention in an ex vivo method of identifying the human Dectin-1 protein, as for example and without limiting the scope of the invention, in: - the identification of cells pathogens activated in autoimmune and / or inflammatory diseases, which indicates the degree of exacerbation of the disease and allows measuring the efficacy of treatments in the early stages of clinical trials with new therapies, - a procedure to determine the susceptibility to infection by pathogens fungal, through the identification of changes in the expression of the endogenous receptor of the human Dectin-1 protein, indicative of the presence of ⁇ -glucan in biological fluids,
- another particular aspect of the present invention is the use of the human anti-Dectin-1 antibody or the therapeutic pharmaceutical composition of the invention in a therapeutic procedure of a disease where the human Dectin-1 protein has an etiopathogenic role.
- the human anti-Dectin-1 monoclonal antibody of the invention could be used for the elaboration of pharmaceutical compositions useful in the treatment of inflammatory pathologies, since it blocks the production of pro-inflammatory cytokines that are induced through of Dectin-1 (for example, the Tumor Necrosis Factor, TNF-alpha) and that are involved in the pathogenesis, for example and without limiting the scope of the invention, of various forms of arthritis.
- dectin-1 for example, the Tumor Necrosis Factor, TNF-alpha
- Another object of the invention is the use of the composition and / or the antibody of the invention in the treatment of inflammatory diseases.
- Another object of the invention is the use of the composition and / or the antibody of the invention in the treatment of infectious and / or autoimmune diseases.
- Another particular object of the invention is the use of the composition and / or the antibody of the invention in an ex vivo procedure for the diagnosis of an autoimmune, infectious and / or inflammatory disease.
- the monoclonal antibody (mAb) MGD3 of the invention recognizes human Dectin-1 on the surface of stable transfectants.
- K562Dectin1aFlag cells stained with the isotype control (in gray) and with the MGD3 antibody (in black) are shown.
- FIG. 2 The MGD3 mAb recognizes human Dectin-1 on the surface of transient transfectants of Dectin-1.
- Human HEK293T cells were transiently transfected with human Dectin-1a and with the empty vector (pcDNA 3.1) as a control.
- pcDNA 3.1 empty vector
- a flow cytometry was performed, staining the cells with the anti-Dectin-1 MGD3 mAb (black line) and with an isotype control (lgG2a, gray histogram).
- the MGD3 mAb of the invention specifically recognizes the Dectin-1 protein in vivo on the surface of monocytes (MO), macrophages (M0) and dendritic cells derived from human peripheral blood monocytes (MDDC).
- the MO were obtained by purification with magnetic balls coupled to CD14 (Miltenyi Biotech).
- M0 the MOs were cultured for 5-7 days in the presence of 1000 U / ml GM-CSF and to obtain MDDCs the cells were cultured with 1000 U / ml GM-CSF + 10 ⁇ g / ml IL-4.
- the gray histograms represent staining with a control mAb (X63) and the black line represents staining with the anti-Dectin-1 mAb MGD3.
- the staining obtained by a specific marker of each cell type used is shown in the lower part of the figure.
- the MGD3 mAb of the invention is capable of recognizing human Dectin-1, in Western Blot assays.
- Total lysates of K562Dectin1aFlag cells treated or not treated with tunicamycin (3 ⁇ M, 16 hours) were subjected to electrophoresis (SDS-PAGE) under non-denaturing conditions (30 ⁇ g total cell protein / lane), transferred to a membrane and revealed with the mAb MGD3.
- the MGD3 recognizes a band of approximately 45 kDa in the lanes of the lysates of the K562Dectin1aFlag cells untreated with tunicamycin, corresponding to the isoform a of the human Dectin-1.
- a band of approximately 43 kDa appears corresponding to this same isoform after the loss of N-glycosylations due to the treatment.
- the MGD3 mAb is capable of recognizing human Dectin-1 in immunofluorescence assays.
- A. K562 WT and K562Dectin1aFlag cells adhered to crystals (10 x 10 4 cells / coverslip) coated with poly-L-lysine were used. The cells were stained with the human anti-Dectin-1 mAbs MGD3 or with a lgG2a isotype control, followed by a secondary goat-anti-immunoglobulin mouse Ab conjugated to Alexa488 (green). The figure shows the images obtained by fluorescence microscopy using the isotype control, and the MGD3 mAb.
- the figure shows the images obtained by fluorescence microscopy with Ab anti-Flag and MGD3. Only cells expressing Dectin-1 show staining.
- Figure 6.- The MGD3 mAb of the invention is capable of inhibiting the binding of Candida albicans to human Dectin-1 in flow cytometry assays in a manner similar to the inhibition caused by soluble ⁇ -glucan laminarin.
- the MGD3 mAb of the invention is capable of inhibiting the production of cytokines secreted by dendritic cells in response to Candida albicans spores.
- Human dendritic cells derived from peripheral blood monocytes were used. The cells were incubated with C. albicans spores (obtained from the CAF2 strain). The spores were previously inactivated with ultraviolet (UV) light to prevent germination.
- UV ultraviolet
- the MGD3 mAb of the invention is capable, in assays of molecular interactions by surface plasmon resonance (SPR), to capture the recombinant human Dectin-1 protein (extracellular domain, amino acids 71-247) present in the medium conditioned by the growth of transfected mammalian cells.
- SPR surface plasmon resonance
- a Biacore 3000 biosensor device (GE Healthcare) and a CM5 model chip were used, to which an anti-immunoglobulin capture antibody was attached mouse, covalently, using standardized cross-linking techniques of amino groups.
- the responses are shown as relative resonance signal (RU) as a function of time in seconds.
- the curves of the figure correspond to the subtraction between the signal obtained for the MGD3 antibody and the signal obtained for the negative control antibody.
- the baseline corresponds to the injection signal of the HBS buffer, followed by the injection of the conditioned media performed between 300 and 1200 seconds. In this phase of association, the progressive capture of the two soluble forms of Dectin-1 by the antibody is observed. Once the injection is finished, the system continues to inject buffer and the dissociation of Dectin-1 and HA-Dectin-1-Flag of the complex formed with the MGD3 antibody can be observed.
- a soluble protein was generated with the amino acid sequence 71-247 of the human Dectin-1 receptor, which comprises the neck and the carbohydrate recognition domain (CTLD) of said receptor (SEQ ID NO2).
- Said protein was obtained by inserting the gene sequence of DNA encoding said soluble peptide (SEQ ID NO1) into mammalian expression vectors (pEF), fused to the HA and Flag domains, transiently transfected by the calcium phosphate technique in Ia human cell line HEK293T.
- hybridoma selection was carried out by enzymatic immuno-absorption assays (Enzyme-Linked ImmunoSorbent Assay, ELISA).
- the antigens immobilized on the solid phase used have been soluble human Dectin-1 proteins, purified and obtained in both mammals and bacteria, as well as various ⁇ -glucans (Dectin-1 receptor ligands).
- the antigens referred to as tapestry were used and as a detection method a mouse anti-immunoglobulin antibody coupled to peroxidase was added followed by the substrate of the colorimetric reaction.
- hybridomas secreting more specific antibodies to the antigen were selected.
- the best hybridomas were selected, among which the MGD3 hybridoma (deposited in the ECACC -European Collection of Animal CeII Culture- crop collection was selected on February 6, 2008 with reference 08020601) which expresses stably immunoglobulins of the lgG2a isotype, corresponding to the human anti-Dectin-1 monoclonal antibody (mAb) MGD3.
- MGD3 hybridoma deposited in the ECACC -European Collection of Animal CeII Culture- crop collection was selected on February 6, 2008 with reference 08020601
- mAb human anti-Dectin-1 monoclonal antibody
- Example 2. The MGD3 monoclonal antibody of the invention recognizes human Dectin-1 on the surface of stable transfectants of human Dectin-1.
- the staining pattern allowed us to verify that the MGD3 mAb specifically recognizes human Dectin-1 as it specifically stains the K562Dectin1aFlag transfected cells and not the parental cells ( Figure 1).
- Example 2 does not rule out the possibility that the MGD3 hybridoma could be secreting antibodies that recognize the FLAG epitope.
- the hybridoma MGD3 produced only anti-Dectin-1 mAb
- other human cells HEK293T, derived from embryonic kidney
- pcDNA 3.1 empty vector
- Example 4.- The monoclonal antibody MGD3 specifically recognizes the ex vivo protein on the surface of monocytes (MO), macrophages (M0) and dendritic cells derived from peripheral blood monocytes (MDDC).
- MO monocytes
- M0 macrophages
- MDDC peripheral blood monocytes
- PBMCs Human peripheral blood mononuclear cells
- Figure 3 Monocytes were obtained by purification with magnetic balls coupled to CD14 (Miltenyi Biotech). To obtain MDDCs and M0, monocytes are cultured for 7 days in the presence of 1000 U / ml GM-CSF for M0, and 1000 U / ml GM-CSF + 10 ⁇ g / ml IL-4 for MDDCs. Due to the great variability of the samples from different individuals, Figure 3 shows the Cytometric profiles of a single representative donor. Staining patterns show a similar expression of Dectin-1 in the membrane of the three cell types. In the lower part the expression of molecular markers characteristic of each cell type is shown.
- Example 5 The MGD3 mAb of the invention is capable of recognizing human Dectin-1 in Western Blot assays.
- the MGD3 mAb of the invention recognized two bands: one of approximately 45 kDa. corresponding to the isoform a of the human Dectin-1 present in the sample of untreated K562Dectin1aFlag transfected cells and another approximately 43 kDa corresponding to this same isoform after tunicamycin treatment ( Figure 4).
- Example 6 The MGD3 mAb is capable of recognizing human Dectin-1 in immunofluorescence assays.
- Figure 5A shows the images obtained by fluorescence microscopy for isotype control and the MGD3 mAb. Only green signal is detected in K562Dectin1aFlag cells stained with the MGD3 Io mAb which indicates that the Dectin-1a -FLAG receptor is being specifically recognized.
- Example 7 The MGD3 mAb is capable of inhibiting the binding of Candida albicans to human Dectin-1 in flow cytometry assays in a manner similar to the inhibition caused by soluble laminarin ⁇ -glucan.
- the binding capacity of Candida albicans (strain CAF-2) to transfectants K562Dectin1aFlag was studied.
- fluorescein-labeled C. albicans spores (FITC) incubated in a cell / spore ratio of 1/10 were used for 30 min.
- the cells were pre-incubated with laminarin (500 ⁇ g / ml), or with the MGD3 mAb of the invention (2 ⁇ g / ml) for 20 min., Before incubation with the spores The analysis of the union and blocking of It was performed by flow cytometry (Figure 6A). The results indicate that the MGD3 antibody totally inhibits the specific binding of C. albicans mediated by Dectin-1 since incubation with the antibody decreases the binding to levels even lower than those presented by the parental cells. In this test, the MGD3 antibody has the same ability to inhibit the binding of spores to Dectin-1 as the laminarin soluble beta-glucan (Figure 6B).
- Example 8.- The MGD3 mAb is capable of inhibiting the production of TNF- ⁇ and IL-10 in human dendritic cells derived from monocytes in response to Candida albicans spores
- MGD3 mAbs of the invention 5 ⁇ g / ml
- isotype control lgG2a, 5 ⁇ g / ml
- the quantification of the cytokines present in the supernatant was performed by ELISA assays. The results indicate that the MGD3 antibody significantly inhibits both the production of TNF- ⁇ Fig.
- the MGD3 mAb is able, in molecular interaction assays by surface plasmon resonance (SPR), to capture recombinant human Dectin-1 present in the medium conditioned by the growth of transfected mammalian cells.
- SPR surface plasmon resonance
- HEK293T cells were transiently transfected either with the extracellular portion (amino acids 71-247, domain Dectin-1 / 71-247) of the human receptor Dectin-1 or with this same extracellular portion fused to the HA and Flag (HA epitopes) -Dectin-1a-Flag) (SEQ ID NO2).
- the collected supernatants were purified by immunoaffinity and chromatographic techniques.
- a Biacore 3000 biosensor device (GE Healthcare) and a CM5 model chip were used, to which a mouse anti-immunoglobulin capture antibody was attached, covalently, using standardized cross-linking techniques of amino groups.
- Two flow cells were used in parallel, in one supernatant containing a nonspecific control antibody was injected and in the other the MGD3 antibody, at a concentration of 15 ⁇ g / ml and at a rate of 5 ⁇ l / min, in HBS buffer (150 mN NaCI, 10 mM Hepes, pH 7.4).
- HBS buffer 150 mN NaCI, 10 mM Hepes, pH 7.4
- Figure 8 shows the responses as a relative resonance signal (RU) as a function of time in seconds.
- the curves of the figure correspond to the subtraction between the signal obtained for the MGD3 antibody and the signal obtained for the negative control antibody.
- the baseline corresponds to the injection signal of the HBS buffer, followed by the injection of the conditioned media performed between 300 and 1200 seconds. In this phase of association, the progressive capture of the two soluble forms of Dectin-1 by the antibody is observed. Once the injection is finished, the system continues to inject buffer and the Ia can be observed dissociation of Dectin-1 and HA-Dectin-1a-Flag from the complex formed with the MGD3 antibody.
- the MGD3 antibody of the invention specifically captures the soluble proteins Dectin-1 and HA-Dectin-1a-Flag present in the culture medium conditioned by the growth of HEK293T transfectants. These results indicate that this antibody can be used, in biosensors based on the SPR technique, as a fundamental component of screening assays and characterization of the interaction between Dectin-1 and different ligands.
