WO2021119858A1 - Method for generating allergen-specific tolerogenic cells, tolerogenic cells obtained, and methods for inducing allergen tolerance by means of autologous immunotherapy - Google Patents
Method for generating allergen-specific tolerogenic cells, tolerogenic cells obtained, and methods for inducing allergen tolerance by means of autologous immunotherapy Download PDFInfo
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- WO2021119858A1 WO2021119858A1 PCT/CL2019/050147 CL2019050147W WO2021119858A1 WO 2021119858 A1 WO2021119858 A1 WO 2021119858A1 CL 2019050147 W CL2019050147 W CL 2019050147W WO 2021119858 A1 WO2021119858 A1 WO 2021119858A1
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- cells
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Classifications
-
- 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
- A61K39/35—Allergens
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- 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
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/464839—Allergens
-
- 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
- A61P37/08—Antiallergic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention in general terms relates to a method for treating various allergic diseases. More particularly, a method is disclosed for the generation of allergen-specific tolerogenic dendritic cells, to be applied as autologous immunotherapy in allergic diseases. Also described are allergen-specific tolerogenic dendritic cells obtained, and methods for inducing tolerance to allergens.
- compositions and methods for treating various allergic diseases are described in the state of the art.
- the prevalence of food allergy is increasing.
- there are no standardized and validated curative therapies for food allergy and strict avoidance of the offending food remains the only therapeutic option.
- the Allergen immunotherapy has been used successfully to treat asthma, allergic rhinitis, and hypersensitivity to bee and wasp venom.
- TI is based on the delivery of increasing doses of specific allergens over time with the aim of inducing desensitization and immune tolerance.
- the patent of invention WO 2019/076477 discloses a pharmaceutical composition for the induction of food tolerance by means of a combination of subcutaneous immunotherapy based on hydrogel (phase A composition) and subsequent oral (OIT) or sublingual (SLIT) immunotherapy ( phase composition B).
- the phase A composition comprises a thermogelling hydrogel for release of embedded components comprising a) one or more allergen peptides which is specific for T cells, b) a tolerance promoting dose of synthetic oligodeoxynucleotides (ODN) containing one or more CpG or GpC or GpG motifs, c) one or more molecules integrated into hydrogel to attract peripheral antigen presenting cells (APC) to the site of the administered hydrogel composition, and d) optionally one or more immune modulators that promote tolerance; wherein the Phase B composition comprises one or more modified or unmodified food allergens modified, including natural and recombinant food allergens or their fragments that serve as epitopes for B cells. Additionally, the application of molecules in hydrogel compositions for Phase A to attract peripheral antigen presenting cells (APCs) including cells is described. dendritic cells (DC) and macrophages, to the injection site.
- ODN synthetic oligodeoxynucleotides
- APC peripheral antigen
- patent application WO 2018/146274 describes a gastrointestinal release capsule to desensitize and / or induce tolerance in a patient with peanut allergy.
- Said capsule comprises a shell and a core, where the core comprises a composition comprising peanuts, at least one oil, at least one first powdered vehicle and optionally at least one second prebiotic excipient.
- the capsule is supplied and swallowed in an unopened form and is not mixed with the food bolus.
- the mucosa of the small intestine possesses numerous specialized antigen-presenting cells, in particular macrophage-like mononuclear phagocytes or dendritic cells.
- the patent application for invention WO 2019/038668 describes a method and composition to induce tolerance to allergens in a patient suffering from atopic allergy or allergic asthma; thus reducing allergy symptoms in these patients, where the invention comprises providing a human milk oligosaccharide for use in inducing allergen tolerance in a patient suffering from atopic allergy and / or allergic asthma, as well as a synthetic composition comprising one or more oligosaccharides from human milk and one or more of an immunotherapeutic allergen and / or an active source of vitamin A.
- compositions that comprises a delivery vehicle that includes an immunoconjugate, in which the immunoconjugate comprises an immunomodulatory agent covalently linked to an antigen, in which said antigen comprises a tumor antigen. Also provided is a composition comprising an antigen covalently linked to an immunomodulatory compound such as a tolerogen or an adjuvant.
- a delivery device comprising the composition of the present invention and a dendritic cell (DC) recruitment composition is provided.
- composition comprising (i) a first population of synthetic nanocarriers that are coupled to immunosuppressants, and (ii) a second population of synthetic nanocarriers that are coupled to MHC Class restricted epitopes. II of an antigen that generates an unwanted humoral immune response. It is specified that "MHC class II restricted epitopes" are epitopes that are presented to immune cells by MHC class II molecules found on antigen presenting cells (APC), eg professional immune cells that present antigen, such as macrophages, B cells, and dendritic cells.
- APC antigen presenting cells
- the patent application for invention WO 2019/160979 discloses a composition that selectively inhibits the interaction between an Fe receptor type I or an Fe receptor type II, FcRn and an immunocomplexed antibody, where the composition comprises a first binding domain that binds specifically to a human Fe type I receptor or a human Fe type II receptor; and a second binding domain that binds specifically to a human FcRn.
- FcRn plays an important role in MFIC class II antigen presentation and MFIC class I cross-presentation of IgG complex antigen.
- dendritic cells When antigen is presented as an immune complex (Cl) containing IgG, dendritic cells that are CD8-CD1 Ib + CDI le + (inflammatory dendritic cells) show significant cross-presentation at low doses of antigen in a highly dependent pathway. of FcRn expression.
- Invention patent US 9,603,800 teaches a method for treating a subject with an inflammatory, allergic or autoimmune disease or disorder that comprises administering to the subject a composition comprising an effective amount of nanolipogels comprising: a polymeric matrix formed by a polymer crosslinked, an amphiphilic polymer, a block copolymer, or a three-block copolymer that has been dispersed therein or covalently bonded to one or more host molecules that reversibly complexed with an agent, where the host molecules are selected from the group consisting of one or more polysaccharides, cryptands, cryptophanes, cavitants, crown ethers, dendrimers, catenanes, carcerands, spherands, carbon nanotubes and fullerenes, for the controlled release of at least one therapeutic, diagnostic or prophylactic agent, and a shell lipid; wherein one or more active agents to reduce, lessen, or alleviate one or more symptoms of inflammatory, allergic, or autoimmune disease or
- the present invention relates to methods for treating various allergic diseases. More particularly, a method is disclosed for the generation of allergen-specific tolerogenic cells, to be applied as immunotherapy. autologous in allergic diseases. Also described are allergen-specific tolerogenic cells, and methods for inducing allergen tolerance to develop an autologous immunotherapy.
- the method described in the present invention comprises the following steps: a) obtaining a blood sample from a subject with an allergy to a certain allergen; b) obtaining mononuclear cells from peripheral blood and culturing said cells in appropriate media; c) adding a sufficient quantity of IL-4 and GM-CSF on the cultured cells; d) stimulating the cultured cells with sufficient amounts of Retinoic Acid and Dexamethasone; e) co-culturing the cells of the previous steps with a sufficient amount of a purified extract of the allergen; f) analyzing the cells from the previous steps; and g) obtaining allergen-specific tolerogenic cells.
- Figure 1 shows that treatment with dexamethasone and retinoic acid does not cause cell death in moDCs.
- FIG. 2 shows a flow cytometric analysis of CD40 and HLA-DR in moDC stimulated with Dexamethasone and Retinoic Acid.
- the graphs show the expression of activation markers in monocyte-derived dendritic cells (moDCs) cultured with dexamethasone (Dex) and retinoic acid (RA), with or without post-stimulation for 48 hours with lipopolysaccharide (LPS).
