Patent Application
20170196950
The present invention is related with the production of tolerogenic dendritic cells for specific antigens designed for using in autoimmune diseases, in particular for the treatment of Systemic Lupus Erythematosus (SLE).
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease of unknown etiology affecting mainly women of childbearing age with a prevalence of 124 cases for each 100,000 persons. From a clinical point of view, lupus affects multiple organs, including kidneys, heart, lungs, skin, and musculo-skeletal and hematological systems, among others. It also presents an unpredictable clinical evolution characterized by reactivations or outbreaks alternated with remission periods. Although mortality of SLE has decreased significantly in the last 50 years, a patient developing SLE at 20 years of age has a 15% chance of dying by 35 years of age due to lupus itself or an associated complication. SLE also shows an important aggregated morbidity. For example, one of the most relevant complications in terms of impact in survival and life quality for patients is lupus nephritis (LN) affecting up to a 50% of lupus patients. In spite of important efforts to optimize the treatment of this manifestation, still 10-30% of patients with LN develops terminal renal insufficiency and therefore requires dialysis and/or renal transplant. Likewise, lupus patient have a mortality between 2 and 5 times higher than general population, presenting up to 23% of work incapacity and in the case of women of childbearing age, this is accompanied by difficulties in conceiving and higher morbidity-mortality rates during pregnancy compared to healthy women.
Likewise, FDA approved in 2011 for the first time in 30 years, a new drug (belimumab) for SLE treatment. Nevertheless, this drug and other usual use drugs for this disease, including mycophenolate mofetil (MMF), cyclophosphamide and steroids, are non-curative and immunosuppressive non-specific treatments associated to important complications such as opportunistic infections. Recently, the research team of project ALMS (NCT00377637) studying the use of MMF in lupus nephritis, published results of the maintenance phase of said study, wherein the drug was compared to azathioprine. The study shows that 33.3% of patients using azathioprine and 23.5% of patients using MMF presented serious adverse effects motivating their withdrawal from the study in a 39.6% and 25.2% of patients respectively. Other biological agents approved for use in other autoimmune pathologies such as rituximab, have shown no efficacy in controlled clinical studies for lupus, and just like traditional immunosuppressant, can also contribute to emergence of opportunistic infections. The present invention consists in a procedure to obtain autologous dendritic cells (DCs) specific for antigens from apoptotic cells for immunotherapy for SLE. In a search for prior art, certain documents related with provision and use of dendritic cells for treatment of immunological diseases were found. The closest document to the technology under study is the patent application WO2001085207 wherein the use of apoptotic cells is proposed to decrease the capacity of DC cells for stimulating a cellular response through induction of T cell anergy. Also, it includes diverse alternatives to modulate apoptotic antigen display by DCs and the use of maturating factors for DCs. In this case, the apoptotic cells are the ones exerting the modulating activity in the function of dendritic cells. The relationship of apoptotic cells and Systemic Lupus Erythematosus is not mentioned in the document. In the present invention, the objective of using apoptotic cells is different, since it is not directed per se to direct modulation of the function of DCs and it is not relevant how the apoptotic antigens are presented by DCs that will be able to induce T cell anergy. The objective of using apoptotic cells is to achieve that the DCs of the present invention are specific to only modulate the response of T cells to antigens contained in apoptotic cells, which have a relevant role in the pathology of Systemic Lupus Erythematosus. Furthermore, during the performed experiments, an evaluation was conducted to demonstrate that apoptotic cells would not induce maturation of DCs, i.e., that they would not increase the immunogenic capacity since it was never in the objectives of use that they modulate immunogenicity of DCs. The modulation of the function of DCs is achieved using Rosiglitazone (RGZ) and Dexamethasone (DEXA).
The publication by Carreño et al. “Induction of Tolerogenic Dendritic Cells by NF-κB Blockade and Fcg Receptor Modulation” describes the use of DCs treated with Rosiglitazone and andrographolide for a murine model treatment for multiple sclerosis and suggests that it can be used for other autoimmune diseases such as Lupus, wherein the antigen is unknown. Protocols are described to treat animals directly with these drugs and generating tolerogenic DCs (tolDCs) with Rosiglitazone and Andrographolide. In the present invention, the use of Rosiglitazone and Dexamethasone together is proposed to generate tolerogenic DCs, which is not disclosed in the publication by Carreño et al. Also, said publication proposes to treat mice directly with NF-κB inhibitory drugs, rosiglitazone and andrographolide, not with DCs treated with these drugs, as it is proposed in the present invention. The treatment with treated DCs is made in a murine model of multiple sclerosis. The only mention to the possibility of treating SLE with tolerogenic DCs is subtle and never includes the idea of including apoptotic cells to induce anergy in T lymphocytes recognizing antigens from these cells. Even more, it is specifically mentioned that the Lupus antigen are unknown, and never proposes that apoptotic cells can be used as a source of antigens. Even this is shown as a potential obstacle.