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Abstract
The invention relates to hybridoma MGD3 and the monoclonal antibody produced thereby (also called MGD3), which specifically recognises the human Dectin-1 membrane receptor. Antibody MGD3 is capable of inhibiting the binding of Dectin-1 to the natural ligand thereof, the β-glucans that are components of the fungal wall. In addition, the aforementioned antibody specifically blocks binding to Candida albicans and the secretion of cytokines induced thereby. The MGD3 antibody obtained enables in vitro, ex vivo and in vivo detection of the human protein Dectin-1. Similarly, said antibody can be used to detect the level of expression of the receptor in human leukocytes, to define the role of this receptor in the pathogenesis of autoimmune illnesses and of conditions characterised by chronic inflammation and to ascertain the therapeutic effect of different anti-inflammatory and immunosuppressor agents. In addition, the MGD3 antibody may have therapeutic potential in chronic inflammatory illnesses.
Description
ANTICUERPO ANTI-DECTIN-1 HUMANO. HIBRIDOMA PRODUCTOR DE DICHO ANTICUERPO Y SUS APLICACIONES ANTI-DECTIN-1 HUMAN ANTIBODY. HYBRIDOMA PRODUCER OF SUCH ANTIBODY AND ITS APPLICATIONS
ESTADO DE LA TÉCNICA La respuesta inmune innata es Ia encargada del reconocimiento y contención inmediatos de Ia invasión microbiana y dirige Ia respuesta adaptativa contra los patógenos a través del control del crecimiento de los mismos, de Ia expresión de moléculas co-estimuladoras en los fagocitos, de Ia presentación antigénica y de Ia producción de citoquinas y quimioquinas. Estos factores regulan el tráfico de los leucocitos iniciando Ia respuesta inflamatoria, activan las capacidades microbicidas de los fagocitos y dirigen Ia polarización de las células T cooperadoras. La respuesta inflamatoria sirve para limitar Ia infección, pero Ia inflamación tiene que resolverse para evitar procesos patológicos. En este sentido, se ha propuesto que Ia producción de citoquinas inhibitorias como Ia interleuquina (IL)-IO por los neutrófilos, macrófagos, células dendríticas (DCs) y células T reguladoras, tiene un papel importante en las fases tardías de Ia infección para prevenir una activación excesiva de Ia respuesta innata y el establecimiento del comensalismo y quizá de Ia latencia y persistencia del patógeno (Romani 2004).STATE OF THE TECHNIQUE The innate immune response is the one in charge of the immediate recognition and containment of the microbial invasion and directs the adaptive response against the pathogens through the control of their growth, of the expression of co-stimulating molecules in the phagocytes, of the antigen presentation and the production of cytokines and chemokines. These factors regulate the leukocyte traffic by initiating the inflammatory response, activating the microbicidal abilities of the phagocytes and directing the polarization of the cooperating T cells. The inflammatory response serves to limit the infection, but the inflammation has to be resolved to avoid pathological processes. In this sense, it has been proposed that the production of inhibitory cytokines such as interleukin (IL) -IO by neutrophils, macrophages, dendritic cells (DCs) and regulatory T cells, has an important role in the late stages of infection to prevent an excessive activation of the innate response and the establishment of commensalism and perhaps of the latency and persistence of the pathogen (Romani 2004).
En Ia respuesta inmune innata, el reconocimiento de los patógenos se lleva a cabo por las células fagocíticas a través de los receptores de patrones de reconocimiento (PRR, Pattern Recognition Receptor) que detectan estructuras microbianas altamente conservadas. El prototipo de PRR son los TLR (Toll-like receptors), pero recientemente se ha demostrado que también receptores de Ia familia de las lectinas como Dectin-1 pueden funcionar como PRRs (Akira y Takeda 2004; Gordon 2002; Brown 2005). El receptor de membrana Dectin-1 humano reconoce polisacáridos con enlaces β(1→3) y/o β(1→6), los llamados β-glucanos, presentes en Ia pared de los hongos (Brown y Gordon 2001 ; Brown et al. 2003). Como consecuencia, Dectin-1 está implicado en Ia respuesta
inmune innata frente a patógenos fúngicos (Brown y Gordon 2003) tanto en humanos como en ratón.In the innate immune response, the recognition of the pathogens is carried out by the phagocytic cells through the pattern recognition receptors (PRR), which detect highly conserved microbial structures. The PRR prototype is the TLR (Toll-like receptors), but recently it has been shown that receptors of the lectin family such as Dectin-1 can function as PRRs (Akira and Takeda 2004; Gordon 2002; Brown 2005). The human Dectin-1 membrane receptor recognizes polysaccharides with β (1 → 3) and / or β (1 → 6) bonds, the so-called β-glucans, present in the fungal wall (Brown and Gordon 2001; Brown et al . 2003). As a consequence, Dectin-1 is involved in the response Innate immune against fungal pathogens (Brown and Gordon 2003) in both humans and mice.
La estructura de este receptor contiene una región extracelular con un único dominio de unión a carbohidratos de tipo CTLD (C-type lectin-like domain) (Weis et al. 1998) conectado al dominio intracelular a través de un cuello y una región transmembrana. Dectin-1 sufre un proceso de procesamiento alternativo que da lugar a varias isoformas, sólo dos de ellas funcionales: Dectin-1a (completa) y Dectin-1 b (predominante, carente del cuello). Aunque parecen funcionar de manera similar in vitro, se expresan de forma diferencial según el tipo celular (Willment et al. 2001 , Heinsbroek et al. 2006). En su tallo citoplasmático posee un motivo semi- ITAM (Immunoreceptor Tyrosine-based Activation Motif) con dos residuos de tirosina potencialmente fosforilables, implicado en activación celular (Gantner et al. 2003; Brown et al. 2003; Gordon 2002). Identificado inicialmente en macrófagos de ratón como el receptor principal de β-glucanos (Brown y Gordon 2001 ; Willment et al. 2005), Dectin-1 media Ia unión, captación y eliminación de patógenos, dispara Ia respuesta oxidativa generando especies reactivas de oxígeno (ROS) y produce citoquinas pro-inflamatorias y quimioquinas. Además, Dectin-1 está implicado en fenómenos de tolerancia y control de Ia respuesta inmune puesto que puede inducir Ia generación de citoquinas inhibitorias (Interleuquina (IL)-IO, IL-2). Estas funciones pueden influenciar Ia respuesta inmune resultante y, en ciertas circunstancias, llevar a procesos patológicos y de autoinmunidad (Haskard y Landi 2002). Aunque se consideraba que Dectin-1 se expresaba de forma mayoritaria en células dendríticas (DCs) inmaduras y en células de Langerhans y que los estímulos de maduración disminuían su expresión (Ariizumi et al .2000; Hermanz-Falcon et al. 2001 ; Sobanov et al. 2001 ; Yokota et al. 2001 ), posteriormente se comprobó que Dectin-1 no está restringido al linaje mieloide, sino que también se expresa en neutrófilos, eosinófilos, células B
y una sub-población de células T (Brown et al. 2002; Taylor et al. 2002; Willment et al. 2005) aunque no en todos los tejidos (Reid. et al. 2004).The structure of this receptor contains an extracellular region with a single carbohydrate binding domain of the CTLD (C-type lectin-like domain) (Weis et al. 1998) connected to the intracellular domain through a neck and a transmembrane region. Dectin-1 undergoes an alternative processing process that results in several isoforms, only two of them functional: Dectin-1a (complete) and Dectin-1 b (predominant, lacking the neck). Although they appear to work similarly in vitro, they are differentially expressed according to cell type (Willment et al. 2001, Heinsbroek et al. 2006). In its cytoplasmic stem it has a semi-ITAM motif (Immunoreceptor Tyrosine-based Activation Motif) with two potentially phosphorylatable tyrosine residues, involved in cellular activation (Gantner et al. 2003; Brown et al. 2003; Gordon 2002). Initially identified in mouse macrophages as the main β-glucan receptor (Brown and Gordon 2001; Willment et al. 2005), Dectin-1 mediates the binding, uptake and elimination of pathogens, triggers the oxidative response generating reactive oxygen species ( ROS) and produces pro-inflammatory cytokines and chemokines. In addition, Dectin-1 is involved in phenomena of tolerance and control of the immune response since it can induce the generation of inhibitory cytokines (Interleukin (IL) -IO, IL-2). These functions can influence the resulting immune response and, in certain circumstances, lead to pathological and autoimmunity processes (Haskard and Landi 2002). Although Dectin-1 was considered to be expressed mostly in immature dendritic cells (DCs) and Langerhans cells and that maturation stimuli decreased their expression (Ariizumi et al. 2000; Hermanz-Falcon et al. 2001; Sobanov et al. 2001; Yokota et al. 2001), it was subsequently found that Dectin-1 is not restricted to myeloid lineage, but is also expressed in neutrophils, eosinophils, B cells and a sub-population of T cells (Brown et al. 2002; Taylor et al. 2002; Willment et al. 2005) although not in all tissues (Reid. et al. 2004).
Dectin-1 es crucial en Ia respuesta inmune frente a patógenos fúngicos vivos como Candida albicans (Gantner et al. 2005; Taylor et al. 2007), Coccidiodes posadasii (Viriyakosol et al. 2005), Pneumocystis carinii (Steele et al. 2003 y 2005) y Aspergillus fumigatus (Hohl et al. 2005; Steele et al. 2005; Gersuk et al. 2006). La relevancia de este receptor se ha establecido gracias a Ia generación de ratones deficientes en el gen que codifica para Dectin-1 , en los cuales se ha visto que aumenta Ia susceptibilidad a C. albicans (Taylor et al. 2007) y a P. carinii (Saijo et al. 2007). No obstante, el papel que desempeña Dectin-1 en el control de Ia infección es aún controvertido. En cualquier caso, se ha demostrado que Dectin-1 puede desencadenar respuestas específicas contra patógenos en colaboración con los TLRs o de forma independiente de ellos. En Ia vía independiente de TLRs Ia unión del ligando provoca una cascada de señalización intracelular: Ia fosforilación de Ia tirosina proximal del motivo de activación ITAM por Ia tirosina quinasa Src, permite reclutar Ia tirosina quinasa de bazo (Syk, Spleen Tyrosine Kinase). Syk es necesaria para Ia señalización a través del receptor específico de antígeno de células T y B, y en células mieloides se ha descrito como crucial para estimular Ia producción de especies de oxígeno reactivas a través de Dectin-1 (Underhill et al. 2005; Rogers et al. 2005). Tras Ia activación de Syk, se produce una cascada de fosforilación que implica entre otros, al factor de transcripción NF-κB (Dennehy. y Gordon 2007; Dennehy et al. 2008). Recientemente, se ha demostrado que Ia señalización vía Dectin-1 (inducida por β-glucano particulado (zymosan) o con esporas de C. albicans) puede modular directamente Ia expresión génica a través de Ia activación del factor de transcripción NFAT, tanto en macrófagos como en DCs, induciendo a su vez los factores de transcripción Egr2, Egr3 y COX-2 (Goodridge. et al. 2007). Además, Dectin-1 participa en una nueva ruta de señalización independiente de TLRs en DCs, mediando directamente Ia
activación de NF-κB vía Ia molécula adaptadora de señalización CARD9 (Gross et al. 2006). La ruta Dectin-1-Syk-CARD9 acopla Ia respuesta inmune y Ia adaptativa, siendo CARD9 necesaria para el desarrollo de una respuesta de tipo linfocito T cooperador (TH-17) frente a patógenos como C. albicans (LeibundGut-Landmann et al. 2007).Dectin-1 is crucial in the immune response against living fungal pathogens such as Candida albicans (Gantner et al. 2005; Taylor et al. 2007), Coccidiodes posadasii (Viriyakosol et al. 2005), Pneumocystis carinii (Steele et al. 2003 and 2005) and Aspergillus fumigatus (Hohl et al. 2005; Steele et al. 2005; Gersuk et al. 2006). The relevance of this receptor has been established thanks to the generation of mice deficient in the gene coding for Dectin-1, in which it has been seen that susceptibility to C. albicans (Taylor et al. 2007) and P. carinii increases. (Saijo et al. 2007). However, the role that Dectin-1 plays in the control of the infection is still controversial. In any case, it has been shown that Dectin-1 can trigger specific responses against pathogens in collaboration with or independently of TLRs. In the independent route of TLRs, ligand binding causes an intracellular signaling cascade: the phosphorylation of the proximal tyrosine of the motive for ITAM activation by Src tyrosine kinase, allows the spleen tyrosine kinase to be recruited (Syk, Spleen Tyrosine Kinase). Syk is necessary for signaling through the specific T and B cell antigen receptor, and in myeloid cells it has been described as crucial to stimulate the production of reactive oxygen species through Dectin-1 (Underhill et al. 2005; Rogers et al. 2005). After the activation of Syk, a phosphorylation cascade is produced that involves, among others, the transcription factor NF-κB (Dennehy. And Gordon 2007; Dennehy et al. 2008). Recently, it has been shown that signaling via Dectin-1 (induced by particulate β-glucan (zymosan) or with C. albicans spores) can directly modulate gene expression through the activation of the NFAT transcription factor, both in macrophages as in DCs, in turn inducing the transcription factors Egr2, Egr3 and COX-2 (Goodridge. et al. 2007). In addition, Dectin-1 participates in a new signaling route independent of TLRs in DCs, directly mediating Ia NF-κB activation via the CARD9 signaling adapter molecule (Gross et al. 2006). The Dectin-1-Syk-CARD9 route combines the immune and adaptive response, being CARD9 necessary for the development of a cooperative T-lymphocyte response (T H -17) against pathogens such as C. albicans (LeibundGut-Landmann et al . 2007).