- Graph A) describes the expression of HLA-DR;
- Graph B) shows the expression of CD40;
- Graph C) shows the expression of CD86; and
- graph D) shows the expression of CD83.
- FIG. 3 shows the secretion of IL-10, TGF-b, IL-4 and IL-12 by moDC stimulated with Dexamethasone and Retinoic acid.
- the graphs show that the secretion of cytokines by dendritic cells derived from monocytes (moDCs) cultured with dexamethasone (Dex) and retinoic acid (AR), with or without subsequent stimulation for 48 hours with lipopolysaccharide (LPS), measured by ELISA of the supernatant of cell cultures.
- Graph A) shows IL-10 secretion;
- Graph B) shows TGF-b secretion;
- the graph shows the C) secretion of IL-4; and
- Graph D) shows IL-12 secretion.
- n 5.
- Figure 4 shows a proliferation assay T cell Na ⁇ 've cocultured with moDC previously stimulated with retinoic acid and dexamethasone.
- Graph A teaches representative Dot-plot naive T cells stained with CFSE and co-cultured with moDC for 5 days. moDC were stimulated with dexamethasone (Dex) and retinoic acid (AR), with or without subsequent stimulation for 48 hours with lipopolysaccharide (LPS).
- the present invention refers to a process for the generation of allergen-specific tolerogenic dendritic cells, to be applied as autologous immunotherapy in allergic diseases.
- Said procedure is characterized by comprising the following steps: a) obtaining a blood sample from a subject with an allergy to a certain allergen; b) obtaining mononuclear cells from peripheral blood and culturing said cells in appropriate media; c) adding a sufficient quantity of IL-4 and GM-CSF on the cultured cells; d) stimulate the cultured cells with sufficient amounts of retinoic acid and
- Dexamethasone e) co-cultivating the cells of the previous steps with a sufficient amount of a purified extract of the allergen; f) analyzing the cells from the previous steps; Y g) obtain allergen-specific tolerogenic cells.
- step a) comprises obtaining a blood sample through venipuncture, phlebotomy or leukapheresis.
- step b) comprises obtaining peripheral blood mononuclear cells by dilution of the blood and subsequent density gradient with ficoll.
- the appropriate media for culturing the cells of step b) are selected from AIM-V culture medium, RPMI RPMI supplemented with human serum, or other means known to a person skilled in the art. technique, in which the culture conditions comprise between 35 to 39 ° C, with 5% CO2.
- the sufficient amount of IL-4 and GM-CSF from step c) comprises between 500-2000 IU / mL.
- the sufficient amounts of Retinoic Acid and Dexamethasone from step d) comprise between 0.5-10 uM of Retinoic Acid and between 0.5-10 uM of Dexamethasone.
- the sufficient amount of the purified extract of the allergen from step e) comprises between 10 to 50 ug / mL.
- the allergen of stage e) is selected from among peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables.
- step f) comprises analyzing the cells obtained by flow cytometry, ELISA, fluorescence microscopy, and confocal microscopy.
- a method for inducing tolerance to allergens by means of autologous immunotherapy, wherein said method comprises injecting the cells obtained from claims 1 to 10, to a subject with an allergy to a certain allergen.
- the allergen is selected from peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables.
- a tolerogenic cell specific to a certain allergen is described to be applied as autologous immunotherapy in allergic diseases, where said cell is obtained by the procedure described in the present invention, and is characterized by having tolerance to a certain allergen, where the allergen It is selected from peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables.
- Example N ° 1 Obtaining tolerogenic cells specific to allergens.
- PBMC peripheral blood mononuclear cells
- PBMC peripheral blood mononuclear cells
- the cells adhered to the plate were incubated for 7 days in AIM-V medium to which IL-4 and GM-CSF were added at a concentration of 1000 IU / mL. Every 2 days 1/3 of the culture medium was removed and replaced by 1/2 fresh medium with the same concentration of IL-4 and GM-CSF.
- cells were stimulated with 1 uM Retinoic Acid (AR) and 1 uM Dexamethasone (Dex).
- AR Retinoic Acid
- Dex Dexamethasone
- the cells were co-cultured with purified Peanut extract (PE) at a concentration of 50ug / mL or 10 ug / ml Ara h2 for 48 hours.
- DCs dendritic cell
- monocyte-derived DCs were then co-cultured with PE or Ara h2 for 2 days, after which they were reanalyzed by Facs for the same markers mentioned above and then re-injected into the patient.
- moDCs monocyte-derived DCs
- LPS lipopolysaccharide
- RA retinoic acid
- Dex dexamethasone
- the measurement of IL-10 shows that moDC LPS secrete a greater amount of IL-10 as well as moDC AR + Dex + LPS, which also secretion of IL-10 is greater than its counterpart moDC AR Dex.
- the TGF-b results show no differences between any of the groups.
- the results of IL-4 and IL-10 and TGF-b in moDC AR Dex LPS, would induce a tolerogenic response for T lymphocytes.
- IL-12 secretion was not detected in moDC AR Dex LPS, indicating that there would be no response to activate other cells.
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Abstract
The present invention relates to methods for treating different allergic diseases. More specifically, a method is disclosed for generating allergen-specific tolerogenic cells for use as an autologous immunotherapy in allergic diseases. Also described are the allergen-specific tolerogenic cells obtained and methods for inducing allergen tolerance by means of autologous immunotherapy.
Description
Procedimiento para la generación de células tolerogénicas específicas a alérgenos, células tolerogénicas obtenidas, y procedimientos para inducir la tolerancia a alérgenos mediante inmunoterapia autóloga ÁMBITO DE LA INVENCIÓN Procedure for the generation of allergen-specific tolerogenic cells, obtained tolerogenic cells, and procedures for inducing allergen tolerance by means of autologous immunotherapy. SCOPE OF THE INVENTION
La presente invención en términos generales, se refiere a un procedimiento para tratar diversas enfermedades alérgicas. Más particularmente, se divulga un procedimiento para la generación de células dendríticas tolerogénicas alérgeno-específicas, para ser aplicadas como inmunoterapia autóloga en enfermedades alérgicas. También se describen células dendríticas tolerogénicas alérgeno-específicas obtenidas, y procedimientos para inducir la tolerancia a alérgenos. The present invention in general terms relates to a method for treating various allergic diseases. More particularly, a method is disclosed for the generation of allergen-specific tolerogenic dendritic cells, to be applied as autologous immunotherapy in allergic diseases. Also described are allergen-specific tolerogenic dendritic cells obtained, and methods for inducing tolerance to allergens.
ANTECEDEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
En el estado de la técnica se describen composiciones y procedimientos para tratar diversas enfermedades alérgicas. En la actualidad, la prevalencia de alergia alimentaria es cada vez mayor. Al día de hoy no existen terapias curativas estandarizadas y validadas para la alergia alimentaria y la evitación estricta del alimento causal sigue siendo la única opción terapéutica. Dada la prevalencia cada vez mayor de alergia alimentaria, la dificultad de evitar los alimentos involucrados, las posibles consecuencias graves de las reacciones accidentales y que los pacientes con alergia alimentaria rara vez superan su alergia, es que en los últimos años ha surgido una importante investigación para lograr inducir la tolerancia inmunológica. La
inmunoterapia con alérgenos (IT) se ha utilizado con éxito para tratar el asma, la rinitis alérgica y la hipersensibilidad al veneno de abejas y avispas. La IT se basa en el suministro de dosis crecientes de alérgenos específicos a lo largo del tiempo con el objetivo de inducir la desensibilización y la tolerancia inmunológica. Sobre la base de la eficacia probada de la IT para otras enfermedades alérgicas, se han probado diferentes tipos de estrategias de IT para el tratamiento de la alergia alimentaria. Los primeros intentos de usar la IT para la alergia alimentaria fue por medio de la IT subcutánea, lo que dio como resultado una alta tasa de reacciones sistémicas, causando que esta estrategia no fuese utilizable. Así, se abandonó este enfoque. Por lo tanto, estudios posteriores se han centrado en rutas alternativas de administración de proteínas alergénicas, que incluyen la inducción de tolerancia oral (OIT), IT sublingual (SLIT) y, más recientemente, IT epicutánea (EPIT). Por ejemplo, la patente de invención WO 2019/076477 divulga una composición farmacéutica para la inducción de tolerancia alimentaria mediante una combinación de inmunoterapia subcutánea a base de hidrogel (composición de fase A) y posterior inmunoterapia oral (OIT) o sublingual (SLIT) (composición de fase B). La composición de fase A comprende un hidrogel termogelificante para liberación de componentes embebidos que comprenden a) uno o más péptidos del alérgeno el cual es específico para células T, b) una dosis promotora de tolerancia de oligodesoxinucleótidos sintéticos (ODN) que contienen uno o más motivos CpG o GpC o GpG, c) una o más moléculas integradas en hidrogel para atraer células presentadoras de antígeno (APC) periféricas al sitio de la composición de hidrogel administrada, y d) opcionalmente uno o más moduladores inmunes que promueven la tolerancia; en donde la composición de Fase B comprende uno o más alérgenos alimentarios modificados o no
modificados, incluidos los alérgenos alimentarios naturales y recombinantes o sus fragmentos que sirven como epítopos para células B. Adicionalmente, se describe la aplicación de moléculas en las composiciones de hidrogel para la Fase A para atraer células presentadoras de antígeno periféricas (APC) que incluyen células dendríticas (DC) y macrófagos, al sitio de inyección. Compositions and methods for treating various allergic diseases are described in the state of the art. At present, the prevalence of food allergy is increasing. To date, there are no standardized and validated curative therapies for food allergy, and strict avoidance of the offending food remains the only therapeutic option. Given the increasing prevalence of food allergy, the difficulty of avoiding the foods involved, the potentially serious consequences of accidental reactions, and that food allergy patients rarely outgrow their allergy, significant research has emerged in recent years. to induce immune tolerance. The Allergen immunotherapy (IT) has been used successfully to treat asthma, allergic rhinitis, and hypersensitivity to bee and wasp venom. TI is based on the delivery of increasing doses of specific allergens over time with the aim of inducing desensitization and immune tolerance. Based on the proven efficacy of IT for other allergic diseases, different types of IT strategies have been tried for the treatment of food allergy. The first attempts to use IT for food allergy were by means of subcutaneous IT, which resulted in a high rate of systemic reactions, rendering this strategy unusable. Thus, this approach was abandoned. Therefore, subsequent studies have focused on alternative routes of administration of allergenic proteins, including oral tolerance induction (OIT), sublingual IT (SLIT) and, more recently, epicutaneous IT (EPIT). For example, the patent of invention WO 2019/076477 discloses a pharmaceutical composition for the induction of food tolerance by means of a combination of subcutaneous immunotherapy based on hydrogel (phase A composition) and subsequent oral (OIT) or sublingual (SLIT) immunotherapy ( phase composition B). The phase A composition comprises a thermogelling hydrogel for release of embedded components comprising a) one or more allergen peptides which is specific for T cells, b) a tolerance promoting dose of synthetic oligodeoxynucleotides (ODN) containing one or more CpG or GpC or GpG motifs, c) one or more molecules integrated into hydrogel to attract peripheral antigen presenting cells (APC) to the site of the administered hydrogel composition, and d) optionally one or more immune modulators that promote tolerance; wherein the Phase B composition comprises one or more modified or unmodified food allergens modified, including natural and recombinant food allergens or their fragments that serve as epitopes for B cells. Additionally, the application of molecules in hydrogel compositions for Phase A to attract peripheral antigen presenting cells (APCs) including cells is described. dendritic cells (DC) and macrophages, to the injection site.
Por otra parte, la solicitud de patente WO 2018/146274, describe una cápsula de liberación gastrointestinal para desensibilizar y / o inducir tolerancia en un paciente con alergia al maní. Dicha cápsula comprende una envoltura y un núcleo, donde el núcleo comprende una composición que comprende maní, al menos un aceite, al menos un primer vehículo en polvo y opcionalmente al menos un segundo excipiente prebiótico. La cápsula se suministra e ingiere en una forma no abierta y no se mezcla con el bolo alimenticio. Se menciona además que la mucosa del intestino delgado posee numerosas células especializadas presentadoras de antígeno, en particular fagocitos mononucleares de tipo macrófago o células dendríticas. Por otra parte, la solicitud de patente de invención WO 2019/038668, describe un método y composición para inducir tolerancia a alérgenos en un paciente que sufre de alergia atópica o asma alérgica; reduciendo así los síntomas de alergia en estos pacientes, donde la invención comprende proporcionar un oligosacárido de leche humana para su uso en la inducción de tolerancia a alérgenos en un paciente que sufre de alergia atópica y / o asma alérgica, así como también se divulga una composición sintética que comprende uno o más oligosacáridos de la leche humana y uno o más de un alérgeno inmunoterapéutico y / o una fuente activa de vitamina A.
La solicitud de patente de invención WO 2016/161372, enseña una composición que comprende un vehículo de suministro que incluye un inmunoconjugado, en el que el inmunoconjugado comprende un agente inmunomodulador unido covalentemente a un antígeno, en el que dicho antígeno comprende un antígeno tumoral. También se proporciona una composición que comprende un antígeno unido covalentemente a un compuesto inmunomodulador tal como un tolerógeno o un adyuvante. En la memoria descriptiva se menciona que se proporciona un dispositivo de administración que comprende la composición de la presente invención y una composición de reclutamiento de células dendríticas (DC). Además, la solicitud de patente WO 2012/149259, describe una composición que comprende (i) una primera población de nanoportadores sintéticos que están acoplados a inmunosupresores, y (ii) una segunda población de nanoportadores sintéticos que están acoplados a epítopos restringidos a MHC Clase II de un antígeno que genera una respuesta inmune humoral no deseada. Se especifica que los "epítopos restringidos de MHC de clase II" son epítopos que se presentan a las células inmunes mediante moléculas de MHC de clase II que se encuentran en las células presentadoras de antígeno (APC), por ejemplo, en las células inmunes profesionales que presentan antígeno, como los macrófagos, las células B, y células dendríticas.On the other hand, patent application WO 2018/146274 describes a gastrointestinal release capsule to desensitize and / or induce tolerance in a patient with peanut allergy. Said capsule comprises a shell and a core, where the core comprises a composition comprising peanuts, at least one oil, at least one first powdered vehicle and optionally at least one second prebiotic excipient. The capsule is supplied and swallowed in an unopened form and is not mixed with the food bolus. It is further mentioned that the mucosa of the small intestine possesses numerous specialized antigen-presenting cells, in particular macrophage-like mononuclear phagocytes or dendritic cells. On the other hand, the patent application for invention WO 2019/038668, describes a method and composition to induce tolerance to allergens in a patient suffering from atopic allergy or allergic asthma; thus reducing allergy symptoms in these patients, where the invention comprises providing a human milk oligosaccharide for use in inducing allergen tolerance in a patient suffering from atopic allergy and / or allergic asthma, as well as a synthetic composition comprising one or more oligosaccharides from human milk and one or more of an immunotherapeutic allergen and / or an active source of vitamin A. The patent application for invention WO 2016/161372, teaches a composition that comprises a delivery vehicle that includes an immunoconjugate, in which the immunoconjugate comprises an immunomodulatory agent covalently linked to an antigen, in which said antigen comprises a tumor antigen. Also provided is a composition comprising an antigen covalently linked to an immunomodulatory compound such as a tolerogen or an adjuvant. In the specification it is mentioned that a delivery device comprising the composition of the present invention and a dendritic cell (DC) recruitment composition is provided. Furthermore, patent application WO 2012/149259, describes a composition comprising (i) a first population of synthetic nanocarriers that are coupled to immunosuppressants, and (ii) a second population of synthetic nanocarriers that are coupled to MHC Class restricted epitopes. II of an antigen that generates an unwanted humoral immune response. It is specified that "MHC class II restricted epitopes" are epitopes that are presented to immune cells by MHC class II molecules found on antigen presenting cells (APC), eg professional immune cells that present antigen, such as macrophages, B cells, and dendritic cells.