The publication of International Patent Application WO2012160200 proposes the use of steroids and Vitamin D to generate mature DCs with tolerogenic properties. The culture time in steroids is 4 days and it explicitly reads “to obtain mature DC”. Afterwards, these cells are cultured in an environment with pro-inflammatory cytokines, wherein mature DCs are identified by the presence of CD14 on their surface and tolerogenic cells are identified by the expression of MERTK. The present invention proposes to obtain immature DCs (iDCs) using dexamethasone (glucocorticoid) and rosiglitazone (thiazolidinedione), which is mentioned in this patent application and it is not similar to Vitamin D. The method of the present invention is different, since the cells are cultured with rosiglitazone and dexamethasone. In no place in this document is suggested or proposed that DCs should have a given specificity for the treatment with some specific antigen. Even more, in no place is suggested that in the case of SLE the source of antigens could be the use of apoptotic cells. The DCs of the present invention are CD14(−), i.e. it is not expressed and the presence of MERTK is not evaluated, since it is about different products originated from different methodologies.
For the generation of immunotherapy based on DCs, the antigenic specificity is relevant, since one of the features of this kind of therapy is decreasing the response against self-antigens without affecting an effective response against external agents. Nevertheless, one of the main obstacles currently complicating the development of immunotherapy in SLE is the lack of knowledge of the specific autoantigen responsible for the development of the disease. Since patients suffering from Lupus present a deficiency for eliminating cellular debris generated in apoptosis process, is that it has been postulated that these would be a source of autoantigens. Therefore, the present invention proposes the use of apoptotic cells as a source of autoantigens for directing the activity of tolerogenic DCs (tolDCs) exclusively towards auto reactive lymphocytes (
The present invention consists in tolerogenic dendritic cells (tolDCs) with specific antigens that reestablish tolerance of immune system to own organs, specific methods that reestablish tolerance of immune system to own organs, method to produce said tolDCs with specific antigens; use of said tolDCs with specific antigens in the production of a therapy for the treatment of Systemic Lupus Erythematosus (SLE).
The present invention considers three main aspects. In a first aspect, the present invention corresponds to a method to produce tolerogenic dendritic cells (tolDCs) with specific antigens. In a second aspect, the present invention corresponds to tolerogenic dendritic cells (tolDCs) with specific antigens that reestablish the tolerance of the immune system to own organs, when administered to a patient in need thereof. Finally, a third aspect of the present invention corresponds to the use of tolerogenic dendritic cells (tolDCs) with specific antigens in a therapy for Systemic Lupus Erythematosus (SLE).
An embodiment of the first aspect of the invention corresponds to a method to produce tolerogenic dendritic cells (tolDCs) with specific antigens. In a particular embodiment, the method to produce tolerogenic dendritic cells (tolDCs) with specific antigens comprises the following steps:
In a particular embodiment, the dendritic cell precursors of step a) are selected among monocytes, bone marrow progenitors, or directly from peripheral blood or umbilical cord blood.
In another particular embodiment, differentiation is performed when culturing precursors and cytokines IL-4 and GM-CSF in conditions between 30 y 45° C., and between 1 and 10% CO2. More specifically at 37° C. and 5% CO2.
In other embodiment, the apoptotic cells of step c) are produced when exposing cells to an apoptotic stimulus selected from ultraviolet B (UV-B) radiation, presence of chemical substances (staurosporine, methotrexate), activation of specific receptors (Fas-Fas ligand interaction) or inhibition of mitochondrial electron transport (heptachlor, rotenone). In another embodiment, the cells from which the apoptotic cells are originated correspond to blood cells, muscular cells, epidermal cells, epithelial cells, stem cells or human cell lines. In a particular embodiment, the blood cells are peripheral blood lymphocytes, platelets, neutrophils, or monocytes. In a more specific embodiment, the blood cells are peripheral blood lymphocytes. In another embodiment, culture of dendritic cells in presence of compounds with anti-inflammatory activity of step d) is performed during a period between 5 and 48 hours. In a more specific embodiment, the compounds with anti-inflammatory activity are selected from rosiglitazone (RGZ) and dexamethasone (DEXA) or a combination thereof. In a more specific embodiment, dendritic cells are cultured in the presence of between 5 and 30 μM RGZ, in presence of between 0.5 and 5 μM de DEXA.
In a specific embodiment, co-culture of dendritic cells of step e) with apoptotic cells is performed considering an amount of apoptotic cells, expressed as DNA content, between 5 and 20 μg/ml. Animal serum-free medium is used to this end, for example AIM-V (GIBCO® AIM V® Medium Grand Island, N.Y., USA).
In other embodiment, co-culture of dendritic cells with apoptotic cells is performed during a period between 5 and 48 hours.