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DESCRIPCIÓN DE LA PATENTEPATENT DESCRIPTION
Descripción brevebrief description
Un objeto de Ia presente invención Io constituye el anticuerpo anti-An object of the present invention constitutes the anti-antibody
Dectin-1 humano MGD3, caracterizado porque se une y reconoce específicamente el dominio 71-247 de Ia proteína humana Dectin-1 (SEQHuman Dectin-1 MGD3, characterized in that it specifically binds and recognizes domain 71-247 of the human protein Dectin-1 (SEQ
ID NO2) ya sea un anticuerpo monoclonal o policlonal, en adelante el anticuerpo de Ia invención.ID NO2) either a monoclonal or polyclonal antibody, hereinafter the antibody of the invention.
Un objeto más particular de Ia invención es el anticuerpo de Ia invención caracterizado porque es monoclonal que es producido por el hibridoma MGD3 (depositado en Ia colección de cultivos ECACC -A more particular object of the invention is the antibody of the invention characterized in that it is monoclonal that is produced by the MGD3 hybridoma (deposited in the ECACC culture collection -
European Collection of Animal CeII Culture- el 6 de Febrero de 2008 con Ia referencia 08020601 ).
Otro objeto de Ia presente invención es Ia secuencia de nucleótidos caracterizada por ser codificante del dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO1 ), y por ser: a) una secuencia de nucleótidos análoga a Ia SEQ ID NO1 b) un fragmento de Ia secuencias de a, y c) una secuencia de nucleótidos que comprende una secuencia cualquiera perteneciente de a) y b).European Collection of Animal CeII Culture- on February 6, 2008 with reference 08020601). Another object of the present invention is the nucleotide sequence characterized by being coding for domain 71-247 of the human protein Dectin-1 (SEQ ID NO1), and for being: a) a nucleotide sequence analogous to SEQ ID NO1 b ) a fragment of the sequences of a, and c) a nucleotide sequence comprising any sequence belonging to a) and b).
Otro objeto de Ia invención Io constituye Ia secuencia de aminoácidos aislada de Ia proteína humana Dectin-1 caracterizada porque se corresponde con el dominio 71-247 de ésta (SEQ ID NO2), y por ser: a) una secuencia de aminoácidos análoga a Ia SEQ ID NO2 b) un fragmento de Ia secuencia de a, y c) una secuencia de aminoácidos que comprende una secuencia cualquiera perteneciente de a) y b).Another object of the invention constitutes the isolated amino acid sequence of the human protein Dectin-1 characterized in that it corresponds to domain 71-247 thereof (SEQ ID NO2), and for being: a) an amino acid sequence analogous to Ia SEQ ID NO2 b) a fragment of the sequence of a, and c) an amino acid sequence comprising any sequence belonging to a) and b).
Un objeto particular de Ia invención es el uso de Ia secuencia de nucleótidos (SEQ ID NO1 ) para Ia producción de anticuerpos anti-Dectin-1 humanos, ya sean monoclonales o policlonales. Un objeto particular de Ia invención es el uso de Ia secuencia de aminoácidos (SEQ ID NO2) para Ia producción de anticuerpos anti-Dectin- 1 humanos, ya sean monoclonales o policlonales.A particular object of the invention is the use of the nucleotide sequence (SEQ ID NO1) for the production of human anti-Dectin-1 antibodies, whether monoclonal or polyclonal. A particular object of the invention is the use of the amino acid sequence (SEQ ID NO2) for the production of human anti-Dectin-1 antibodies, whether monoclonal or polyclonal.
Un objeto de Ia presente invención es el hibridoma caracterizado porque produce un anticuerpo específico del dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO2).An object of the present invention is the hybridoma characterized in that it produces an antibody specific for domain 71-247 of the human protein Dectin-1 (SEQ ID NO2).
Un objeto particular de Ia invención es el hibridoma de Ia invención caracterizado porque es el hibridoma MGD3 productor del anticuerpo de Ia invención (depositado en Ia colección de cultivos ECACC -European Collection of Animal CeII Culture-e\ 6 de Febrero de 2008 con Ia referenciaA particular object of the invention is the hybridoma of the invention characterized in that it is the hybridoma MGD3 producing the antibody of the invention (deposited in the ECACC-European Collection of Animal CeII Culture-e \ 6 February 2008 crop collection with reference
08020601 ).
Otro objeto más particular de Ia invención es el uso de hibridoma de Ia invención para Ia producción del anticuerpo monoclonal anti-Dectin-1 humano, preferentemente el anticuerpo monoclonal de Ia invención.08020601). Another more particular object of the invention is the use of hybridoma of the invention for the production of the human anti-Dectin-1 monoclonal antibody, preferably the monoclonal antibody of the invention.
Un objeto de Ia presente invención es una composición biotecnológica, farmacéutica, diagnostica o terapéutica caracterizada porque contiene el anticuerpo de Ia invención.An object of the present invention is a biotechnological, pharmaceutical, diagnostic or therapeutic composition characterized in that it contains the antibody of the invention.
Uso del anticuerpo de Ia invención para Ia elaboración de una composición biotecnológica, farmacéutica diagnóstica o terapéutica útil para Ia realización de: - ensayos de detección, identificación y/o cuantificación de Ia proteína Dectin-1 humana,Use of the antibody of the invention for the elaboration of a biotechnological, pharmaceutical diagnostic or therapeutic composition useful for the performance of: - detection, identification and / or quantification assays of the human Dectin-1 protein,
- tratamiento de enfermedades infecciosas, autoinmunes y/o inflamatorias, y- treatment of infectious, autoimmune and / or inflammatory diseases, and
- diagnóstico de enfermedades infecciosas, autoinmunes y/o inflamatorias.- diagnosis of infectious, autoimmune and / or inflammatory diseases.
Otro objeto de Ia invención es Ia composición biotecnológica, farmacéutica diagnostica o terapéutica caracterizada porque comprende el anticuerpo de Ia invención.Another object of the invention is the biotechnological, pharmaceutical or diagnostic pharmaceutical composition characterized in that it comprises the antibody of the invention.
Otro objeto particular de Ia invención es el uso de Ia composición de Ia invención y del anticuerpo de Ia invención en un procedimiento ex vivo de detección, identificación y/o cuantificación de Ia proteína Dectin-1 humana.Another particular object of the invention is the use of the composition of the invention and the antibody of the invention in an ex vivo method of detection, identification and / or quantification of the human Dectin-1 protein.
Otro objeto particular de Ia invención es el uso de Ia composición de Ia invención y del anticuerpo de Ia invención en un procedimiento ex vivo para el diagnóstico de una enfermedad autoinmune, infecciosa y/o inflamatoria.Another particular object of the invention is the use of the composition of the invention and the antibody of the invention in an ex vivo procedure for the diagnosis of an autoimmune, infectious and / or inflammatory disease.
Otro objeto particular de Ia invención es el uso de Ia composición de Ia invención y del anticuerpo de Ia invención en un procedimiento terapéutico de una enfermedad humana donde Ia proteína Dectin-1 humana presenta un papel etiopatogénico.
Otro objeto más particular de Ia invención es el uso de Ia composición de Ia invención y del anticuerpo de Ia invención donde Ia enfermedad a Ia que se Ie aplica el procedimiento terapéutico es una enfermedad inflamatoria, infecciosa y/o autoinmune.Another particular object of the invention is the use of the composition of the invention and the antibody of the invention in a therapeutic procedure of a human disease where the human Dectin-1 protein has an etiopathogenic role. Another more particular object of the invention is the use of the composition of the invention and the antibody of the invention where the disease to which the therapeutic procedure is applied is an inflammatory, infectious and / or autoimmune disease.
Descripción DetalladaDetailed description
La presente invención se basa en que los inventores han observado que un anticuerpo específico, concretamente un anticuerpo monoclonal y más concretamente el anticuerpo monoclonal MGD3, generado por los inventores al inmunizar ratones con una proteína de fusión que comprende Ia región entre los aminoácidos 71-247 (dominio Dectin-1/71-247) de Ia proteína Dectin-1 humana (SEQ ID NO2), presenta una serie de características que Ie confieren una serie de ventajas técnicas con respecto a los anticuerpos descritos en el estado de Ia técnica, por Io que el anticuerpo MGD3 representa una nueva herramienta biotecnológica útil en el campo de Ia investigación biomédica y en el campo asistencial médico, tanto en el área del diagnóstico como en el ámbito terapéutico.The present invention is based on the fact that the inventors have observed that a specific antibody, specifically a monoclonal antibody and more specifically the monoclonal antibody MGD3, generated by the inventors by immunizing mice with a fusion protein comprising the region between amino acids 71-247 (Dectin-1 / 71-247 domain) of the human Dectin-1 protein (SEQ ID NO2), presents a series of characteristics that confer a series of technical advantages with respect to the antibodies described in the state of the art, by Therefore, the MGD3 antibody represents a new biotechnological tool useful in the field of biomedical research and in the medical care field, both in the area of diagnosis and in the therapeutic field.
Las ventajas técnicas que posee el anticuerpo MGD3 de Ia invención son las siguientes: es capaz de inhibir Ia unión del ligando natural a su receptor - en ensayos de citometría de flujo (Figura 6)- de manera análoga a los β-glucanos solubles (laminarina). También es capaz de inhibir de forma significativa Ia producción de citoquinas pro- y antiinflamatorias como TNF-α e interleuquina (IL)-IO, respectivamente, en células dendríticas derivadas de monocitos en respuesta a Ia interacción con esporas de C. albicans (Figuras 7A y 7B). Puede emplearse para detectar cambios en los niveles de expresión del receptor Dectin-1 en leucocitos humanos, Io que podría indicar Ia capacidad de estas células para detectar Ia presencia de patógenos fúngicos como Candida albicans, Coccidiodes posadasii, Pneumocystis carínii y/o Aspergillus fumigatus y por tanto, Ia susceptibilidad hacia estos. Son útiles como marcadores de Ia proteína Dectin-1 humana in vivo en Ia superficie de monocitos,
macrófagos, células de Langerhans y células dendríticas derivadas de monocitos de sangre periférica (Figura 3), leucocitos en reposo y activados en procesos infecciosos, por ejemplo por agentes fúngicos, así como en patologías autoinmunes y/o inflamatorias y podrían servir para definir el papel de este receptor en Ia patogenia de estas enfermedades así como para definir Ia susceptibilidad hacia diferentes patologías inflamatorias. Finalmente, los anticuerpos MGD3 de Ia invención pueden ser utilizados como indicadores del efecto terapéutico de distintos agentes antiinflamatorios e inmunosupresores. Además, el anticuerpo MGD3 de Ia invención:The technical advantages of the MGD3 antibody of the invention are the following: it is capable of inhibiting the binding of the natural ligand to its receptor - in flow cytometry tests (Figure 6) - analogously to soluble β-glucans (laminarin ). It is also capable of significantly inhibiting the production of pro and anti-inflammatory cytokines such as TNF-α and interleukin (IL) -IO, respectively, in dendritic cells derived from monocytes in response to the interaction with spores of C. albicans (Figures 7A and 7B). It can be used to detect changes in Dectin-1 receptor expression levels in human leukocytes, which could indicate the ability of these cells to detect the presence of fungal pathogens such as Candida albicans, Coccidiodes posadasii, Pneumocystis carínii and / or Aspergillus fumigatus and therefore, the susceptibility to these. They are useful as markers of human Dectin-1 protein in vivo on the surface of monocytes, macrophages, Langerhans cells and dendritic cells derived from peripheral blood monocytes (Figure 3), leukocytes at rest and activated in infectious processes, for example by fungal agents, as well as in autoimmune and / or inflammatory pathologies and could serve to define the role of this receptor in the pathogenesis of these diseases as well as to define the susceptibility to different inflammatory pathologies. Finally, the MGD3 antibodies of the invention can be used as indicators of the therapeutic effect of different anti-inflammatory and immunosuppressive agents. In addition, the MGD3 antibody of the invention:
1. reconoce Ia proteína Dectin-1 in vivo en células mieloides con una elevada calidad en Ia relación señal-fondo (ver Figura 3).1. recognizes the Dectin-1 protein in vivo in myeloid cells with a high quality in the signal-to-background ratio (see Figure 3).
2. es el único anticuerpo monoclonal anti-Dectin-1 humano que bloquea Ia unión de Candida albicans en ensayos in vitro con transfectantes estables de Ia proteína.2. It is the only human anti-Dectin-1 monoclonal antibody that blocks the binding of Candida albicans in in vitro assays with stable protein transfectants.
3. es el único anticuerpo monoclonal anti-Dectin-1 humano que reconoce Ia proteína Dectin-1 humana (SEC ID NO2) con alta afinidad tanto en ensayos de citometría de flujo como de Western Blot (ver Figuras 1 , 2, 3 y 4). 4. es capaz de capturar Ia proteína soluble DECTIN-1-FLAG en ensayos de interacciones moleculares mediante SPR en el biosensor Biacore 3000, Io que indica una gran afinidad por el ligando (Figura 7).3. It is the only human anti-Dectin-1 monoclonal antibody that recognizes the human Dectin-1 protein (SEQ ID NO2) with high affinity in both flow cytometry and Western Blot assays (see Figures 1, 2, 3 and 4 ). 4. It is capable of capturing the soluble protein DECTIN-1-FLAG in molecular interaction assays by means of SPR in the Biacore 3000 biosensor, which indicates a great affinity for the ligand (Figure 7).
5. es capaz de inhibir de forma significativa Ia producción de TNF-α en DCs derivadas de monocitos que han estado en contacto con esporas de C. albicans (Figura 7A).5. It is capable of significantly inhibiting the production of TNF-α in DCs derived from monocytes that have been in contact with C. albicans spores (Figure 7A).
6. es capaz de inhibir de forma significativa Ia producción de IL-10 en DCs derivadas de monocitos que han estado en contacto con esporas de C. albicans (Figura 7B).6. It is able to significantly inhibit the production of IL-10 in DCs derived from monocytes that have been in contact with C. albicans spores (Figure 7B).
