Method for producing antigen-specific T lymphocytes

Abstract

The present invention relates to a novel method for producing antigen-specific T lymphocytes (CellTrAg) in vitro, wherein the lymphocytes are marked with an intracellular dye, expanded in the presence of antigen-loaded monocytes, and subsequently sorted based on a low-intensity intracellular dye, wherein the low-intensity fluorescence is a marker of antigen-specificity in that the loss of fluorescence correlates with the intensity of proliferation. Antigen-specificity is evaluated in a functional test in which antigen-specific lymphocytes sorted based on low fluorescence are more active than non-specific lymphocytes having high fluorescence during sorting; activity is defined as the inhibition of effector lymphocyte function in the case of regulatory T lymphocytes, and as the enhancement of cell characteristics, e.g., proliferation, cytokine production, and cytotoxic factors in the case of T effector lymphocytes.

Description

Technology Field

[0001] The present invention relates to a method for producing antigen-specific T lymphocytes (CellTrAg) for clinical use in immunotherapy. CellTrAg produced in this manner is suitable for the treatment of autoimmune diseases, including, for example, multiple sclerosis, rheumatoid arthritis, and type 1 diabetes, and for the suppression of inverse immune responses, for example, transplant rejection, allergic reactions, and graft-versus-host disease (GVHD). Background Technology

[0002] Treg lymphocytes constitute about 1% of all peripheral blood lymphocytes, but they are important for maintaining tolerance to one's own tissues (Trzonkowski P 2009) (Vignali DA 2008) (Yi S 2012). A deficiency of regulatory T cells leads to a number of autoimmune diseases and hypersensitivity, as seen in patients with X-linked immunodeficiency syndrome, polyendocrine disorders, and enteropathy (IPEX) (Gambineri E 2003). While Treg lymphocytes can be called "intelligent steroids" because they suppress inflammatory responses and act immunosuppressively as steroids, the physiological inhibitory effect of Treg cells is related only to pathological responses (e.g., their instruction to their own tissues). The results of clinical trials, including observations by the inventors, indicate that treatment with Treg lymphocytes is safe and does not impair the immune response to external dangerous antigens (viruses, bacteria, cancer cells) (Marek-Trzonkowska N 2012)(Marek-Trzonkowska N 2014) (Martelli MF 2014)(Bluestone JA 2015).

[0003] The inventors' research team has been conducting research on the biological and clinical uses of Treg lymphocytes for over 10 years. The inventors are the first to use in vitro amplified Treg cells in the treatment of graft-versus-host disease (GvHD) in adults (NKEBN / 458-310 / 2008) (Trzonkowski P 2009), in type 1 diabetes mellitus (T1D) in children (TregVAC ISRCTN06128462; TregVAC2.0EudraCT:2014-004319-35) (Marek-Trzonkowska N 2012) (Marek-Trzonkowska N 2014), and in multiple sclerosis (TregSM EudraCT:2320-25Trkzon). Currently, clinical trials using Treg cells are being conducted at various centers around the world and are related to the therapy / prevention of GVHD (Di Ianni M 2011), the treatment of T1D in adults (NCT01210664), and the induction of resistance in kidney (ia NCT02091232 and NCT02129881) and liver (ThRIL NCT02166177 and NCT01624077) transplants. In recent years, dynamic development of cell therapies using Treg lymphocytes as a therapeutic tool has begun. Currently, approximately 40 clinical trials involving these cells are underway globally. All of these projects proceed under a single goal: intelligent immunosuppression, which suppresses unwanted immune responses without compromising physiological immune responses (Trzonkowski P 2015, Gliwinski M 2017).

[0004] During the above studies, Treg lymphocytes were obtained from the patient's peripheral blood or umbilical cord blood units. The best isolation method is to use a sorter, which yields a very pure (97 to 100%) population of the cells. Generally, lymphocytes with the CD3+CD4+CD25high phenotype or CD3+CD4+CD25highCD127- or CD3+CD4+CD25highCD127low are isolated (Patent Application P. 399447)(Trzonkowski P 2009)(Marek-Trzonkowska N 2012)(Marek-Trzonkowska N 2014)(Trzonkowska P 2015). The isolated cells are subsequently activated for vigorous proliferation for 10 to 14 days, yielding a sufficient amount for administration to the patient. Effective expansion must be carried out under conditions that maintain the full phenotype, including particularly high expression of the FoxP3 factor as measured by specific laboratory activity (Marek N 2011, Gołab K 2013, Marek-Trzonkowska N 2017). In addition, since the production of Treg lymphocytes is classified as an Advanced Therapy Medicinal Product (ATMP) and is subject to pharmaceutical laws and European Parliament Regulation No. 1394 / 2007 on "Advanced Therapy Medicinal Products," the expansion of this plan for clinical application must be carried out in accordance with Good Manufacturing Practice (GMP) standards.