La solicitud de patente de invención WO 2019/160979, divulga una composición que inhibe selectivamente la interacción entre un receptor Fe tipo I o un receptor Fe tipo II, FcRn y un anticuerpo inmunocomplejado, donde la composición comprende un primer dominio de unión que se une específicamente a un receptor Fe tipo I humano o un receptor Fe tipo II humano; y un segundo dominio de unión que se une
específicamente a un FcRn humano. En la memoria descriptiva se señala que FcRn juega un papel importante en la presentación del antígeno MFIC de clase II y la presentación cruzada de MFIC clase I del antígeno complejo IgG. Cuando el antígeno se presenta como un complejo inmunitario (Cl) que contiene IgG, las células dendríticas que son CD8-CD1 Ib + CDI le + (células dendríticas inflamatorias) muestran una presentación cruzada significativa a bajas dosis de antígeno en una vía que depende mucho de la expresión de FcRn. The patent application for invention WO 2019/160979, discloses a composition that selectively inhibits the interaction between an Fe receptor type I or an Fe receptor type II, FcRn and an immunocomplexed antibody, where the composition comprises a first binding domain that binds specifically to a human Fe type I receptor or a human Fe type II receptor; and a second binding domain that binds specifically to a human FcRn. In the specification it is noted that FcRn plays an important role in MFIC class II antigen presentation and MFIC class I cross-presentation of IgG complex antigen. When antigen is presented as an immune complex (Cl) containing IgG, dendritic cells that are CD8-CD1 Ib + CDI le + (inflammatory dendritic cells) show significant cross-presentation at low doses of antigen in a highly dependent pathway. of FcRn expression.
La patente de invención US 9.603.800 enseña un método para tratar a un sujeto con una enfermedad o trastorno inflamatorio, alérgico o autoinmune que comprende administrar al sujeto una composición que comprende una cantidad eficaz de nanolipogeles que comprende: una matriz polimérica formada por un polímero reticulado, un polímero anfifílico, un copolímero de bloque o un copolímero de tres bloques que se ha dispersado en el mismo o unido covalentemente a uno o más moléculas huésped que forman un complejo de forma reversible con un agente, donde las moléculas huésped se seleccionan del grupo que consiste de uno o más polisacáridos, criptandos, criptofanos, cavitantes, éteres corona, dendrímeros, catenanos, carcerands, spherands, nanotubos de carbono y fullerenos, para la liberación controlada de al menos un agente terapéutico, diagnóstico o profiláctico, y una cubierta lipídica; en donde uno o más agentes activos para reducir, disminuir o aliviar uno o más síntomas de la enfermedad o trastorno inflamatorio, alérgico o autoinmune se complejizan o se unen de manera reversible a las moléculas huésped, se dispersan dentro de la matriz polimérica, se dispersan o se unen a la cubierta lipídica, o combinaciones de los mismos.
Por otra parte, el documento no patente titulado “Role of dendritic cells in peanut allerg y”, divulga factores relacionados con la adquisición de tolerancia oral a las proteínas de maní, enfocando la discusión a células dendríticas intestinales y los factores que determinan el desarrollo de la alergia al maní, así como la evidencia reciente del efecto de nuevos enfoques de tratamiento en las células dendríticas.Invention patent US 9,603,800 teaches a method for treating a subject with an inflammatory, allergic or autoimmune disease or disorder that comprises administering to the subject a composition comprising an effective amount of nanolipogels comprising: a polymeric matrix formed by a polymer crosslinked, an amphiphilic polymer, a block copolymer, or a three-block copolymer that has been dispersed therein or covalently bonded to one or more host molecules that reversibly complexed with an agent, where the host molecules are selected from the group consisting of one or more polysaccharides, cryptands, cryptophanes, cavitants, crown ethers, dendrimers, catenanes, carcerands, spherands, carbon nanotubes and fullerenes, for the controlled release of at least one therapeutic, diagnostic or prophylactic agent, and a shell lipid; wherein one or more active agents to reduce, lessen, or alleviate one or more symptoms of inflammatory, allergic, or autoimmune disease or disorder become complexed or reversibly bind to host molecules, disperse within the polymeric matrix, disperse or they bind to the lipid shell, or combinations thereof. On the other hand, the non-patent document entitled "Role of dendritic cells in peanut allerg y", discloses factors related to the acquisition of oral tolerance to peanut proteins, focusing the discussion on intestinal dendritic cells and the factors that determine the development of peanut allergy, as well as recent evidence of the effect of new treatment approaches on dendritic cells.