Todas las características descritas y enumeradas hasta ahora confieren al anticuerpo MGD3 de Ia invención las ventajas técnicas que
hacen del mismo un anticuerpo único y ventajoso sobre el resto de anticuerpos anti-Dectin-1 humanos descritos previamente.All the characteristics described and enumerated so far confer on the MGD3 antibody of the invention the technical advantages that they make it a unique and advantageous antibody over the rest of human anti-Dectin-1 antibodies described previously.
La invención, también se refiere a un hibridoma clonado (MGD3) (depositado en Ia colección de cultivos ECACC -European Collection of Animal CeII Culture- el 6 de Febrero de 2008 con Ia referencia 08020601 ) que produce un anticuerpo monoclonal, denominado en adelante MGD3, que reconoce específicamente el receptor de membrana Dectin-1 humano. Dicho clon se obtuvo por procedimientos estándar de fusión celular entre un linfocito B del bazo de un ratón BALB/c inmunizado y células de mieloma (X63.Ag.653 NP3) (Ejemplo 1 ).The invention also relates to a cloned hybridoma (MGD3) (deposited in the ECACC-European Collection of Animal CeII Culture crop collection on February 6, 2008 with reference 08020601) that produces a monoclonal antibody, hereinafter referred to as MGD3 , which specifically recognizes the human Dectin-1 membrane receptor. Said clone was obtained by standard procedures of cell fusion between a B lymphocyte of the spleen of an immunized BALB / c mouse and myeloma cells (X63.Ag.653 NP3) (Example 1).
Por Io tanto, un aspecto de Ia presente invención es un anticuerpo anti-Dectin-1 humano, en adelante anticuerpo de Ia invención, que se une y reconoce específicamente el dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO2), ya sea un anticuerpo monoclonal o policlonal. Un aspecto más particular de Ia invención Io constituye el anticuerpo monoclonal de Ia invención y producido por el hibridoma MGD3 (depositado en Ia colección de cultivos ECACC -European Collection of Animal CeII Culture- el 6 de Febrero de 2008 con Ia referencia 08020601 ), en adelante anticuerpo monoclonal MGD3 de Ia invención. Tal como se utiliza en Ia presente invención el término "anticuerpo" se refiere a un anticuerpo recombinante o minianticuerpo que mantienen su capacidad de unión a antígeno. El término "minianticuerpo" se define como fragmentos derivados de anticuerpos construidos por tecnología de ADN recombinante, que, pese a su menor tamaño, conservan Ia capacidad de unión al antígeno ya que mantienen al menos un dominio variable de inmunoglobulina donde residen las zonas de unión a antígenos, e incluye, a título ilustrativo y sin que limite el alcance de Ia invención, al siguiente grupo: Fab, F(ab')2, scFv, y anticuerpos recombinantes monodominio (dAbs). En el marco de Ia presente invención, se entiende por anticuerpos recombinantes monodominio y/o dominios tipo inmunoglobulina con capacidad de unión y reconocimiento independiente, tanto a los dominios
variables de cadena pesada (VH), a los dominios variables de cadena ligera (VL), a los anticuerpos recombinantes de camélidos (VHH), los anticuerpos recombinantes de camélidos humanizados, los anticuerpos recombinantes de otras especies camelizados, los anticuerpos monodominio IgNAR de peces cartilaginosos; es decir, que se incluyen tanto dominios que de forma natural son monodominio (caso de VHH e IgNAR), como anticuerpos que por ingeniería se han alterado para que por sí solos sean capaces de interaccionar con el antígeno y mejorar sus propiedades de estabilidad y solubilidad. Se incluye en esta definición cualquier modificación de los anticuerpos recombinantes como su multimerización o Ia fusión a cualquier molécula (p.ej. toxinas, enzimas, antígenos, otros fragmentos de anticuerpos, etc.).Therefore, one aspect of the present invention is a human anti-Dectin-1 antibody, hereinafter antibody of the invention, which specifically binds and recognizes domain 71-247 of the human protein Dectin-1 (SEQ ID NO2) , either a monoclonal or polyclonal antibody. A more particular aspect of the invention constitutes the monoclonal antibody of the invention and produced by the hybridoma MGD3 (deposited in the ECACC culture collection - European Collection of Animal CeII Culture - on February 6, 2008 with reference 08020601), in below monoclonal antibody MGD3 of the invention. As used in the present invention, the term "antibody" refers to a recombinant antibody or mini-antibody that maintains its antigen binding capacity. The term "mini-antibody" is defined as fragments derived from antibodies constructed by recombinant DNA technology, which, despite their smaller size, retain the antigen binding capacity since they maintain at least one variable immunoglobulin domain where the binding areas reside to antigens, and includes, by way of illustration and without limiting the scope of the invention, the following group: Fab, F (ab ') 2, scFv, and recombinant monodomain antibodies (dAbs). Within the framework of the present invention, it is understood as recombinant monodomain antibodies and / or immunoglobulin-like domains with independent binding and recognition capacity, both to the domains heavy chain variables (VH), to light chain variable domains (VL), to recombinant camelid antibodies (VHH), recombinant antibodies to humanized camelids, recombinant antibodies to other camelized species, fish monodomain antibodies IgNAR cartilaginous; that is, that both domains that are naturally monodomain (case of VHH and IgNAR) are included, as well as engineering antibodies that have been altered so that by themselves they are able to interact with the antigen and improve its stability and solubility properties . Any modification of the recombinant antibodies such as their multimerization or fusion to any molecule (eg toxins, enzymes, antigens, other antibody fragments, etc.) is included in this definition.
El anticuerpo puede ser obtenido de un ser humano o un animal (p.ej. camellos, llamas, vicuñas, ratones, ratas, conejos, caballos, tiburones nodriza, etc.) o mediante técnicas de DNA recombinante o síntesis química de genes, o bien desde o selección in vitro o in vivo de genotecas de anticuerpos.The antibody can be obtained from a human being or an animal (eg camels, llamas, vicunas, mice, rats, rabbits, horses, nurse sharks, etc.) or by recombinant DNA techniques or chemical gene synthesis, or either from or in vitro or in vivo selection of antibody libraries.
Otro aspecto de Ia presente invención es Ia secuencia de nucleótidos caracterizada por ser codificante del dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO1 ), así como cualquier secuencia de nucleótidos análoga a ésta. En el sentido utilizado en esta descripción, el término "análoga" pretende incluir cualquier secuencia de nucleótidos que pueda ser aislada o construida en base a Ia secuencia de nucleótidos mostrada en Ia presente memoria, por ejemplo, mediante Ia introducción de sustituciones de nucleótidos conservativos o no conservativos, incluyendo Ia inserción de uno o más nucleótidos, Ia adición de uno o más nucleótidos en cualquiera de los extremos de Ia molécula o Ia deleción de uno o más nucleótidos, en cualquier extremo o en el interior de Ia secuencia, y que constituya una secuencia codificante o péptido con actividad similar a Ia secuencia de Ia proteína humana Dectin-1.
En general, una secuencia de nucleótidos análoga es sustancialmente homologa a Ia secuencia de aminoácidos comentada anteriormente. En el sentido utilizado en esta descripción, Ia expresión "sustancialmente homologa" significa que las secuencias de nucleótidos en cuestión tienen un grado de identidad de, al menos, un 40%, preferentemente de, al menos, un 85%, o más preferentemente de, al menos, un 95%.Another aspect of the present invention is the nucleotide sequence characterized by being coding for domain 71-247 of the human protein Dectin-1 (SEQ ID NO1), as well as any nucleotide sequence analogous to it. In the sense used in this description, the term "analogous" is intended to include any nucleotide sequence that can be isolated or constructed based on the nucleotide sequence shown herein, for example, by introducing conservative nucleotide substitutions or non-conservative, including the insertion of one or more nucleotides, the addition of one or more nucleotides at any of the ends of the molecule or the deletion of one or more nucleotides, at any end or inside the sequence, and constituting a coding sequence or peptide with activity similar to the sequence of the human protein Dectin-1. In general, an analogous nucleotide sequence is substantially homologous to the amino acid sequence discussed above. In the sense used in this description, the expression "substantially homologous" means that the nucleotide sequences in question have a degree of identity of at least 40%, preferably of at least 85%, or more preferably of At least 95%.
Esta secuencia de nucleótidos puede ser utilizada bien en su totalidad o bien de forma parcial mediante fragmentos de Ia misma para Ia expresión del péptido/s correspondientes en distintas células para sus posteriores usos biotecnológicos.This nucleotide sequence can be used either in its entirety or partially by means of fragments thereof for the expression of the corresponding peptide / s in different cells for subsequent biotechnological uses.
Otro aspecto de Ia presente invención es Ia secuencia de aminoácidos aislada de Ia proteína humana Dectin-1 correspondiente al dominio 71-247 de Ia misma (SEQ ID NO2), así como cualquier secuencia de aminoácidos análoga a ésta. En el sentido utilizado en esta descripción, el término "análoga" pretende incluir cualquier secuencia de aminoácidos que pueda ser aislada o construida en base a Ia secuencia de aminoácidos mostrada en Ia presente memoria, por ejemplo, mediante Ia introducción de sustituciones de aminoácidos conservativas o no conservativas, incluyendo Ia inserción de uno o más aminoácidos, Ia adición de uno o más aminoácidos en cualquiera de los extremos de Ia molécula o Ia deleción de uno o más aminoácidos, en cualquier extremo o en el interior de Ia secuencia, y que constituya un péptido con actividad similar a Ia secuencia de Ia proteína humana Dectin-1. En general, una secuencia de aminoácidos análoga es sustancialmente homologa a Ia secuencia de aminoácidos comentada anteriormente. En el sentido utilizado en esta descripción, Ia expresión "sustancialmente homologa" significa que las secuencias de aminoácidos en cuestión tienen un grado de identidad de, al menos, un 40%, preferentemente de, al menos, un 85%, o más preferentemente de, al menos, un 95%.
Otro aspecto de Ia invención es el uso de Ia secuencia nucleótidos y de aminoácidos - SEC ID NO1 y SEC ID NO2, respectivamente- para producir anticuerpos anti-Dectin-1 humanos, ya sean monoclonales y policlonales. Otro aspecto de Ia invención es un hibridoma, en adelante hibridoma de Ia invención, que produce un anticuerpo específico del dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO2).Another aspect of the present invention is the amino acid sequence isolated from the human protein Dectin-1 corresponding to domain 71-247 thereof (SEQ ID NO2), as well as any amino acid sequence analogous to it. In the sense used in this description, the term "analogous" is intended to include any amino acid sequence that can be isolated or constructed based on the amino acid sequence shown herein, for example, by introducing conservative amino acid substitutions or non-conservative, including the insertion of one or more amino acids, the addition of one or more amino acids at any of the ends of the molecule or the deletion of one or more amino acids, at any end or inside the sequence, and constituting a peptide with activity similar to the sequence of the human protein Dectin-1. In general, an analogous amino acid sequence is substantially homologous to the amino acid sequence discussed above. In the sense used in this description, the expression "substantially homologous" means that the amino acid sequences in question have a degree of identity of at least 40%, preferably of at least 85%, or more preferably of At least 95%. Another aspect of the invention is the use of the nucleotide and amino acid sequence - SEQ ID NO1 and SEQ ID NO2, respectively - to produce human anti-Dectin-1 antibodies, both monoclonal and polyclonal. Another aspect of the invention is a hybridoma, hereinafter hybridoma of the invention, which produces an antibody specific for domain 71-247 of the human protein Dectin-1 (SEQ ID NO2).
Otro aspecto más particular de Ia invención Io constituye el hibridoma de Ia invención denominado hibridoma MGD3 y productor del anticuerpo MGD3 (depositado en Ia colección de cultivos ECACC -Another more particular aspect of the invention constitutes the hybridoma of the invention called the MGD3 hybridoma and producer of the MGD3 antibody (deposited in the ECACC culture collection -
European Collection of Animal CeII Culture-e\ 6 de Febrero de 2008 con Ia referencia 08020601 ).European Collection of Animal CeII Culture-e \ February 6, 2008 with reference 08020601).
Otro aspecto de Ia invención Io constituye el uso del anticuerpo de Ia invención en Ia elaboración de una composición biotecnológica o una composición farmacéutica diagnóstica o terapéutica útil para Ia realización de ensayos de identificación de Ia proteína Dectin-1 humana o para el tratamiento y/o diagnóstico de enfermedades autoinmunes y/o inflamatorias.Another aspect of the invention constitutes the use of the antibody of the invention in the elaboration of a biotechnological composition or a diagnostic or therapeutic pharmaceutical composition useful for performing tests to identify the human Dectin-1 protein or for the treatment and / or diagnosis of autoimmune and / or inflammatory diseases.
Otro aspecto de Ia invención Io constituye una composición biotecnológica, composición farmacéutica diagnóstica o terapéutica que comprende el anticuerpo anti-Dectin-1 de Ia invención.Another aspect of the invention constitutes a biotechnological composition, diagnostic or therapeutic pharmaceutical composition comprising the anti-Dectin-1 antibody of the invention.
Otro aspecto de Ia invención Io constituye el uso del anticuerpo o deAnother aspect of the invention constitutes the use of the antibody or
Ia composición farmacéutica de Ia invención en un procedimiento ex vivo de detección, identificación y/o cuantificación de Ia proteína anti-Dectin-1 humana, ya sea con fines de investigación o con fines de diagnóstico de una enfermedad humana.The pharmaceutical composition of the invention in an ex vivo method of detection, identification and / or quantification of the human anti-Dectin-1 protein, either for research purposes or for diagnostic purposes of a human disease.