In other embodiment of the invention, identification of tolDCs of step f) is performed by evaluating: i) production of cytokines IL-6, and IL-12p70 which must decrease compared to mature immunogenic DCs; and ii) absence or reduced expression of surface markers compared to mature immunogenic DCs, wherein the surface markers are selected from CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof. In a more specific embodiment, the evaluation of the production of IL-6, and IL-12p70 and expression of surface markers CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof is performed using a technique selected from ELISA, flow cytometry, Western blot, and also level of transcription of messenger RNA through RT-PCR.
A second aspect of the invention, corresponds to tolerogenic dendritic cells (tolDCs) with specific antigens obtained using the previously exposed method. In a specific embodiment, the specific antigens are autoantigens, and not because they come from a patient, since in autoimmune diseases the immunological response is against a self-element, and therefore, the antigen is an autoantigen. In a specific embodiment of the invention, dendritic cells come from: monocytes, bone marrow progenitors, or directly from peripheral blood or umbilical cord blood. In another embodiment, the specific antigens come from apoptotic cells. In a more specific embodiment, the apoptotic cells come from cells that have been subjected to an apoptotic stimulus. In a specific embodiment, the apoptotic stimulus to which the cells are subjected to, is selected from ultraviolet B (UV-B) radiation, in the presence of chemical substances (staurosporine, methotrexate), activation of specific receptors (Fas-Fas ligand interaction) or inhibition of mitochondrial electron transport (heptachlor, rotenone). More specifically, the stimulus is ultraviolet type B (UV-B) radiation.
In other embodiment, the cells from which the apoptotic cells come from, correspond to blood cells, muscular cells, epidermal cells, epithelial cells, stem cells, or human cell lines. In a particular embodiment, the blood cells are peripheral blood cells, platelets, neutrophils, or monocytes.
In a specific embodiment, tolerogenic dendritic cells (tolDCs) of the invention are identified through phenotypic evaluation. In a more specific embodiment, the identification of tolDCs is made by evaluating: i) production of cytokines IL-6, y IL-12p70 which must decrease compared to immunogenic mature DCs; and ii) absence or reduced expression of surface markers compared to mature immunogenic DCs, wherein the surface markers are selected from CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof. In a more specific embodiment, the evaluation of production of IL-6, and IL-12p70 and expression of surface markers CD40, CD80, CD83, CD86, HLA-DR, or combinations thereof, is performed using a technique selected from ELISA, flow cytometry, Western blot, and also transcription level of messenger RNA using RT-PCR.
In a third aspect of the invention, the use of tolerogenic dendritic cells (tolDCs) with specific antigens is described in the production of a therapy for the treatment of Systemic Lupus Erythematous (SLE).
In a specific embodiment, the invention describes the use of tolerogenic dendritic cells (tolDCs) with specific antigens, which can be used in the preparation of a medicine useful in the treatment of Systemic Lupus Erythematous (SLE).
Apoptosis Induction Using Ultraviolet Type 8 (UV-8) Radiation in Peripheral Blood Lympchotytes
In this stage, different exposition times to UV-B radiation were evaluated to obtain apoptotic cells from lymphocytes from peripheral blood from initially healthy individuals and afterwards from patients with SLE. To corroborate the apoptosis induction, Propidium Iodide (PI) and Annexin V (Anex) were used as dyes and the samples were analyzed using flow cytometry. Said cells in apoptosis status are later used to pulse DCs with the aim to provide autoantigens (
Evaluation of the Capacity of DCs of Patients with SLE to Endocytose Apoptotic Cells.
Afterwards, it was evaluated if DCs generated in the previous Example had the capacity to endocytose apoptotic cells to process antigens and then present them to self-reactive T lymphocytes. Apoptotic cells generated through exposition to UV-B radiation were labeled with cellular dye carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Carlsbad, Calif., USA) to detect them using flow cytometry. DCs were incubated for 24 hours with apoptotic cells labeled with CFSE. This experiment selected a population CD11c+ allowing to identify DCs. CFSE detection in a CD11c+ population (39.5%) is indicative of phagocytosis of apoptotic cells by DCs (
Efecto de Co-Cultivo De Células Apoptóticas y DCs en Inmunofenotipo de DCs.
It is widely reported that DNA molecules play a role as autoantigen in lupus patients and that are exposed to extracellular space during apoptosis associated to proteins, such as histones in the form of nucleosomes (1). Therefore, determining DNA constitutes a manner to quantify apoptotic cells, a method previously used by other researchers (2).
For the results shown in the present invention, a total of 14 SLE patients were included, whose clinical characteristics are detailed in Table 1, from whom DCs were generated and the sixth day were pulsed with apoptotic cells (12.5 μg/ml of DNA content) for 24 hours prior to the analysis. The maturation state of resulting DCs was analyzed by flow cytometry using conjugated antibodies anti CD40, CD80, CD83, CD86 and HLA-DR (
RGZ and DEXA Effect on DC Pulsed with Apoptotic Cells.