Sin embargo, ninguno de los documentos de la técnica anterior, describe un procedimiento para la generación de células dendríticas tolerogénicas específicas a alérgenos, para ser aplicadas como inmunoterapia autóloga en enfermedades alérgicas. Actualmente no existe tratamiento para la alergia alimentaria, por lo que la presente invención busca resolver este problema, mediante un procedimiento para la generación de células dendríticas tolerogénicas específicas a alérgenos, particularmente para tratar en forma autóloga alergia alimentaria a maní u otros alimentos, utilizando células dendríticas autólogas tolerogénicas. Adicionalmente, no existen estudios que demuestren el efecto combinado de dos compuestos como es el ácido retinoico y la dexametasona, para la generación de células dendríticas tolerogénicas como una inmunoterapia en enfermedades alérgicas, y específicamente en alergia al maní, no han sido desarrolladas utilizando la mezcla de ambos compuestos. RESUMEN DE LA INVENCIÓN However, none of the prior art documents describe a process for the generation of allergen-specific tolerogenic dendritic cells, to be applied as autologous immunotherapy in allergic diseases. Currently there is no treatment for food allergy, so the present invention seeks to solve this problem, by means of a process for the generation of allergen-specific tolerogenic dendritic cells, particularly to autologously treat food allergy to peanuts or other foods, using cells tolerogenic autologous dendritic. Additionally, there are no studies that demonstrate the combined effect of two compounds such as retinoic acid and dexamethasone, for the generation of tolerogenic dendritic cells as an immunotherapy in allergic diseases, and specifically in peanut allergy, they have not been developed using the mixture. of both compounds. SUMMARY OF THE INVENTION
La presente invención se refiere a procedimientos para tratar diversas enfermedades alérgicas. Más particularmente, se divulga un procedimiento para la generación de células tolerogénicas específicas a alérgenos, para ser aplicadas como inmunoterapia
autóloga en enfermedades alérgicas. También se describen células tolerogénicas específicas a alérgenos, y procedimientos para inducir la tolerancia a alérgenos para desarrollar una inmunoterapia autóloga. El procedimiento descrito en la presente invención, comprende las siguientes etapas: a) obtener una muestra de sangre de un sujeto con alergia a un determinado alérgeno; b) obtener células mononucleares de sangre periférica y cultivar dichas células en medios apropiados; c) añadir una cantidad suficiente de IL-4 y GM-CSF sobre las células cultivadas; d) estimular las células cultivadas con cantidades suficientes de Ácido retinoico y Dexametasona; e) co-cultivar las células de los pasos anteriores con una cantidad suficiente de un extracto purificado del alérgeno; f) analizar las células de los pasos anteriores; y g) obtener células tolerogénicas alérgeno-específicas. The present invention relates to methods for treating various allergic diseases. More particularly, a method is disclosed for the generation of allergen-specific tolerogenic cells, to be applied as immunotherapy. autologous in allergic diseases. Also described are allergen-specific tolerogenic cells, and methods for inducing allergen tolerance to develop an autologous immunotherapy. The method described in the present invention comprises the following steps: a) obtaining a blood sample from a subject with an allergy to a certain allergen; b) obtaining mononuclear cells from peripheral blood and culturing said cells in appropriate media; c) adding a sufficient quantity of IL-4 and GM-CSF on the cultured cells; d) stimulating the cultured cells with sufficient amounts of Retinoic Acid and Dexamethasone; e) co-culturing the cells of the previous steps with a sufficient amount of a purified extract of the allergen; f) analyzing the cells from the previous steps; and g) obtaining allergen-specific tolerogenic cells.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
La Figura 1 muestra que el tratamiento con dexametasona y ácido retinoico no causa muerte celular en moDCs. Los gráficos enseñan que la viabilidad celular evaluada mediante citometría de flujo utilizando la sonda LIVE/DEAD cell viability de células
dendríticas derivadas de monocitos (moDCs) cultivadas con dexametasona (Dex) y ácido retinoico (RA), con o sin estímulo posterior por 48 horas con lipopolisacárido (LPS). Error estándar (SEM) p<0,05. n=5. Figure 1 shows that treatment with dexamethasone and retinoic acid does not cause cell death in moDCs. The graphs show that cell viability assessed by flow cytometry using the LIVE / DEAD cell viability probe of cells dendritic cells derived from monocytes (moDCs) cultured with dexamethasone (Dex) and retinoic acid (RA), with or without subsequent stimulation for 48 hours with lipopolysaccharide (LPS). Standard error (SEM) p <0.05. n = 5.
La Figura 2 muestra un análisis de citometría de flujo de CD40 y HLA-DR en moDC estimuladas con Dexametasona y Ácido retinoico. Los gráficos enseñan que la expresión de marcadores de activación en células dendríticas derivadas de monocitos (moDCs) cultivadas con dexametasona (Dex) y ácido retinoico (RA), con o sin estímulo posterior por 48 horas con lipopolisacárido (LPS). El gráfico A) describe la expresión de HLA-DR; El gráfico B) muestra la expresión de CD40; El gráfico C) enseña la expresión de CD86; y el gráfico D) muestra la expresión de CD83. Expresado como las veces de aumento de la intensidad de fluorescencia (MFI) en relación a las moDCs sin estímulo (SE) (fold change), medido por citometría flujo (FACS). Error estándar (SEM) *p<0,05, **p<0,01. n=5. Figure 2 shows a flow cytometric analysis of CD40 and HLA-DR in moDC stimulated with Dexamethasone and Retinoic Acid. The graphs show the expression of activation markers in monocyte-derived dendritic cells (moDCs) cultured with dexamethasone (Dex) and retinoic acid (RA), with or without post-stimulation for 48 hours with lipopolysaccharide (LPS). Graph A) describes the expression of HLA-DR; Graph B) shows the expression of CD40; Graph C) shows the expression of CD86; and graph D) shows the expression of CD83. Expressed as the times of increase in fluorescence intensity (MFI) in relation to moDCs without stimulus (SE) (fold change), measured by flow cytometry (FACS). Standard error (SEM) * p <0.05, ** p <0.01. n = 5.
La Figura 3 muestra la secreción de IL-10, TGF-b, IL-4 e IL-12 por moDC estimuladas con Dexametasona y Ácido retinoico. Los gráficos enseñan que la secreción de citoquinas por células dendríticas derivadas de monocitos (moDCs) cultivadas con dexametasona (Dex) y ácido retinoico (AR), con o sin estímulo posterior por 48 horas con lipopolisacárido (LPS), medido mediante ELISA del sobrenadante de los cultivos celulares. El gráfico A) enseña la secreción de IL-10; El gráfico B) muestra la secreción de TGF-b; El gráfico enseña la C) secreción de IL-4; y El gráfico D) muestra secreción de IL-12. Error estándar (SEM) * p<0,05. n=5. Figure 3 shows the secretion of IL-10, TGF-b, IL-4 and IL-12 by moDC stimulated with Dexamethasone and Retinoic acid. The graphs show that the secretion of cytokines by dendritic cells derived from monocytes (moDCs) cultured with dexamethasone (Dex) and retinoic acid (AR), with or without subsequent stimulation for 48 hours with lipopolysaccharide (LPS), measured by ELISA of the supernatant of cell cultures. Graph A) shows IL-10 secretion; Graph B) shows TGF-b secretion; The graph shows the C) secretion of IL-4; and Graph D) shows IL-12 secretion. Standard error (SEM) * p <0.05. n = 5.
La Figura 4 muestra un ensayo de proliferación de células T naí've co-cultivadas con moDC previamente estimuladas con Dexametasona y Acido retinoico. El gráfico A)
enseña Dot-plot representativos células T naive teñidas con CFSE y co-cultivadas con moDC por 5 días. moDC fueron estimuladas con dexametasona (Dex) y ácido retinoico (AR), con o sin estímulo posterior por 48 horas con lipopolisacárido (LPS). El gráfico B) muestra la proliferación en unidades arbitrarias (a.u) y normalizado respecto al sin estimulo (SE). SE y LPS, n=3.DEX+AR y DEX+AR+LPS, n=2. Figure 4 shows a proliferation assay T cell Naí've cocultured with moDC previously stimulated with retinoic acid and dexamethasone. Graph A) teaches representative Dot-plot naive T cells stained with CFSE and co-cultured with moDC for 5 days. moDC were stimulated with dexamethasone (Dex) and retinoic acid (AR), with or without subsequent stimulation for 48 hours with lipopolysaccharide (LPS). Graph B) shows proliferation in arbitrary units (au) and normalized with respect to that without stimulus (SE). SE and LPS, n = 3.DEX + AR and DEX + AR + LPS, n = 2.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La presente invención se refiere a un procedimiento para la generación de células dendríticas tolerogénicas alergeno-específicas, para ser aplicadas como inmunoterapia autóloga en enfermedades alérgicas. Dicho procedimiento se caracteriza por comprender las siguientes etapas: a) obtener una muestra de sangre de un sujeto con alergia a un determinado alérgeno; b) obtener células mononucleares de sangre periférica y cultivar dichas células en medios apropiados; c) añadir una cantidad suficiente de IL-4 y GM-CSF sobre las células cultivadas; d) estimular las células cultivadas con cantidades suficientes de Ácido retinoico yThe present invention refers to a process for the generation of allergen-specific tolerogenic dendritic cells, to be applied as autologous immunotherapy in allergic diseases. Said procedure is characterized by comprising the following steps: a) obtaining a blood sample from a subject with an allergy to a certain allergen; b) obtaining mononuclear cells from peripheral blood and culturing said cells in appropriate media; c) adding a sufficient quantity of IL-4 and GM-CSF on the cultured cells; d) stimulate the cultured cells with sufficient amounts of retinoic acid and
Dexametasona; e) co-cultivar las células de los pasos anteriores con una cantidad suficiente un extracto purificado del alérgeno; f) analizar las células de los pasos anteriores; y
g) obtener células tolerogénicas alergeno-específicas. Dexamethasone; e) co-cultivating the cells of the previous steps with a sufficient amount of a purified extract of the allergen; f) analyzing the cells from the previous steps; Y g) obtain allergen-specific tolerogenic cells.