Otro aspecto más particular de Ia presente invención es el uso del anticuerpo anti-Dectin-1 humano o de Ia composición biotecnológica de Ia invención, en un procedimiento biotecnológico ex vivo donde se requiera Ia identificación de Ia proteína Dectin-1 humana, como por ejemplo y sin que ello limite el alcance de Ia invención, en:
- ensayos de inmunofluorescencia e inmunohistoquímicaAnother more particular aspect of the present invention is the use of the human anti-Dectin-1 antibody or of the biotechnological composition of the invention, in an ex vivo biotechnological procedure where the identification of the human Dectin-1 protein is required, as for example and without limiting the scope of the invention, in: - immunofluorescence and immunohistochemical tests
- procesos de inmunoprecipitación de Ia proteína Dectin-1- Immunoprecipitation processes of the Dectin-1 protein
- ensayos in vivo para reconocer Ia proteína Dectin-1 humana en Ia superficie de monocitos, macrófagos, células de Langerhans y células dendríticas derivadas de monocitos de sangre periférica y que permitan identificar o seleccionar poblaciones que expresan Dectin-1 de poblaciones que no Ia expresan.- in vivo assays to recognize the human Dectin-1 protein on the surface of monocytes, macrophages, Langerhans cells and dendritic cells derived from peripheral blood monocytes and that allow to identify or select populations that express Dectin-1 from populations that do not express it .
- ensayos in vitro de inhibición de Ia unión de hongos, por ejemplo de Candida albicans, Saccharomyces cerevisiae, Coccidiodes posadasii, Pneumocystis carínii y Aspergillus fumigatus, al receptor- in vitro assays for the inhibition of fungal binding, for example of Candida albicans, Saccharomyces cerevisiae, Coccidiodes posadasii, Pneumocystis carínii and Aspergillus fumigatus, to the recipient
Dectin-1 expresado en transfectantes estables o transitorios de dicha proteína o en células primarias.Dectin-1 expressed in stable or transient transfectants of said protein or in primary cells.
- ensayos in vitro de inhibición de Ia producción de citoquinas mediada por Dectin-1 (por ejemplo TNF-a, IL-6, IL-10, IL-2, etc) Otro aspecto más particular de Ia presente invención es el uso del anticuerpo anti-Dectin-1 humano o de Ia composición farmacéutica diagnóstica de Ia invención en un procedimiento ex vivo de identificación de Ia proteína Dectin-1 humana, como por ejemplo y sin que limite el alcance de Ia invención, en: - Ia identificación de células patogénicas activadas en enfermedades autoinmunes y/o inflamatorias, que indique el grado de exacerbación de Ia enfermedad y permita medir Ia eficacia de los tratamientos en las fases tempranas de ensayos clínicos con nuevas terapias, - un procedimiento para determinar Ia susceptibilidad a Ia infección por patógenos fúngicos, mediante Ia identificación de cambios en Ia expresión del receptor endógeno de Ia proteína Dectin-1 humana, indicativos de Ia presencia de β-glucano en fluidos biológicos,- In vitro assays for inhibition of the production of cytokines mediated by Dectin-1 (for example TNF-a, IL-6, IL-10, IL-2, etc.) Another more particular aspect of the present invention is the use of the antibody anti-human Dectin-1 or the diagnostic pharmaceutical composition of the invention in an ex vivo method of identifying the human Dectin-1 protein, as for example and without limiting the scope of the invention, in: - the identification of cells pathogens activated in autoimmune and / or inflammatory diseases, which indicates the degree of exacerbation of the disease and allows measuring the efficacy of treatments in the early stages of clinical trials with new therapies, - a procedure to determine the susceptibility to infection by pathogens fungal, through the identification of changes in the expression of the endogenous receptor of the human Dectin-1 protein, indicative of the presence of β-glucan in biological fluids,
- Ia detección y cuantificación por citometría de Ia expresión del receptor de Ia proteína Dectin-1 humana en fagocitos activados en
patologías autoinmunes o condiciones de inflamación crónica, como- The detection and quantification by cytometry of the expression of the human Dectin-1 protein receptor in phagocytes activated in autoimmune pathologies or conditions of chronic inflammation, such as
Ia artritis psoriásica.Ia psoriatic arthritis.
Así, otro aspecto particular de Ia presente invención es el uso del anticuerpo anti-Dectin-1 humano o de Ia composición farmacéutica terapéutica de Ia invención en un procedimiento terapéutico de una enfermedad donde Ia proteína Dectin -1 humana presenta un papel etiopatogénico.Thus, another particular aspect of the present invention is the use of the human anti-Dectin-1 antibody or the therapeutic pharmaceutical composition of the invention in a therapeutic procedure of a disease where the human Dectin-1 protein has an etiopathogenic role.
El anticuerpo monoclonal anti-Dectin-1 humano de Ia invención, más concretamente el anticuerpo MGD3, podría utilizarse para Ia elaboración de composiciones farmacéuticas útiles en el tratamiento de patologías inflamatorias, ya que bloquea Ia producción de citoquinas pro- inflamatorias que se inducen a través de Dectin-1 (por ejemplo el Factor de Necrosis Tumoral, TNF-alfa) y que están involucradas en Ia patogenia, por ejemplo y sin que limite el alcance de Ia invención, de diversas formas de artritis.The human anti-Dectin-1 monoclonal antibody of the invention, more specifically the MGD3 antibody, could be used for the elaboration of pharmaceutical compositions useful in the treatment of inflammatory pathologies, since it blocks the production of pro-inflammatory cytokines that are induced through of Dectin-1 (for example, the Tumor Necrosis Factor, TNF-alpha) and that are involved in the pathogenesis, for example and without limiting the scope of the invention, of various forms of arthritis.
Así, otro objeto de Ia invención es el uso de Ia composición y/o del anticuerpo de Ia invención en el tratamiento de enfermedades inflamatorias.Thus, another object of the invention is the use of the composition and / or the antibody of the invention in the treatment of inflammatory diseases.
Otro objeto de Ia invención es el uso de Ia composición y/o del anticuerpo de Ia invención en el tratamiento de enfermedades infecciosas y/o autoinmunes.Another object of the invention is the use of the composition and / or the antibody of the invention in the treatment of infectious and / or autoimmune diseases.
Otro objeto particular de Ia invención es el uso de Ia composición y/o del anticuerpo de Ia invención en un procedimiento ex vivo para el diagnóstico de una enfermedad autoinmune, infecciosa y/o inflamatoria.Another particular object of the invention is the use of the composition and / or the antibody of the invention in an ex vivo procedure for the diagnosis of an autoimmune, infectious and / or inflammatory disease.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
Figura 1.- El anticuerpo monoclonal (mAb) MGD3 de Ia invención reconoce Dectin-1 humano en Ia superficie de transfectantes estables. Células de Ia línea celular linfoblástica humana K562 transfectadas establemente con DectiniaFlag (denominadas de ahora en adelante K562Dectin1aFlag) y células de Ia línea parental, fueron teñidas
con el mAb anti-Dectin-1 MGD3 y con el control de isotipo (lgG2a) y analizadas por citometría de flujo. En Ia figura solo se muestran las células K562Dectin1aFlag teñidas con el control de isotipo (en gris) y con el anticuerpo MGD3 (en negro). Figura 2.- El mAb MGD3 reconoce Dectin-1 humano en Ia superficie de transfectantes transitorios de Dectin-1. Las células humanas HEK293T se transfectaron transitoriamente con Dectin-1a humano y con el vector vacío (pcDNA 3.1 ) como control. A las 48 horas se realizó una citometría de flujo, tiñendo las células con el mAb anti- Dectin-1 MGD3 (línea negra) y con un control de isotipo (lgG2a, histograma gris).Figure 1.- The monoclonal antibody (mAb) MGD3 of the invention recognizes human Dectin-1 on the surface of stable transfectants. Cells of the human lymphoblastic cell line K562 stably transfected with DectiniaFlag (hereinafter referred to as K562Dectin1aFlag) and cells of the parental line, were stained with the anti-Dectin-1 MGD3 mAb and with the isotype control (lgG2a) and analyzed by flow cytometry. In the figure only K562Dectin1aFlag cells stained with the isotype control (in gray) and with the MGD3 antibody (in black) are shown. Figure 2.- The MGD3 mAb recognizes human Dectin-1 on the surface of transient transfectants of Dectin-1. Human HEK293T cells were transiently transfected with human Dectin-1a and with the empty vector (pcDNA 3.1) as a control. At 48 hours a flow cytometry was performed, staining the cells with the anti-Dectin-1 MGD3 mAb (black line) and with an isotype control (lgG2a, gray histogram).
Figura 3.- El mAb MGD3 de Ia invención reconoce específicamente a Ia proteína Dectin-1 in vivo en Ia superficie de monocitos (MO), macrófagos (M0) y células dendríticas derivadas de monocitos de sangre periférica humana (MDDC). Los MO se obtuvieron mediante purificación con bolas magnéticas acopladas a CD14 (Miltenyi Biotech). Para Ia obtención de M0, se cultivaron los MO durante 5 - 7días en presencia de 1000 U/ml GM-CSF y para obtener MDDCs se cultivaron las células con 1000 U/ml GM-CSF + 10 μg/ml IL-4. Los histogramas grises representan Ia tinción con un mAb control (X63) y Ia línea negra representan Ia tinción con el mAb anti-Dectin-1 MGD3. En Ia parte inferior de Ia figura se muestra Ia tinción obtenida por un marcador específico de cada tipo celular utilizado.Figure 3.- The MGD3 mAb of the invention specifically recognizes the Dectin-1 protein in vivo on the surface of monocytes (MO), macrophages (M0) and dendritic cells derived from human peripheral blood monocytes (MDDC). The MO were obtained by purification with magnetic balls coupled to CD14 (Miltenyi Biotech). To obtain M0, the MOs were cultured for 5-7 days in the presence of 1000 U / ml GM-CSF and to obtain MDDCs the cells were cultured with 1000 U / ml GM-CSF + 10 μg / ml IL-4. The gray histograms represent staining with a control mAb (X63) and the black line represents staining with the anti-Dectin-1 mAb MGD3. The staining obtained by a specific marker of each cell type used is shown in the lower part of the figure.
Figura 4.- El mAb MGD3 de Ia invención es capaz de reconocer Dectin-1 humano, en ensayos de Western Blot. Lisados totales de células K562Dectin1aFlag tratadas o no con tunicamicina (3 μM, 16 horas) fueron sometidos a electroforesis (SDS-PAGE) en condiciones no desnaturalizantes (30 μg de proteína celular total/carril), transferido a una membrana y revelado con el mAb MGD3. El MGD3 reconoce una banda de aproximadamente 45 kDa en los carriles de los lisados de las células K562Dectin1aFlag sin tratar con tunicamicina, correspondiente a Ia isoforma a del Dectin-1 humano. En los carriles dónde Ia muestra ha sido
tratada con tunicamicina, aparece una banda de aproximadamente 43 kDa que corresponde a esta misma isoforma tras Ia pérdida de N- glicosilaciones debidas al tratamiento.Figure 4.- The MGD3 mAb of the invention is capable of recognizing human Dectin-1, in Western Blot assays. Total lysates of K562Dectin1aFlag cells treated or not treated with tunicamycin (3 μM, 16 hours) were subjected to electrophoresis (SDS-PAGE) under non-denaturing conditions (30 μg total cell protein / lane), transferred to a membrane and revealed with the mAb MGD3. The MGD3 recognizes a band of approximately 45 kDa in the lanes of the lysates of the K562Dectin1aFlag cells untreated with tunicamycin, corresponding to the isoform a of the human Dectin-1. In the lanes where the sample has been treated with tunicamycin, a band of approximately 43 kDa appears corresponding to this same isoform after the loss of N-glycosylations due to the treatment.