[0005] The use of cells generated according to the above plan implies multispecificity, and since this is a set of lymphocytes specific to many different antigens, its effect is limited after administration. Of course, the fact that all Treg lymphocytes have an affinity for the site of inflammation, the ability to regulate the 'bystander' type, and the ability to convert other cells into a regulatory phenotype based on infection resistance affect the multispecific (polyclonal) product. Nevertheless, the effect of this product can be enhanced by targeting cells to specific antigens. In this way, the antigen-specific Treg lymphocytes can migrate only to the site where the expression of the specific antigen occurs and can selectively inhibit the activity of pathological effector cells that recognize the antigen only in the site of the inflammatory response induced by the stimulation of the antigen. In the case of autoimmune diseases, it is possible to inhibit the destruction of affected organs (e.g., insulin-producing pancreatic islets in type 1 diabetes or myelin in multiple sclerosis). At the same time, it will limit systemic side effects of Treg lymphocytes, which will show affinity only to sites expressing specific sensitized antigens instead of circulating throughout the entire lymphatic system.

[0006] The development of a safe, easy-to-use, and simultaneously economically viable method for multiplying stable Treg lymphocytes with specific antigen specificity and high inhibitory potential is of critical importance for the development and success of clinical trials using Treg cells as a therapeutic tool.

[0007] Another apparent problem is an excessive immune response, also known as hypersensitivity to allergens. Most of these disorders are successfully treated symptomatically using available medications. However, some forms of hypersensitivity can cause complications leading to severe disability and even death. The course of the disease often progresses over time, and intensified inflammation causes permanent structural changes in the airways, leading to the discontinuation of the effects of medications (Panettieri RA Jr 2008) (Barbaro MP 2014). In such situations, antigen-specific Treg lymphocytes can also serve as modern anti-allergic drugs.

[0008] In conclusion, an effective and safe drug used for the treatment of autoimmune diseases, allergies, and transplant recipients is one that selectively modulates the immune system response to well-defined antigens responsible for unwanted immune responses (e.g., autoimmune diseases or allergies, organ rejection, graft-versus-host disease) while not impairing the physiological immune response to foreign, dangerous antigens. The potential for such intelligent immunosuppression lies precisely in antigen-specific Treg lymphocytes. However, a condition for the success of clinical therapy using Treg cells is the development of a patient-safe protocol for the expansion of said lymphocytes, which ensures a large number of antigen-specific cells while maintaining stability and inhibitory activity throughout the culture period (Tang Q 2013). The method disclosed in this application satisfies the requirements described above.

[0009] In particular, the present invention relates to a method for producing antigen-specific T lymphocytes that are marked and classified with a monoclonal antibody, wherein the lymphocytes are

[0010] a) generated using autologous monocytes loaded with the above antigen;

[0011] b) The T regulatory lymphocytes or T effector lymphocytes to be generated are suspended in PBS and intracellularly stained with a fluorescent dye;

[0012] c) Subsequently, the above lymphocytes are cultured in a dark place;

[0013] d) Subsequently, the lymphocyte cells are intensively washed several times with culture medium;

[0014] e) T regulatory lymphocytes or T effector lymphocytes stained with an intracellular fluorescent dye are suspended in a culture medium containing antigen-loaded gamma-irradiated autoCD14+ monocytes;

[0015] f) A co-culture of the above T regulatory lymphocytes or T effector lymphocytes and CD14+ monocytes is co-incubated with anti-CD154 antibody and anti-CD28 antibody;

[0016] g) The above co-culture is incubated in a culture medium;

[0017] h) A method is provided in which antigen-specific T lymphocytes after incubation are classified based on the low intensity of an intracellular dye, and the low fluorescence intensity serves as a marker of antigen specificity in that the loss of fluorescence correlates with the proliferation intensity.

[0018] In the method defined above, T regulatory lymphocytes or T effector lymphocytes are 1 x 10 6 It is desirable to suspend at a concentration of cell / ml PBS.

[0019] In the method defined above, it is preferable that the lymphocytes be stained with one of CFSE or violet blue fluorescent dyes having a final concentration of 1 to 5 μM.

[0020] In the method defined above, it is preferable that the lymphocytes be incubated at room temperature or 37°C for 20 minutes.

[0021] In the method defined above, it is preferable that the autologous monocytes be added to the co-culture at a final monocyte:lymphocyte ratio of 1:1.

[0022] In the method defined above, it is preferable that the monocytes be irradiated with gamma rays.

[0023] In the method defined above, it is preferable that the co-culture of the monocytes and lymphocytes be cultured at a temperature with an anti-CD154 antibody at a final concentration of 5 μg / ml and an anti-CD28 antibody at a final concentration of 5 μg / ml.

[0024] In the method defined above, it is preferable that the co-culture be cultured in a constant temperature at 37°C in 5% CO2.