En una de las modalidades de la presente invención, la etapa a) comprende obtener una muestra de sangre a través de venopunción, flebotomía o leucoferesis. In one of the embodiments of the present invention, step a) comprises obtaining a blood sample through venipuncture, phlebotomy or leukapheresis.
En una de las modalidades de la presente invención, la etapa b) comprende obtener células mononucleares de sangre periférica mediante dilución de la sangre y posterior gradiente de densidad con ficoll. In one of the embodiments of the present invention, step b) comprises obtaining peripheral blood mononuclear cells by dilution of the blood and subsequent density gradient with ficoll.
En una de las modalidades de la presente invención, los medios apropiados para cultivar las células de la etapa b) se seleccionan entre medio de cultivo AIM-V, RPMI RPMI complementado con suero humano, u otros medios conocidos por una persona versada en la materia técnica, en el cual las condiciones de cultivo comprenden entre 35 a 39 °C, con un 5% de CO2. In one of the embodiments of the present invention, the appropriate media for culturing the cells of step b) are selected from AIM-V culture medium, RPMI RPMI supplemented with human serum, or other means known to a person skilled in the art. technique, in which the culture conditions comprise between 35 to 39 ° C, with 5% CO2.
En una de las modalidades de la presente invención, la cantidad suficiente de IL-4 y GM-CSF de la etapa c) comprende entre 500 - 2000 UI/mL. In one of the embodiments of the present invention, the sufficient amount of IL-4 and GM-CSF from step c) comprises between 500-2000 IU / mL.
En una de las modalidades de la presente invención, las cantidades suficientes de Ácido retinoico y Dexametasona de la etapa d) comprende entre 0,5 - 10 uM de Ácido retinoico y entre 0,5 - 10 uM de Dexametasona. In one of the embodiments of the present invention, the sufficient amounts of Retinoic Acid and Dexamethasone from step d) comprise between 0.5-10 uM of Retinoic Acid and between 0.5-10 uM of Dexamethasone.
En una de las modalidades de la presente invención, la cantidad suficiente del extracto purificado del alérgeno de la etapa e) comprende entre 10 a 50 ug/mL. In one of the embodiments of the present invention, the sufficient amount of the purified extract of the allergen from step e) comprises between 10 to 50 ug / mL.
En una de las modalidades de la presente invención, el alérgeno de la etapa e) se selecciona entre maní, huevo, leche, trigo, gluten, soya, frutos secos (nueces, almendras), pescados, mariscos, látex, lupino, frutas, verduras.
En una de las modalidades de la presente invención, la etapa f) comprende analizar las células obtenidas por citometría de flujo, ELISA, microscopía de fluorescencia, microscopía confocal. In one of the embodiments of the present invention, the allergen of stage e) is selected from among peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables. In one of the embodiments of the present invention, step f) comprises analyzing the cells obtained by flow cytometry, ELISA, fluorescence microscopy, and confocal microscopy.
Además, se describe un procedimiento para inducir la tolerancia a alérgenos mediante inmunoterapia autóloga, donde dicho procedimiento comprende inyectar las células obtenidas de las reivindicaciones 1 a 10, a un sujeto con alergia a un determinado alérgeno. En una de las modalidades de la presente invención, el alérgeno se selecciona entre maní, huevo, leche, trigo, gluten, soya, frutos secos (nueces, almendras), pescados, mariscos, látex, lupino, frutas, verduras. Finalmente, se describe una célula tolerogénica específica a un determinado alérgeno para ser aplicada como inmunoterapia autóloga en enfermedades alérgicas, donde dicha célula es obtenida por el procedimiento descrito en la presente invención, y se caracteriza por tener tolerancia a un determinado alérgeno, donde el alérgeno se selecciona entre maní, huevo, leche, trigo, gluten, soya, frutos secos (nueces, almendras), pescados, mariscos, látex, lupino, frutas, verduras. Furthermore, a method is described for inducing tolerance to allergens by means of autologous immunotherapy, wherein said method comprises injecting the cells obtained from claims 1 to 10, to a subject with an allergy to a certain allergen. In one of the embodiments of the present invention, the allergen is selected from peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables. Finally, a tolerogenic cell specific to a certain allergen is described to be applied as autologous immunotherapy in allergic diseases, where said cell is obtained by the procedure described in the present invention, and is characterized by having tolerance to a certain allergen, where the allergen It is selected from peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables.
EJEMPLOS EXAMPLES
Ejemplo N° 1 : Obtención de células tolerogénicas específicas a alérgenos. Example N ° 1: Obtaining tolerogenic cells specific to allergens.
En primer lugar, se procedió a obtener muestras de sangre por punción venosa de un paciente con alergia al maní. First, blood samples were obtained by venipuncture from a patient with a peanut allergy.
Posteriormente, se diluyó la sangre en buffer fosfato salino 1x (PBS 1x) en una proporción de 1 :1 , a esta dilución se realizó un gradiente de densidad con ficoll a una
proporción 1 :3, se centrifugó a 1000 g x 25 min para obtener células mononucleares de sangre periférica (PBMC). Subsequently, the blood was diluted in 1x phosphate saline buffer (1x PBS) in a ratio of 1: 1, at this dilution a density gradient with ficoll was performed at a 1: 3 ratio, centrifuged at 1000 x 25 min to obtain peripheral blood mononuclear cells (PBMC).
Se recuperaron los PBMC ubicadas en la interfase del gradiente y se cultivaron en una placa de 6 pocilios a una concentración de 10.000.000 células/mL por 2 horas a 37QC y 5% CO2 en medio de cultivo AIM-V. Luego se retiraron las células que no se adhirieron a la placa. PBMC were recovered located at the interface of the gradient and cultured in a 6 - well plate at a concentration of 10 million cells / mL for 2 hours at 37 Q C and 5% CO2 in culture medium AIM-V. Cells that did not adhere to the plate were then removed.
Las células adheridas a la placa (monocitos) fueron incubados por 7 días en medio AIM-V al cual se le agregó IL-4 y GM-CSF a una concentración de 1000 UI/mL. Cada 2 días se retiró 1/3 del medio de cultivo y fue reemplazado por 1/2 de medio fresco con igual concentración de IL-4 y GM-CSF. El día 4, las células fueron estimuladas con 1 uM Acido retinoico (AR) y 1 uM de Dexametasona (Dex). El día 5 de cultivo las células fueron co-cultivadas con extracto purificado de Maní (PE) a una concentración de 50ug/mL o 10 ug/ml Ara h2 por 48 horas. The cells adhered to the plate (monocytes) were incubated for 7 days in AIM-V medium to which IL-4 and GM-CSF were added at a concentration of 1000 IU / mL. Every 2 days 1/3 of the culture medium was removed and replaced by 1/2 fresh medium with the same concentration of IL-4 and GM-CSF. On day 4, cells were stimulated with 1 uM Retinoic Acid (AR) and 1 uM Dexamethasone (Dex). On day 5 of culture, the cells were co-cultured with purified Peanut extract (PE) at a concentration of 50ug / mL or 10 ug / ml Ara h2 for 48 hours.