Figura 5.- El mAb MGD3 es capaz de reconocer Dectin-1 humano en ensayos de inmunofluorescencia. A.- Se utilizaron células K562 WT y K562Dectin1aFlag adheridas a cristales (10 x 104 céls/cubreobjeto) recubiertos con poli-L-lisina. Las células se tiñeron con los mAb anti- Dectin-1 humano MGD3 o con un control de isotipo lgG2a, seguidos de un Ab secundario de cabra-anti-inmunoglobulinas de ratón conjugado a Alexa488 (verde). En Ia figura se muestran las imágenes obtenidas mediante microscopía de fluorescencia utilizando el control de isotipo, y el mAb MGD3. En paralelo se muestra Ia tinción de núcleos con el colorante DAPI (azul), Ia imagen del contraste diferencial (DIC) de Nomarsky y el resultado de solapar las imágenes. En verde tan sólo se observa tinción cuando se utiliza el mAb MGD3 en las células K562Dectin1aFlag. B.- Células HeLa WT y transfectadas transitoriamente con Dectin-1a-Flag, (50 x 103 células/cubreobjeto), se tiñeron con el anticuerpo anti-Dectin-1 humano MGD3, seguido de un Ab secundario de cabra-anti- inmunoglobulinas de ratón conjugado a Alexa488. Del mismo modo, se utilizó un mAb anti-Flag como control positivo de Ia expresión de Ia proteína transfectada. En Ia figura se muestran las imágenes obtenidas mediante microscopía de fluorescencia con el Ab anti-Flag y MGD3. Únicamente muestran tinción las células que expresan Dectin-1. Figura 6.- El mAb MGD3 de Ia invención es capaz de inhibir Ia unión de Candida albicans a Dectin-1 humano en ensayos de citometría de flujo de manera similar a Ia inhibición causada por el β-glucano soluble laminarina. Se estudió Ia capacidad de unión de Candida albicans a células K562 WT y a los transfectantes K562Dectin1aFlag, para Io que se incubaron las células con laminarina (500 μg/ml, SIGMA) o con el mAb MGD3 (2 μg/ml), previamente a Ia incubación con las esporas marcadas con fluoresceína. A.- Análisis por citometría de flujo del bloqueo de Ia unión de esporas a células
K562Dectin1aFlag. La línea gris oscura representa el bloqueo por laminarina y Ia línea negra representa el bloqueo por el anticuerpo monoclonal MGD3. En gris aparece Ia unión total, en ausencia de competidor. Se muestra un experimento representativo. B.- Análisis de siete experimentos de citometría realizados en los que se muestra Ia cuantificación de Ia unión (binding) y los bloqueos con el MGD3 y Ia laminarina.Figure 5.- The MGD3 mAb is capable of recognizing human Dectin-1 in immunofluorescence assays. A.- K562 WT and K562Dectin1aFlag cells adhered to crystals (10 x 10 4 cells / coverslip) coated with poly-L-lysine were used. The cells were stained with the human anti-Dectin-1 mAbs MGD3 or with a lgG2a isotype control, followed by a secondary goat-anti-immunoglobulin mouse Ab conjugated to Alexa488 (green). The figure shows the images obtained by fluorescence microscopy using the isotype control, and the MGD3 mAb. In parallel, the staining of nuclei with the DAPI dye (blue), the image of the differential contrast (DIC) of Nomarsky and the result of overlapping the images is shown. In green only staining is observed when using the MGD3 mAb in K562Dectin1aFlag cells. B.- HeLa WT cells and transiently transfected with Dectin-1a-Flag, (50 x 10 3 cells / coverslip), were stained with the human anti-Dectin-1 antibody MGD3, followed by a secondary goat-anti-immunoglobulin Ab mouse conjugated to Alexa488. In the same way, an anti-Flag mAb was used as a positive control of the expression of the transfected protein. The figure shows the images obtained by fluorescence microscopy with Ab anti-Flag and MGD3. Only cells expressing Dectin-1 show staining. Figure 6.- The MGD3 mAb of the invention is capable of inhibiting the binding of Candida albicans to human Dectin-1 in flow cytometry assays in a manner similar to the inhibition caused by soluble β-glucan laminarin. The ability to bind Candida albicans to K562 WT cells and K562Dectin1aFlag transfectants was studied, for which the cells were incubated with laminarin (500 μg / ml, SIGMA) or with the MGD3 mAb (2 μg / ml), prior to incubation with fluorescein-labeled spores. A.- Analysis by flow cytometry of blocking the binding of spores to cells K562Dectin1aFlag. The dark gray line represents the laminarin block and the black line represents the blockade by the MGD3 monoclonal antibody. The total union appears in gray, in the absence of a competitor. A representative experiment is shown. B.- Analysis of seven cytometry experiments performed in which the quantification of binding (binding) and blockages with MGD3 and laminarin are shown.
Figura 7.- El mAb MGD3 de Ia invención es capaz de inhibir Ia producción de citoquinas secretadas por células dendríticas en respuesta a esporas de Candida albicans. Se utilizaron células dendríticas humanas derivadas de monocitos de sangre periférica. Las células fueron incubadas con esporas de C. albicans (obtenidas a partir de Ia cepa CAF2). Las esporas fueron previamente inactivadas con luz ultravioleta (U. V.) para evitar su germinación. Los tratamientos de inhibición con 5 μg/ml de los anticuerpos purificados y libres de endotoxinas, anti-Dectin-1 MGD3 (blanco) y el control de isotipo lgG2a (gris), o en ausencia de tratamiento (negro), se pre-incubaron 30 min a 37° C antes de añadir las esporas. Como controles de las condiciones experimentales se utilizaron zymosan (200 μg/ml, SIGMA) y lipopolisacárido (LPS, 100 ng/ml, SIGMA). Se recogieron los sobrenadantes y se cuantificó Ia producción de TNF-α e IL-10 mediante ELISA (Immunotools y R&D respectivamente). (A) Secreción de TNF-a las 6 horas B) producción de IL-10 a las 18 horas. **P<0.01 células no tratadas versus tratadas con el anticuerpo MGD3. Los valores de P fueron calculados por el método de Mann Whitney's two tailed analysis. Los datos representan Ia media ± S. E. de los duplicados de seis diferentes experimentos. Figura 8.- El mAb MGD3 de Ia invención es capaz, en ensayos de interacciones moleculares mediante resonancia de plasmón de superficie (SPR), de capturar Ia proteína Dectin-1 recombinante humana (dominio extracelular, aminoácidos 71-247) presente en el medio condicionado por el crecimiento de células de mamífero transfectadas. Se utilizó un equipo biosensor Biacore 3000 (GE Healthcare) y un chip modelo CM5, al que se unió un anticuerpo de captura anti-inmunoglobulinas
de ratón, de forma covalente, utilizando técnicas estandarizadas de entrecruzamiento de grupos amino. Se muestran las respuestas como señal relativa de resonancia (RU) en función del tiempo en segundos. Las curvas de Ia figura corresponden a Ia sustracción entre Ia señal obtenida para el anticuerpo MGD3 y Ia señal obtenida para el anticuerpo control negativo. La línea base corresponde a Ia señal de inyección del tampón HBS, seguida por Ia inyección de los medios condicionados realizada entre los 300 y 1200 segundos. En esta fase de asociación se observa Ia captura progresiva de las dos formas solubles de Dectin-1 por el anticuerpo. Una vez finalizada Ia inyección, el sistema continúa inyectando tampón y puede observarse Ia disociación de Dectin-1 y de HA-Dectin-1-Flag del complejo formado con el anticuerpo MGD3.Figure 7.- The MGD3 mAb of the invention is capable of inhibiting the production of cytokines secreted by dendritic cells in response to Candida albicans spores. Human dendritic cells derived from peripheral blood monocytes were used. The cells were incubated with C. albicans spores (obtained from the CAF2 strain). The spores were previously inactivated with ultraviolet (UV) light to prevent germination. Inhibition treatments with 5 μg / ml of purified and endotoxin-free antibodies, anti-Dectin-1 MGD3 (white) and lgG2a isotype control (gray), or in the absence of treatment (black), were pre-incubated 30 min at 37 ° C before adding spores. As controls of the experimental conditions, zymosan (200 μg / ml, SIGMA) and lipopolysaccharide (LPS, 100 ng / ml, SIGMA) were used. Supernatants were collected and the production of TNF-α and IL-10 was quantified by ELISA (Immunotools and R&D respectively). (A) TNF-secretion at 6 o'clock B) IL-10 production at 6 o'clock. ** P <0.01 untreated cells versus treated with the MGD3 antibody. P values were calculated by the method of Mann Whitney 's two tailed analysis. The data represent the mean ± SE of the duplicates of six different experiments. Figure 8.- The MGD3 mAb of the invention is capable, in assays of molecular interactions by surface plasmon resonance (SPR), to capture the recombinant human Dectin-1 protein (extracellular domain, amino acids 71-247) present in the medium conditioned by the growth of transfected mammalian cells. A Biacore 3000 biosensor device (GE Healthcare) and a CM5 model chip were used, to which an anti-immunoglobulin capture antibody was attached mouse, covalently, using standardized cross-linking techniques of amino groups. The responses are shown as relative resonance signal (RU) as a function of time in seconds. The curves of the figure correspond to the subtraction between the signal obtained for the MGD3 antibody and the signal obtained for the negative control antibody. The baseline corresponds to the injection signal of the HBS buffer, followed by the injection of the conditioned media performed between 300 and 1200 seconds. In this phase of association, the progressive capture of the two soluble forms of Dectin-1 by the antibody is observed. Once the injection is finished, the system continues to inject buffer and the dissociation of Dectin-1 and HA-Dectin-1-Flag of the complex formed with the MGD3 antibody can be observed.
EJEMPLOS DE LA INVENCIÓN Ejemplo 1.- Obtención del anticuerpo monoclonal (mAb) MGD3EXAMPLES OF THE INVENTION Example 1.- Obtaining the monoclonal antibody (mAb) MGD3
En una primera etapa, se generó una proteína soluble con Ia secuencia de aminoácidos 71-247 del receptor Dectin-1 humano, que comprende el cuello y el dominio de reconocimiento de carbohidratos (CTLD) de dicho receptor (SEQ ID NO2). Dicha proteína se obtuvo mediante inserción de Ia secuencia génica de ADN codificante para dicho péptido soluble (SEQ ID NO1 ) en vectores de expresión de mamífero (pEF), fusionados a los dominios HA y Flag, transitoriamente transfectados mediante Ia técnica de fosfato calcico en Ia línea celular humana HEK293T. La recolección de los sobrenadantes de cultivo de dichas células eucariotas y su posterior purificación mediante técnicas de inmunoafinidad y cromatográficas, permitió obtener cantidad suficiente como para realizar Ia inmunización de ratones de Ia cepa BALB/c, siguiendo protocolos estándar de inmunización.In a first stage, a soluble protein was generated with the amino acid sequence 71-247 of the human Dectin-1 receptor, which comprises the neck and the carbohydrate recognition domain (CTLD) of said receptor (SEQ ID NO2). Said protein was obtained by inserting the gene sequence of DNA encoding said soluble peptide (SEQ ID NO1) into mammalian expression vectors (pEF), fused to the HA and Flag domains, transiently transfected by the calcium phosphate technique in Ia human cell line HEK293T. The collection of the culture supernatants of said eukaryotic cells and their subsequent purification by immunoaffinity and chromatographic techniques, allowed obtaining enough quantity to perform the immunization of mice of the BALB / c strain, following standard immunization protocols.
Tras Ia fusión, Ia selección de hibridomas se realizó por ensayos de inmuno-absorción enzimática (Enzyme-Linked ImmunoSorbent Assay, ELISA). En este caso, los antígenos inmovilizados sobre Ia fase sólida
utilizados han sido proteínas solubles de Dectin-1 humanas, purificadas y obtenidas tanto en mamíferos como en bacterias, así como diversos β- glucanos (ligandos del receptor Dectin-1 ). En estos ensayos se utilizaron los antígenos referidos como tapiz y como método de detección se añadió un anticuerpo anti-inmunoglobulinas de ratón acoplado a peroxidasa seguidos del sustrato de Ia reacción colorimétrica. De esta forma, se seleccionaron los hibridomas secretores de anticuerpos más específicos para el antígeno. Todos los hibridomas fueron testados y confirmados en paralelo por ensayos de citometría de flujo sobre transfectantes estables de Dectin-1 humano en Ia línea linfoblastoide K562 versus Ia línea parental. Del mismo modo se testó su capacidad de reconocer células humanas obtenidas a partir de sangre periférica (monocitos y derivados de ellos, células dendríticas, macrófagos y células de Langerhans). Como resultado de los tres tipos de análisis realizados se seleccionaron los mejores hibridomas entre los que destacó el hibridoma MGD3 (depositado en Ia colección de cultivos ECACC -European Collection of Animal CeII Culture- el 6 de Febrero de 2008 con Ia referencia 08020601 ) que expresa establemente inmunoglobulinas del isotipo lgG2a, correspondientes al anticuerpo monoclonal (mAb) anti-Dectin-1 humano MGD3.After the fusion, hybridoma selection was carried out by enzymatic immuno-absorption assays (Enzyme-Linked ImmunoSorbent Assay, ELISA). In this case, the antigens immobilized on the solid phase used have been soluble human Dectin-1 proteins, purified and obtained in both mammals and bacteria, as well as various β-glucans (Dectin-1 receptor ligands). In these tests the antigens referred to as tapestry were used and as a detection method a mouse anti-immunoglobulin antibody coupled to peroxidase was added followed by the substrate of the colorimetric reaction. Thus, hybridomas secreting more specific antibodies to the antigen were selected. All hybridomas were tested and confirmed in parallel by flow cytometry assays on stable transfectants of human Dectin-1 in the K562 lymphoblast line versus the parental line. Similarly, their ability to recognize human cells obtained from peripheral blood (monocytes and derivatives thereof, dendritic cells, macrophages and Langerhans cells) was tested. As a result of the three types of analysis, the best hybridomas were selected, among which the MGD3 hybridoma (deposited in the ECACC -European Collection of Animal CeII Culture- crop collection was selected on February 6, 2008 with reference 08020601) which expresses stably immunoglobulins of the lgG2a isotype, corresponding to the human anti-Dectin-1 monoclonal antibody (mAb) MGD3.
Ejemplo 2.- El anticuerpo monoclonal MGD3 de Ia invención reconoce Dectin-1 humano en Ia superficie de transfectantes estables de Dectin-1 humano.Example 2.- The MGD3 monoclonal antibody of the invention recognizes human Dectin-1 on the surface of stable transfectants of human Dectin-1.
Se utilizaron células de Ia línea celular linfoblástica humana K562 transfectadas establemente con un vector con el cDNA de Dectin-1a- humano acoplado al epítopo reportero FLAG (K562Dectin1aFlag) y células parentales, fueron teñidas con el mAb anti-Dectin-1 MGD3 y con control de isotipo (lgG2a) y analizadas por citometría de flujo. El patrón de tinción permitió comprobar que el mAb MGD3 reconoce específicamente Dectin-1 humano ya que tiñe específicamente las células transfectadas K562Dectin1aFlag y no las células parentales (Figura 1 ).