DCs derived from monocytes obtained from SLE patients and control individuals were treated for 24 hours with RGZ (10 μM) and DEXA (1 μM) and then co-cultured with apoptotic cells (12.5 μg/ml of DNA content) for 24 additional hours and the expression of markers CD40, CD80, CD83, CD86 and HLA-DR was evaluated in a total of 14 SLEpatients (Table 1).
Determination of Activation of DCs: Proinflammatory and Antiinflammatory Cytokines Secretion.
With the purpose to corroborate the induction of a tolerogenic state in DCs and having more elements allowing the most complete characterization possible and a functional approach, a quantification of some relevant cytokines in supernatants of previous experimental supernatants was performed, such as interleukin 6 (IL-6) and interleukin 12p70 (IL-12p70), secreted to extracellular medium by tolerogenic dendritic cells, using Enzyme-Linked Immunosorbent Assay (ELISA).
Table 2 summarizes the obtained results and shows that the product tolDC (tolerogenic DCs treated with RGZ and DEXA and pulsed with apoptotic cells) presents a decrease in most of the relevant surface markers associated to maturity and an immunogenic phenotype when challenged with LPS and a decrease in production of pro-inflammatory cytokines when compared to DC that were not treated with RGZ and DEXA.
Toxicity and Cell Viability Studies for SLE Therapy with Autologous Tolerogenic DCs.
With the objective to evaluate the effects of the drugs on viability and cell metabolism, an XTT viability assay was performed, a colorimetric assay estimating the metabolic cellular activity. Its basis consists in reduction of tetrazolium XTT salt, which is transformed to formazan due to the activity of mitochondrial dehydrogenase enzymatic activity of metabolically active cells, generating as a product a colored compound, which is only generated in viable cells and which amount is produced in a proportional amount to the number of viable cells in the sample.
Following the same previous line, an experiment was performed to evaluate if generated DCs secreted any potentially toxic substance to extracellular medium which could affect viability of the cells of the organism that would receive the therapy. To perform this assay, propidium iodide (PI) was used together with Annexin V (Anex V) which were used to label peripheral blood lymphocytes (PBL) of a control individual that were previously treated with the supernatants of DCs cultured in the conditions of our protocol using immunomodulating drugs, but without subjecting them to LPS challenge, since they came from another control individual, during a period of 24 hours.
On the other hand, it was determined if these supernatants from generated DCs were able to produce in the lymphocytes an activation response, and for that, markers CD69 (early activation marker) and CD71 (late activation marker) were determined, which are expressed in response to activation stimulus in lymphoid cell lines.
In the previous example, the qualities and stability of the immunophenotype of tolDCs was characterized as a measure to evaluate their potential as therapy and having a functional approach when studying cytokine secretion. To determine their success in a pre-clinical phase, in vitro functional analyses as such are required directed to evaluate the modulating capacity of generated tolDCs on CD4+ T lymphocytes.
Therefore, a Mixed Lymphocyte Reaction (MLR) allogenic assay was performed using DCs and CD4+ T cells from different healthy individuals (
tolDCs from an individual and T cells from a different individual previously labeled with CFSE were cultured in 200 μL of medium RPMI 1640+10% FBS (ratio 1:5) for 5 days. As a control, T lymphocytes co-cultured with immature DCs (iDCs), mature DCs or without DCs were used until the endpoint of the experiment. The activation of T lymphocytes was evaluated at the fifth day using flow cytometry controlling the expression of CD25 and CD71 (
The same assay was performed with tolDCs from a SLE patient and T cells from a healthy individual previously labeled with CFSE and cultured in 200 μL of medium RPMI 1640+10% FBS (ratio 1:5) for 5 days. As a control, T lymphocytes co-cultured with immature DCs, mature DCs or without DCs were used until the endpoint of the experiment. Proliferation and activation of T lymphocytes on the fifth day were evaluated using flow cytometry by measuring CFSE dilution and CD71 expression (
The expression of CD71 (T lymphocyte activation marker) in T cells cultured in the presence of tolDCs induced for each one of the drugs or both simultaneously, is inferior than the one observed for cells cultured with immature DCs. Additionally, it was observed that T lymphocytes proliferate less in the presence of tolDCs than mature DCs.
A. Isolation of Peripheral Blood or Buffy Coat should not be Greater than 8 Hours and Supplemented with Heparin
B. Obtaining Peripheral Blood Lymphocytes and Differentiation Towards a DC.
C. Apoptotic Cell Generation.
D. Co-Culture of DCs and Apoptotic Cells (Day 7)
E. Induction of Tolerogenic DC (Day 6)
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2015/054267 | 6/5/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62008319 | Jun 2014 | US |