Luego, una proporción de las células que se mantuvieron en cultivo fueron analizadas por Citometría de flujo (Facs) para comprobar si obtuvieron un fenotipo de célula dendrítica (DCs) tolerogénica, mediante la determinación de la expresión de las moléculas CD11c y CD14 (como marcadores de DCs) y de las moléculas HLA-DR, CD40, CD83 y CD86 (como marcadores de maduración y activación de las DCs).Then, a proportion of the cells that were kept in culture were analyzed by flow cytometry (Facs) to check if they obtained a tolerogenic dendritic cell (DCs) phenotype, by determining the expression of the CD11c and CD14 molecules (as markers of DCs) and of the HLA-DR, CD40, CD83 and CD86 molecules (as markers of maturation and activation of DCs).
Las células obtenidas, DCs derivadas de monocitos (moDCs) luego fueron co- cultivadas con PE o Ara h2 por 2 días, luego de lo cual fueron reanalizadas mediante Facs para los mismos marcadores antes mencionados y luego re-inyectadas al paciente.
Para comprobar que las células dendríticas autólogas derivadas de monocitos obtenidas desde sangre periférica (moDCs), generaron un efecto tolerogénico frente a estímulos externos, se procedió a enfrentar a moDCs con lipopolisacarido (LPS), post tratamiento con Ácido retinoico (AR) y Dexametasona (Dex) por 48 horas. Los resultados demuestran que al comparar moDC no estimuladas (SE) y moDC+ LPS, estas responden al estímulo expresando mayor número de moléculas co estimuladoras, tales como CD83, CD86 y CD40 (Figura 1), moléculas necesarias para una respuesta inmune efectora. Por otra parte, al evaluar moDC tratadas previamente con AR y Dex, los resultados muestran que estas son similares a las moDC SE (Figura 1). Interesantemente moDCs tratadas con AR y Dex y además LPS (para inducir respuesta inmune) no responden como moDC+ LPS, si no que son similares a moDC SE, indicando que su respuesta es menor y que mantienen características de células tolerogenicas (Figura 2). Además, utilizando los medios de cultivo en el que se mantuvieron por todo el tiempo las moDC, se midió la secreción de citoquinas como interluequina (IL)-4, IL-10. IL-12 y factor crecimiento transformante beta (TGF-b) (moléculas solubles involucradas en la comunicación de moDC con otros tipos celulares como linfocitos T). Estos ensayos muestran que la secreción de IL-4 al comparar moDC SE, moDC LPS no muestran diferencias entre ambos grupos. Similarmente moDC AR Dex y moDC AR Dex LPS, tampoco muestran diferencias entre los grupos y tampoco respecto a moDC SE y moDC LPS (Figura 3). Por otro lado, la medición de IL-10 muestra que el moDC LPS secretan mayor cantidad de IL- 10 al igual que moDC AR+Dex+LPS, el cual además la secreción de IL-10 es mayor que su contraparte moDC AR Dex. Los resultados TGF-b no muestran diferencias entra ninguno de los grupos. Los resultados de IL-4 e IL-10 y TGF-b en moDC AR
Dex LPS, inducirían una respuesta tolerogenicas para los linfocitos T. Por otra parte la secreción de IL-12 no fue detectada en moDC AR Dex LPS, indicando que no existiría una respuesta para activar otras células. The cells obtained, monocyte-derived DCs (moDCs) were then co-cultured with PE or Ara h2 for 2 days, after which they were reanalyzed by Facs for the same markers mentioned above and then re-injected into the patient. To verify that autologous dendritic cells derived from monocytes obtained from peripheral blood (moDCs), generated a tolerogenic effect against external stimuli, we proceeded to confront moDCs with lipopolysaccharide (LPS), post-treatment with retinoic acid (RA) and dexamethasone ( Dex) for 48 hours. The results demonstrate that when comparing unstimulated moDC (SE) and moDC + LPS, they respond to the stimulus by expressing a greater number of co-stimulatory molecules, such as CD83, CD86 and CD40 (Figure 1), molecules necessary for an effector immune response. On the other hand, when evaluating moDC previously treated with AR and Dex, the results show that these are similar to moDC SE (Figure 1). Interestingly, moDCs treated with AR and Dex and also LPS (to induce immune response) do not respond as moDC + LPS, but are similar to moDC SE, indicating that their response is lower and that they maintain characteristics of tolerogenic cells (Figure 2). Furthermore, using the culture media in which the moDCs were kept all the time, the secretion of cytokines such as interluequine (IL) -4, IL-10 was measured. IL-12 and transforming growth factor beta (TGF-b) (soluble molecules involved in the communication of moDC with other cell types such as T lymphocytes). These tests show that IL-4 secretion when comparing moDC SE, moDC LPS do not show differences between both groups. Similarly, moDC AR Dex and moDC AR Dex LPS did not show differences between the groups, nor did they show differences between moDC SE and moDC LPS (Figure 3). On the other hand, the measurement of IL-10 shows that moDC LPS secrete a greater amount of IL-10 as well as moDC AR + Dex + LPS, which also secretion of IL-10 is greater than its counterpart moDC AR Dex. The TGF-b results show no differences between any of the groups. The results of IL-4 and IL-10 and TGF-b in moDC AR Dex LPS, would induce a tolerogenic response for T lymphocytes. On the other hand, IL-12 secretion was not detected in moDC AR Dex LPS, indicating that there would be no response to activate other cells.
Por lo tanto, los resultados obtenidos permiten demostrar que el procedimiento descrito en la presente invención, permite obtener células tolerogénicas específicas a alérgenos, para ser aplicadas como inmunoterapia autóloga en enfermedades alérgicas. Therefore, the results obtained make it possible to demonstrate that the procedure described in the present invention makes it possible to obtain allergen-specific tolerogenic cells, to be applied as autologous immunotherapy in allergic diseases.
Mientras esta invención ha sido descrita bajo las modalidades señaladas anteriormente, podría parecer evidente que otras alternativas, modificaciones o variaciones entregarían los mismos resultados. Consecuentemente, las modalidades de la invención pretenden ser ilustrativas, no limitantes. Varios cambios pueden ser realizados sin alejarse del espíritu y alcance de la invención como se define en las siguientes reivindicaciones. While this invention has been described under the modalities outlined above, it might seem obvious that other alternatives, modifications, or variations would deliver the same results. Consequently, the embodiments of the invention are intended to be illustrative, not limiting. Various changes can be made without departing from the spirit and scope of the invention as defined in the following claims.
Todas las patentes, solicitudes de patentes, artículos científicos y otros documentos públicos que en conocimiento del solicitante constituyen el estado del arte, han sido adecuadamente citados en la presente solicitud.
All patents, patent applications, scientific articles and other public documents that, to the applicant's knowledge, constitute the state of the art, have been adequately cited in this application.
Claims
1. Un procedimiento para la generación de células tolerogénicas específicas a alérgenos, para ser aplicadas como inmunoterapia autóloga en enfermedades alérgicas, CARACTERIZADO porque el procedimiento comprende las siguientes etapas: a) obtener una muestra de sangre de un sujeto con alergia a un determinado alérgeno; b) obtener células mononucleares de sangre periférica y cultivar dichas células en medios apropiados; c) añadir una cantidad suficiente de IL-4 y GM-CSF sobre las células cultivadas; d) estimular las células cultivadas con cantidades suficientes de Ácido retinoico y Dexametasona; e) co-cultivar las células de los pasos anteriores con una cantidad suficiente un extracto purificado del alérgeno; f) analizar las células de los pasos anteriores; y g) obtener células tolerogénicas específicas al alérgeno. 1. A procedure for the generation of allergen-specific tolerogenic cells, to be applied as autologous immunotherapy in allergic diseases, CHARACTERIZED in that the procedure comprises the following steps: a) obtaining a blood sample from a subject with allergy to a certain allergen; b) obtaining mononuclear cells from peripheral blood and culturing said cells in appropriate media; c) adding a sufficient quantity of IL-4 and GM-CSF on the cultured cells; d) stimulating the cultured cells with sufficient amounts of Retinoic Acid and Dexamethasone; e) co-cultivating the cells of the previous steps with a sufficient amount of a purified extract of the allergen; f) analyzing the cells from the previous steps; and g) obtaining allergen-specific tolerogenic cells.
2. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque la etapa a) comprende obtener una muestra de sangre a través de venopunción, flebotomía, o leucoferesis. 2. The method according to claim 1, CHARACTERIZED in that step a) comprises obtaining a blood sample through venipuncture, phlebotomy, or leukapheresis.
3. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque la etapa b) comprende obtener células mononucleares de sangre periférica mediante dilución de la sangre y posterior gradiente de densidad con ficoll.
3. The method according to claim 1, CHARACTERIZED in that step b) comprises obtaining peripheral blood mononuclear cells by dilution of the blood and subsequent density gradient with ficoll.
4. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque los medios apropiados para cultivar la células de la etapa b) se seleccionan entre medio de cultivo AIM-V, RPMI, en el cual las condiciones de cultivo comprenden entre 35 a 39 °C, con un 5% de CO2. 4. The method according to claim 1, CHARACTERIZED in that the appropriate media to culture the cells of step b) are selected from AIM-V culture medium, RPMI, in which the culture conditions comprise between 35 to 39 ° C, with 5% CO2.
5. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque la cantidad suficiente de IL-4 y GM-CSF de la etapa c) comprende entre 500 - 2000 UI/mL. The method according to claim 1, CHARACTERIZED in that the sufficient amount of IL-4 and GM-CSF from step c) comprises between 500-2000 IU / mL.
6. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque las cantidades suficientes de Ácido retinoico y Dexametasona de la etapa d) comprende entre 0,5 - 10 uM de Ácido retinoico y entre 0,5 - 10 uM de Dexametasona. 6. The process according to claim 1, CHARACTERIZED in that the sufficient amounts of retinoic acid and dexamethasone of step d) comprise between 0.5-10 uM of retinoic acid and between 0.5-10 uM of dexamethasone.
7. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque la cantidad suficiente del extracto purificado del alérgeno de la etapa e) comprende entre 10 a 50 ug/mL. 7. The method according to claim 1, CHARACTERIZED in that the sufficient amount of the purified extract of the allergen from step e) comprises between 10 to 50 ug / mL.
8. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque el alérgeno de la etapa e) se selecciona entre maní, huevo, leche, trigo, gluten, soya, frutos secos, pescados, mariscos, látex, lupino, frutas, verduras. 8. The method according to claim 1, CHARACTERIZED in that the allergen of step e) is selected from among peanuts, eggs, milk, wheat, gluten, soy, nuts, fish, shellfish, latex, lupine, fruits, vegetables.
9. El procedimiento de acuerdo con la reivindicación 1 , CARACTERIZADO porque la etapa f) comprende analizar las células obtenidas por Citometría de flujo, ELISA, microscopía de fluorescencia, microscopía confocal. 9. The method according to claim 1, CHARACTERIZED in that step f) comprises analyzing the cells obtained by flow cytometry, ELISA, fluorescence microscopy, confocal microscopy.
10. Un procedimiento para inducir la tolerancia a alérgenos mediante inmunoterapia autóloga, CARACTERIZADO porque comprende inyectar las células obtenidas de las reivindicaciones 1 a 10, a un sujeto con alergia a un determinado alérgeno.
10. A method for inducing tolerance to allergens by means of autologous immunotherapy, CHARACTERIZED in that it comprises injecting the cells obtained from claims 1 to 10, to a subject with an allergy to a certain allergen.
11. El procedimiento de acuerdo con la reivindicación 10, CARACTERIZADO porque el alérgeno se selecciona entre maní, huevo, leche, trigo, gluten, soya, frutos secos (nueces, almendras), pescados, mariscos, látex, lupino, frutas, verduras. 11. The method according to claim 10, CHARACTERIZED in that the allergen is selected from among peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits, vegetables.
12. Una célula tolerogénica específica a un determinado alérgeno para ser aplicada como inmunoterapia autóloga en enfermedades alérgicas, CARACTERIZADA porque es obtenida por el procedimiento descrito en las reivindicaciones 1 a 9. 12. A tolerogenic cell specific to a certain allergen to be applied as an autologous immunotherapy in allergic diseases, CHARACTERIZED in that it is obtained by the method described in claims 1 to 9.
13. La célula tolerogénica de acuerdo con la reivindicación 12, CARACTERIZADA porque dichas células tienen tolerancia a alérgenos seleccionados entre maní, huevo, leche, trigo, gluten, soya, frutos secos (nueces, almendras), pescados, mariscos, látex, lupino, frutas, y verduras.
13. The tolerogenic cell according to claim 12, CHARACTERIZED in that said cells have tolerance to allergens selected from among peanuts, eggs, milk, wheat, gluten, soy, nuts (walnuts, almonds), fish, shellfish, latex, lupine, fruits and vegetables.
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| WO2012160200A1 (en) * | 2011-05-25 | 2012-11-29 | Hospital Clínic De Barcelona | Tolerogenic dendritic cells and their use in cell therapy |
| WO2013036298A1 (en) * | 2011-09-06 | 2013-03-14 | Selecta Biosciences, Inc. | Allergen-specific induced tolerogenic dendritic cells for allegy therapy |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012160200A1 (en) * | 2011-05-25 | 2012-11-29 | Hospital Clínic De Barcelona | Tolerogenic dendritic cells and their use in cell therapy |
| WO2013036298A1 (en) * | 2011-09-06 | 2013-03-14 | Selecta Biosciences, Inc. | Allergen-specific induced tolerogenic dendritic cells for allegy therapy |
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| AGUILERA-INSUNZA, R. ET AL.: "Role of dendritic cells in peanut allergy", EXPERT REVIEW OF CLINICAL IMMUNOLOGY, vol. 14, no. 5, 2018, pages 367 - 378, DOI: 10.1080/1744666X.2018.1467757 * |
| DAWICKI, WOJCIECH; LI CHUNYAN; TOWN JENNIFER; ZHANG XIAOBEI; GORDON JOHN R: "Therapeutic reversal of food allergen sensitivity by mature retinoic acid -differentiated dendritic cell induction of LAG3+ CD 49b-Foxp3- regulatory T cells", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 139, no. 5, 2017, pages 1608 - 1620, XP029997466, DOI: 10.1016/j.jaci.2016.07.042 * |
| KIM, SANG-HYUN, JUNG HO-HYUN, LEE CHONG-KIL: "Generation, characteristics and clinical trials of ex vivo generated tolerogenic dendritic cells", YONSEI MEDICAL JOURNAL, vol. 59, no. 7, 2018, pages 807 - 815, XP055836171, DOI: 10.3349/ymj.2018.59.7.807 * |
| SIM, WEN JING, MALINARICH FRANO, FAIRHURST ANNA-MARIE, CONNOLLY JOHN EDWARD: "Generation of Immature, Mature and Toierogenic Dendritic Cells with Differing Metabolic Phenotypes", JOURNAL OF VISUALIZED EXPERIMENTE, vol. 112, no. e54128, 2016, pages 12, XP055836168, DOI: 10.3791/54128 * |
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