Ejemplo 3.- El anticuerpo monoclonal MGD3 de Ia invención reconoce Dectin-1 humano en Ia superficie de transfectantes transitorios de Dectin-1 humano.K562 human lymphoblastic cell line cells stably transfected with a vector with the human Dectin-1a-cDNA coupled to the FLAG reporter epitope (K562Dectin1aFlag) and parental cells were stained with the anti-Dectin-1 MGD3 mAb and with control of isotype (lgG2a) and analyzed by flow cytometry. The staining pattern allowed us to verify that the MGD3 mAb specifically recognizes human Dectin-1 as it specifically stains the K562Dectin1aFlag transfected cells and not the parental cells (Figure 1). Example 3.- The MGD3 monoclonal antibody of the invention recognizes human Dectin-1 on the surface of transient transfectants of human Dectin-1.
El resultado obtenido en el ejemplo 2 no descarta Ia posibilidad de que el hibridoma MGD3 pudiera estar secretando anticuerpos que reconozcan el epítopo FLAG. Para comprobar que el hibridoma MGD3 tan solo producía mAb anti-Dectin-1 se utilizó otras células humanas (HEK293T, derivadas de riñon embrionario) que se transfectaron transitoriamente con el cDNA completo de Dectin-1a humano sin FLAG o con el vector vacío (pcDNA 3.1 ) como control. A las 48 horas se realizó una citometría de flujo, tiñendo las células con el mAb anti- Dectin-1 MGD3 y con un control de isotipo. El patrón de tinción de membrana obtenido, tanto en los transfectantes estables de Dectin-1 con el epítopo Flag, como en los transfectantes transitorios que carecen de él, permitió comprobar que el mAb MGD3 de Ia invención reconoce específicamente Dectin-1 humano y no reconoce el epítopo Flag (Figura 2).The result obtained in Example 2 does not rule out the possibility that the MGD3 hybridoma could be secreting antibodies that recognize the FLAG epitope. To verify that the hybridoma MGD3 produced only anti-Dectin-1 mAb, other human cells (HEK293T, derived from embryonic kidney) were used that were transiently transfected with the complete human Dectin-1a cDNA without FLAG or with the empty vector (pcDNA 3.1) as control. At 48 hours a flow cytometry was performed, staining the cells with the anti-Dectin-1 MGD3 mAb and with an isotype control. The membrane staining pattern obtained, both in the stable transfectants of Dectin-1 with the Flag epitope, and in the transient transfectants that lack it, allowed to verify that the MGD3 mAb of the invention specifically recognizes human Dectin-1 and does not recognize the Flag epitope (Figure 2).
Ejemplo 4.- El anticuerpo monoclonal MGD3 reconoce específicamente Ia proteína ex vivo en Ia superficie de monocitos (MO), macrófagos (M0) y células dendríticas derivadas de monocitos de sangre periférica (MDDC).Example 4.- The monoclonal antibody MGD3 specifically recognizes the ex vivo protein on the surface of monocytes (MO), macrophages (M0) and dendritic cells derived from peripheral blood monocytes (MDDC).
Se aislaron células mononucleares de sangre periférica (PBMCs) humanas a partir de concentrados de células sanguíneas exentos de plaquetas {buffy coats) de donantes sanos mediante gradientes de densidad obtenidos utilizando medios de separación de linfocitos (Figura 3). Los monocitos se obtuvieron mediante purificación con bolas magnéticas acopladas a CD14 (Miltenyi Biotech). Para Ia obtención de MDDCs y M0, se cultivan los monocitos durante 7 días en presencia de 1000 U/ml GM-CSF para los M0, y 1000 U/ml GM-CSF + 10 μg/ml IL-4 para las MDDCs. Debido a Ia gran variabilidad de las muestras procedentes de diferentes individuos, en Ia figura 3 se muestran los
perfiles de citometría de un solo donante representativo. Los patrones de tinción muestran una expresión semejante de Dectin-1 en Ia membrana de los tres tipos celulares. En Ia parte inferior se muestra Ia expresión de marcadores moleculares característicos de cada tipo celular.Human peripheral blood mononuclear cells (PBMCs) were isolated from platelet-free blood cells concentrates from healthy donors by density gradients obtained using lymphocyte separation media (Figure 3). Monocytes were obtained by purification with magnetic balls coupled to CD14 (Miltenyi Biotech). To obtain MDDCs and M0, monocytes are cultured for 7 days in the presence of 1000 U / ml GM-CSF for M0, and 1000 U / ml GM-CSF + 10 μg / ml IL-4 for MDDCs. Due to the great variability of the samples from different individuals, Figure 3 shows the Cytometric profiles of a single representative donor. Staining patterns show a similar expression of Dectin-1 in the membrane of the three cell types. In the lower part the expression of molecular markers characteristic of each cell type is shown.
Ejemplo 5.- El mAb MGD3 de Ia invención es capaz de reconocer Dectin-1 humano en ensayos de Western BlotExample 5.- The MGD3 mAb of the invention is capable of recognizing human Dectin-1 in Western Blot assays.
Se realizó un Western Blot en condiciones no reductoras con usados totales de Ia línea celular humana establemente transfectada K562Dectin1aFlag y el correspondiente control con las células parentales. Para inhibir las N-glicosilaciones las células fueron tratadas con tunicamicina 3 μM durante 16 horas. Los usados (30 μg de proteína celular total/carril), fueron sometidos a electroforesis (SDS-PAGE) en condiciones no desnaturalizantes, transferidos a una membrana de nitrocelulosa. Tras el bloqueo de Ia membrana (TBS-T ween 0,1 %, 5% leche desnatada en polvo y 2% BSA) se procedió a Ia hibridación de Ia misma durante Ia noche a 40C con el mAb anti-Dectin-1 humano MGD3 de Ia invención a 2 μg/ml. Tras los lavados correspondientes, Ia membrana se incubó con un Ab secundario de cabra-anti-inmunoglobulinas de ratón acoplado a peroxidasa y se reveló con un sustrato quimioluminiscente. El mAb MGD3 de Ia invención reconoció dos bandas: una de 45 kDa aprox. correspondiente a Ia isoforma a del Dectin-1 humano presente en Ia muestra de células transfectadas K562Dectin1aFlag sin tratar y otra de 43 kDa aproximadamente que corresponde a esta misma isoforma tras el tratamiento con tunicamicina (Figura 4).Western blotting was performed under non-reducing conditions with total used of the stably transfected human cell line K562Dectin1aFlag and the corresponding control with the parental cells. To inhibit N-glycosylations the cells were treated with 3 μM tunicamycin for 16 hours. The used ones (30 μg of total cellular protein / lane) were subjected to electrophoresis (SDS-PAGE) under non-denaturing conditions, transferred to a nitrocellulose membrane. After blocking the membrane (TBS-T ween 0.1%, 5% skimmed milk powder and 2% BSA), it was hybridized overnight at 4 0 C with the anti-Dectin-1 mAb Human MGD3 of the invention at 2 μg / ml. After the corresponding washes, the membrane was incubated with a secondary goat-anti-immunoglobulin mouse Ab coupled to peroxidase and revealed with a chemiluminescent substrate. The MGD3 mAb of the invention recognized two bands: one of approximately 45 kDa. corresponding to the isoform a of the human Dectin-1 present in the sample of untreated K562Dectin1aFlag transfected cells and another approximately 43 kDa corresponding to this same isoform after tunicamycin treatment (Figure 4).
Ejemplo 6.- El mAb MGD3 es capaz de reconocer Dectin-1 humano en ensayos de inmunofluorescencia.Example 6.- The MGD3 mAb is capable of recognizing human Dectin-1 in immunofluorescence assays.
Se utilizaron células K562Dectin1 aFlag en suspensión, adheridas a cristales (10 x 103 céls/cubreobjeto) recubiertos de poli-L-lisina. Las células se fijaron con paraformaldehído 3,7% y tras los lavados y bloqueos
pertinentes se tiñeron con el anticuerpo anti-Dectin-1 humano MGD3, seguido de un Ab de cabra-anti-inmunoglobulinas de ratón conjugado a Alexa488. Del mismo modo, se usó un mAb de isotipo lgG2a como control. La figura 5A muestra las imágenes obtenidas mediante microscopía de fluorescencia para el control de isotipo y el mAb MGD3. Tan solo se detecta señal en verde en las células K562Dectin1aFlag teñidas con el mAb MGD3 Io que indica que el receptor Dectin-1a -FLAG está siendo reconocido específicamente.K562Dectin1 aFlag cells in suspension, adhered to crystals (10 x 10 3 cells / coverslip) coated with poly-L-lysine, were used. The cells were fixed with 3.7% paraformaldehyde and after washing and blocking relevant stained with the human anti-Dectin-1 antibody MGD3, followed by a goat Ab-mouse anti-immunoglobulin conjugated to Alexa488. Similarly, a lgG2a isotype mAb was used as a control. Figure 5A shows the images obtained by fluorescence microscopy for isotype control and the MGD3 mAb. Only green signal is detected in K562Dectin1aFlag cells stained with the MGD3 Io mAb which indicates that the Dectin-1a -FLAG receptor is being specifically recognized.
Estos datos se confirmaron utilizando otra línea celular humana adherente (HeLa, epiteliales de ovario), transfectada transitoriamente con Dectin-1a-Flag. Las células fueron crecidas sobre cristales (50 x 103 células/cubreobjeto) y fijadas con paraformaldehído 3,7%. Tras los lavados y bloqueos pertinentes se tiñeron con el anticuerpo anti-Dectin-1 humano MGD3 o con el mAb anti-Flag que reconoce el epítopo FLAG como control positivo, seguidos de un Ab secundario cabra-anti-inmunoglobulinas de ratón acoplado a Alexa488. En Ia Figura 5B aparecen las imágenes obtenidas con el microscopio de fluorescencia donde se comprueba como el mAb MGD3 tiñe Dectin-1 con similar intensidad que el Ab anti-Flag el epítopo FLAG.These data were confirmed using another adherent human cell line (HeLa, ovarian epithelial), transiently transfected with Dectin-1a-Flag. The cells were grown on crystals (50 x 10 3 cells / coverslip) and fixed with 3.7% paraformaldehyde. After the relevant washes and blockages, they were stained with the human anti-Dectin-1 antibody MGD3 or with the anti-Flag mAb that recognizes the FLAG epitope as a positive control, followed by a secondary goat-anti-immunoglobulin mouse Ab coupled to Alexa488. In Figure 5B the images obtained with the fluorescence microscope appear where it is checked how the MGD3 mAb stains Dectin-1 with similar intensity as the Ab anti-Flag FLAG epitope.
Ejemplo 7.- El mAb MGD3 es capaz de inhibir Ia unión de Candida albicans a Dectin-1 humano en ensayos de citometría de flujo de manera similar a Ia inhibición causada por el β-glucano soluble laminarina. Se estudió Ia capacidad de unión de Candida albicans (cepa CAF-2) a los transfectantes K562Dectin1aFlag. Para ello se utilizaron esporas de C. albicans marcadas con fluoresceína (FITC) incubadas en una relación célula/espora de 1/10 durante 30 min. Para determinar Ia capacidad del mAb MGD3 de bloquear esta unión, se preincubaron las células con laminarina (500 μg/ml), o con el mAb MGD3 de Ia invención (2 μg/ml) durante 20 min., antes de Ia incubación con las esporas. El análisis de Ia unión y bloqueo de
Ia misma se realizó por citometría de flujo (Figura 6A). Los resultados indican que el anticuerpo MGD3 inhibe totalmente Ia unión especifica de C. albicans mediada por Dectin-1 puesto que Ia incubación con el anticuerpo disminuye Ia unión a niveles aún inferiores a los que presentan las células parentales. En este ensayo el anticuerpo MGD3 tiene Ia misma capacidad de inhibir Ia unión de esporas a Dectin-1 que el beta-glucano soluble laminarina (Figura 6B).Example 7.- The MGD3 mAb is capable of inhibiting the binding of Candida albicans to human Dectin-1 in flow cytometry assays in a manner similar to the inhibition caused by soluble laminarin β-glucan. The binding capacity of Candida albicans (strain CAF-2) to transfectants K562Dectin1aFlag was studied. For this, fluorescein-labeled C. albicans spores (FITC) incubated in a cell / spore ratio of 1/10 were used for 30 min. To determine the ability of the MGD3 mAb to block this binding, the cells were pre-incubated with laminarin (500 μg / ml), or with the MGD3 mAb of the invention (2 μg / ml) for 20 min., Before incubation with the spores The analysis of the union and blocking of It was performed by flow cytometry (Figure 6A). The results indicate that the MGD3 antibody totally inhibits the specific binding of C. albicans mediated by Dectin-1 since incubation with the antibody decreases the binding to levels even lower than those presented by the parental cells. In this test, the MGD3 antibody has the same ability to inhibit the binding of spores to Dectin-1 as the laminarin soluble beta-glucan (Figure 6B).
Ejemplo 8.- El mAb MGD3 es capaz de inhibir Ia producción de TNF-α e IL-10 en células dendríticas humanas derivadas de monocitos en respuesta a esporas de Candida albicansExample 8.- The MGD3 mAb is capable of inhibiting the production of TNF-α and IL-10 in human dendritic cells derived from monocytes in response to Candida albicans spores
Para estimular Ia producción de citoquinas, se incubaron las MDDC con esporas de C .albicans inactivadas por luz U. V. Para determinar Ia capacidad del mAb MGD3 de bloquear dicha secreción, se preincubaron las células con los mAb MGD3 de Ia invención (5 μg/ml) o con el control de isotipo (lgG2a, 5 μg/ml) durante 30 min., antes de Ia incubación con las esporas. Se recogió sobrenadante a las 6 y a las 18 horas. La cuantificación de las citoquinas presentes en el sobrenadante se realizó mediante ensayos de ELISA. Los resultados indican que el anticuerpo MGD3 inhibe significativamente tanto Ia producción de TNF-α Fig. 7 Acornó Ia de IL-1 OFig. 7B inducidas por las esporas de C. albicans en las MDDC. Esta inhibición es específica puesto que el control de isotipo (lgG2a) no produce una disminución significativa en Ia secreción de ninguna de las dos citoquinas analizadas. En cuánto a los controles positivos, el anticuerpo MGD3 no bloquea de forma significativa Ia secreción de TNF-q producida por zymosan seguramente debido a que este estímulo induce elevados niveles de secreción de TNF-α mediada por Ia existencia de otros receptores (ej. TLR2 y receptor de mañosa) que también reconocen esta partícula (constituida además de por β-glucanos, por chitina, mananos y lípidos). En el caso del LPS Ia disminución de Ia secreción tampoco es significativa, resultado esperado puesto que el receptor implicado es el TLR4, no Dectin-1.
Ejemplo 9.- El mAb MGD3 es capaz, en ensayos de interacciones moleculares mediante resonancia de plasmón de superficie (SPR), de capturar Dectin-1 humano recombinante presente en el medio condicionado por el crecimiento de células de mamífero transfectadas. Se estudió Ia capacidad de interacción entre el anticuerpo MGD3 deTo stimulate the production of cytokines, MDDCs were incubated with C. Albicans spores inactivated by UV light. To determine the ability of the MGD3 mAb to block said secretion, the cells were pre-incubated with the MGD3 mAbs of the invention (5 μg / ml) or with the isotype control (lgG2a, 5 μg / ml) for 30 min., before incubation with the spores. Supernatant was collected at 6 and 18 hours. The quantification of the cytokines present in the supernatant was performed by ELISA assays. The results indicate that the MGD3 antibody significantly inhibits both the production of TNF-α Fig. 7 Closed Ia of IL-1 OFig. 7B induced by C. albicans spores in the MDDC. This inhibition is specific since the isotype control (lgG2a) does not produce a significant decrease in the secretion of either of the two cytokines analyzed. As for the positive controls, the MGD3 antibody does not significantly block the secretion of TNF-q produced by zymosan surely because this stimulus induces high levels of TNF-α secretion mediated by the existence of other receptors (eg TLR2 and mañosa receptor) that also recognize this particle (constituted in addition to β-glucans, chitin, mannan and lipids). In the case of LPS, the decrease in secretion is not significant, an expected result since the receptor involved is TLR4, not Dectin-1. Example 9.- The MGD3 mAb is able, in molecular interaction assays by surface plasmon resonance (SPR), to capture recombinant human Dectin-1 present in the medium conditioned by the growth of transfected mammalian cells. The interaction capacity between the MGD3 antibody of
Ia invención y proteínas solubles de Dectin-1. Para ello, se transfectaron transitoriamente células HEK293T bien con Ia porción extracelular (aminoácidos 71-247, dominio Dectin-1/71-247) del receptor humano Dectin- 1 o bien con esta misma porción extracelular fusionada a los epítopos HA y Flag (HA-Dectin-1a-Flag) (SEQ ID NO2). Los sobrenadantes recolectados se purificaron mediante técnicas de inmunoafinidad y cromatográficas.The invention and soluble proteins of Dectin-1. For this, HEK293T cells were transiently transfected either with the extracellular portion (amino acids 71-247, domain Dectin-1 / 71-247) of the human receptor Dectin-1 or with this same extracellular portion fused to the HA and Flag (HA epitopes) -Dectin-1a-Flag) (SEQ ID NO2). The collected supernatants were purified by immunoaffinity and chromatographic techniques.
Se utilizó un equipo biosensor Biacore 3000 (GE Healthcare) y un chip modelo CM5, al que se unió un anticuerpo de captura anti-inmunoglobulinas de ratón, de forma covalente, utilizando técnicas estandarizadas de entrecruzamiento de grupos amino. Se utilizaron en paralelo dos celdas de flujo, en una se inyectó sobrenadante que contenía un anticuerpo control inespecífico y en Ia otra el anticuerpo MGD3, a una concentración de 15 μg/ml y a una velocidad de 5 μl/min, en tampón HBS (150 mN NaCI, Hepes 10 mM, pH 7.4). En un nuevo ciclo, realizado a continuación, se inyectó el sobrenadante de células transfectadas con los plásmidos control "mock" (verde), Dectin-1 (azul) y Dectin-1-Flag+HA (roja).A Biacore 3000 biosensor device (GE Healthcare) and a CM5 model chip were used, to which a mouse anti-immunoglobulin capture antibody was attached, covalently, using standardized cross-linking techniques of amino groups. Two flow cells were used in parallel, in one supernatant containing a nonspecific control antibody was injected and in the other the MGD3 antibody, at a concentration of 15 μg / ml and at a rate of 5 μl / min, in HBS buffer (150 mN NaCI, 10 mM Hepes, pH 7.4). In a new cycle, performed next, the supernatant of cells transfected with the control plasmids "mock" (green), Dectin-1 (blue) and Dectin-1-Flag + HA (red) was injected.
En Ia Figura 8 se muestran las respuestas como señal relativa de resonancia (RU) en función del tiempo en segundos. Las curvas de Ia figura corresponden a Ia sustracción entre Ia señal obtenida para el anticuerpo MGD3 y Ia señal obtenida para el anticuerpo control negativo. La línea base corresponde a Ia señal de inyección del tampón HBS, seguida por Ia inyección de los medios condicionados realizada entre los 300 y 1200 segundos. En esta fase de asociación se observa Ia captura progresiva de las dos formas solubles de Dectin-1 por el anticuerpo. Una vez finalizada Ia inyección, el sistema continúa inyectando tampón y puede observarse Ia
disociación de Dectin-1 y de HA-Dectin-1a-Flag del complejo formado con el anticuerpo MGD3.Figure 8 shows the responses as a relative resonance signal (RU) as a function of time in seconds. The curves of the figure correspond to the subtraction between the signal obtained for the MGD3 antibody and the signal obtained for the negative control antibody. The baseline corresponds to the injection signal of the HBS buffer, followed by the injection of the conditioned media performed between 300 and 1200 seconds. In this phase of association, the progressive capture of the two soluble forms of Dectin-1 by the antibody is observed. Once the injection is finished, the system continues to inject buffer and the Ia can be observed dissociation of Dectin-1 and HA-Dectin-1a-Flag from the complex formed with the MGD3 antibody.
Los resultados indican que el anticuerpo MGD3 de Ia invención captura de forma específica a las proteínas solubles Dectin-1 y HA-Dectin- 1a-Flag presentes en el medio de cultivo condicionado por el crecimiento de los transfectantes HEK293T. Estos resultados indican que este anticuerpo puede ser utilizado, en biosensores basados en Ia técnica SPR, como componente fundamental de ensayos de rastreo y caracterización de Ia interacción entre Dectin-1 y diferentes ligandos.
The results indicate that the MGD3 antibody of the invention specifically captures the soluble proteins Dectin-1 and HA-Dectin-1a-Flag present in the culture medium conditioned by the growth of HEK293T transfectants. These results indicate that this antibody can be used, in biosensors based on the SPR technique, as a fundamental component of screening assays and characterization of the interaction between Dectin-1 and different ligands.
Claims
1.- Anticuerpo anti-Dectin-1 humano caracterizado porque se une y reconoce específicamente el dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO2) ya sea un anticuerpo monoclonal o policlonal.1.- Human anti-Dectin-1 antibody characterized in that it specifically binds and recognizes domain 71-247 of the human protein Dectin-1 (SEQ ID NO2) either a monoclonal or polyclonal antibody.
2.- Anticuerpo según las reivindicación 1 caracterizado porque es monoclonal y es producido por el hibridoma MGD3 (depositado en Ia colección de cultivos ECACC -European Collection of Animal CeII Culture- el 6 de Febrero de 2008 con Ia referencia 08020601 ).2. Antibody according to claim 1 characterized in that it is monoclonal and is produced by the MGD3 hybridoma (deposited in the ECACC culture collection - European Collection of Animal CeII Culture - on February 6, 2008 with reference 08020601).
3.- Secuencia de nucleótidos caracterizada por ser codificante del dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO1 ) y por ser seleccionada del siguiente grupo: d) una secuencia de nucleótidos análoga a Ia SEQ ID NO1 , e) un fragmento de Ia secuencias de a, y f) una secuencia de nucleótidos que comprende una secuencia cualquiera perteneciente de a) y b).3.- Nucleotide sequence characterized by being coding for domain 71-247 of the human protein Dectin-1 (SEQ ID NO1) and being selected from the following group: d) a nucleotide sequence analogous to SEQ ID NO1, e) a fragment of the sequences of a, and f) a nucleotide sequence comprising any sequence belonging to a) and b).
4.- Secuencia de aminoácidos aislada de Ia proteína humana Dectin-1 caracterizada porque se corresponde con el dominio 71-247 de ésta (SEQ ID NO2) y por ser seleccionada del siguiente grupo: d) una secuencia de aminoácidos análoga a Ia SEQ ID NO2 e) un fragmento de Ia secuencia de a, y f) una secuencia de aminoácidos que comprende una secuencia cualquiera perteneciente de a) y b).4.- Isolated amino acid sequence of the human protein Dectin-1 characterized in that it corresponds to domain 71-247 thereof (SEQ ID NO2) and for being selected from the following group: d) an amino acid sequence analogous to SEQ ID NO2 e) a fragment of the sequence of a, and f) an amino acid sequence comprising any sequence belonging to a) and b).
5.- Uso de Ia secuencia de nucleótidos según Ia reivindicación 3 en Ia producción de anticuerpos anti-Dectin-1 humanos, ya sean monoclonales o policlonales. 5. Use of the nucleotide sequence according to claim 3 in the production of human anti-Dectin-1 antibodies, whether monoclonal or polyclonal.
6.- Uso de Ia secuencia de aminoácidos según Ia reivindicación 5 en Ia producción de anticuerpos anti-Dectin-1 humanos, ya sean monoclonales6. Use of the amino acid sequence according to claim 5 in the production of human anti-Dectin-1 antibodies, whether monoclonal
0 policlonales.0 polyclonal.
7.- Hibridoma caracterizado porque produce un anticuerpo específico del dominio 71-247 de Ia proteína humana Dectin-1 (SEQ ID NO2).7.- Hybridoma characterized in that it produces an antibody specific for domain 71-247 of the human protein Dectin-1 (SEQ ID NO2).
8.- Hibridoma según Ia reivindicación 7 caracterizado porque es el hibridoma MGD3 productor del anticuerpo MGD3 (depositado en Ia colección de cultivos ECACC -European Collection of Animal CeII Culture- el 6 de Febrero de 2008 con Ia referencia 08020601 ).8. Hybridoma according to claim 7 characterized in that it is the MGD3 hybridoma producing the MGD3 antibody (deposited in the ECACC culture collection -European Collection of Animal CeII Culture- on February 6, 2008 with reference 08020601).
9.- Uso de hibridoma según las reivindicaciones 7 y 8 para Ia producción del anticuerpo monoclonal anti-Dectin-1 humano, preferentemente el anticuerpo monoclonal .MGD3.9. Use of hybridoma according to claims 7 and 8 for the production of the human anti-Dectin-1 monoclonal antibody, preferably the .MGD3 monoclonal antibody.
10.- Uso del anticuerpo según reivindicaciones 1 y 2 para Ia elaboración de una composición biotecnológica, farmacéutica diagnóstica o terapéutica útil para Ia realización de: - ensayos de detección, identificación y/o cuantificación de Ia proteína Dectin-1 humana,10.- Use of the antibody according to claims 1 and 2 for the elaboration of a biotechnological, pharmaceutical diagnostic or therapeutic composition useful for the performance of: - detection, identification and / or quantification assays of the human Dectin-1 protein,
- tratamiento de enfermedades infecciosas, autoinmunes y/o inflamatorias, y- treatment of infectious, autoimmune and / or inflammatory diseases, and
- diagnóstico de enfermedades infecciosas, autoinmunes y/o inflamatorias.- diagnosis of infectious, autoimmune and / or inflammatory diseases.
11.- Composición biotecnológica, farmacéutica diagnostica o terapéutica caracterizada porque comprende un anticuerpo según las reivindicaciones11.- Biotechnological, pharmaceutical or diagnostic pharmaceutical composition characterized in that it comprises an antibody according to the claims
1 y 2. 1 and 2.
12.- Uso de Ia composición según Ia reivindicación 11 y del anticuerpo según Ia reivindicación 1 y 2 en un procedimiento ex vivo de detección, identificación y/o cuantificación de Ia proteína Dectin-1 humana.12. Use of the composition according to claim 11 and the antibody according to claim 1 and 2 in an ex vivo method of detection, identification and / or quantification of the human Dectin-1 protein.
13.- Uso de Ia composición según Ia reivindicación 11 y del anticuerpo según Ia reivindicación 1 y 2 en un procedimiento terapéutico de una enfermedad humana donde Ia proteína Dectin-1 humana presenta un papel etiopatogénico.13.- Use of the composition according to claim 11 and the antibody according to claim 1 and 2 in a therapeutic procedure of a human disease wherein the human Dectin-1 protein has an etiopathogenic role.
14.- Uso según Ia reivindicación 13 caracterizado porque Ia enfermedad es una enfermedad inflamatoria, infecciosa y/o autoinmune.14. Use according to claim 13 characterized in that the disease is an inflammatory, infectious and / or autoimmune disease.
15.- Uso de Ia composición según Ia reivindicación 11 y del anticuerpo según Ia reivindicación 1 y 2 en un procedimiento ex vivo para el diagnóstico de una enfermedad autoinmune, infecciosa y/o inflamatoria. 15.- Use of the composition according to claim 11 and the antibody according to claim 1 and 2 in an ex vivo procedure for the diagnosis of an autoimmune, infectious and / or inflammatory disease.
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