[0025] In the method defined above, specificity for the antigen is preferably evaluated in a functional test in which antigen-specific T lymphocytes, particularly T lymphocytes classified based on low fluorescence of an intracellular dye, are more active than non-specific T lymphocytes, particularly T lymphocytes classified based on conserved high fluorescence of an intracellular dye, wherein, for T regulatory lymphocytes, activity is defined as the inhibition of functional T effector lymphocytes, whereas for T effector lymphocytes, activity is defined as increased proliferation intensity and increased cytokine and cytotoxic factor production intensity. Brief explanation of the drawing

[0026] Fig. 1 - The experimental plan is presented. Buffy coats were separated into monocytes and lymphocytes. Lymphocytes were classified into T regulatory cells (Treg) and T effector cells (Eff), stained with CFSE, and stimulated with irradiated monocytes pre-loaded with peptides (insulin or peptide 9-23). ​​Polyclonal Tregs and Eff were induced using anti-CD3 / anti-CD28 beads. After 7 days of co-culture, cells were classified into antigen-specific and non-specific cells based on the fluorescence of diluted CFSE using the protocols shown in Figures 2c and 2d. The polyclonal Tregs obtained (Treg poly ) subset, antigen-specific (Treg spec ) subset and non-specific (Treg unspec A subset of ) was tested in a functional test in which the responder cells were purple-stained autologous T cell effectors, and said effectors were polyclonal (Eff poly) effector or antigen-specific (Eff spec ) is an effector. Eff spec It was prepared using stimulation of irradiated monocytes pre-loaded with antigen. Fig. 2 - This illustrates the classification method for antigen-specific regulatory T cells. Tregs are stained with a fluorescent dye (b) to distinguish them from other cells (a), particularly antigen-presenting monocytes used during stimulation. After the end of co-culture, Treg lymphocytes that recognized antigens aligned in the reduced fluorescence (P1 gate) region were classified into a separate antigen-specific population. Fig. 3 - Represents the percentage of Treg cells responding to the antigen in co-cultures with monocytes. The analysis included Treg cells co-cultured with the antigen-presenting monocytes insulin or peptide 9-23. In some cultures, the monoclonal antibodies anti-CD28 and anti-CD154 (mab28 / 154) were added to provide a second signal. The percentage of proliferative (antigen-specific) Tregs (a), the percentage of Tregs expressing high intensity of FoxP3 expression (high), and proliferative (light blue) or non-proliferative (dark blue) (b) were evaluated. Results are presented as mean + / - SD. Examples of the analysis are shown in the dot-plots of Chart c from co-cultures stimulated with insulin-loaded monocytes and Chart d from co-cultures stimulated with peptide 9-23-loaded monocytes. The gate of the dot-plot CD4 versus violet is displayed on the proliferative (SPEC) antigen-specific Tregs on the left and the non-proliferative (UNSPEC) non-specific Tregs on the right. Arrows connect the corresponding dot-plots showing FoxP3 expression. The top gate is FoxP3 in the cells high and lower FoxP3 low Represents a subset. Fig. 4- Indicates the clonal nature of Treg lymphocytes. The clonal nature of Treg lymphocytes is measured by the expression of individual classes of TCR receptors (Vβ chains). An example of Treg lymphocyte clonal analysis analyzes proliferating cells ('Treg SPECIFIC') and non-proliferating cells ('Treg UNSPECIFIC') before culture ('Treg POLY') and after co-culture with antigen-presenting monocytes (insulin or 9-23 peptide) classified according to proliferative capacity. In the arrows, clones were identified as being preferentially stimulated to proliferate during co-culture (the percentage increased noticeably after co-culture compared to 'Treg POLY'). In each culture performed, there were clones with different Vβ expressions, and in each culture, one or fewer clones did not preferentially increase, and the growth rate did not exceed a few percent. Fig. 5 - Functional Test - Indicates the inhibition of T effector lymphocyte proliferation. A subset of generated Tregs (Tregs), e.g., polyclonal (Tregs POLY) cells, antigen-specific (Tregs SPEC) cells for insulin or peptide 9-23, and non-specific (Treg UNSPEC) cells, were co-cultured with non-autologous T effector cells (Effector) given at the bottom of the figure. The effectors were treated with polyclonal (a top chart) or antigen-specific (b bottom chart) reaction cells. Subsequently, the co-cultures were stimulated with irradiated monocytes pre-loaded with insulin or peptide 9-23. The reading was the inhibition of reaction cell proliferation. Results are indexed only for cultures of reaction cells where proliferation was always considered 100%. Results are expressed as mean + / - min-max. Significant differences are indicated by / * and p-values. Fig. 6- Functional Test - Represents the inhibition of IFN-gamma production by T effector lymphocytes (ELISA). Subsets of generated Tregs (Tregs), e.g., polyclonal (Tregs POLY) cells, antigen-specific (Tregs SPEC) cells for insulin or peptide 9-23, and non-specific (Treg UNSPEC) cells, were co-cultured with non-autologous T effector cells (Effector) given at the bottom of the figure. Effectors were treated with polyclonal (a top chart) or antigen-specific (b bottom chart) reaction cells. Subsequently, the co-cultures were stimulated with irradiated monocytes pre-loaded with insulin or peptide 9-23. The reading was the inhibition of IFN-gamma production by the reaction cells. Results are indexed only for cultures of reaction cells where proliferation was always considered 100%. Results are expressed as mean + / - min-max. Significant differences are indicated by / * and p-values. Fig. 7 - Functional Test - Inhibition of IFN-gamma production by T effector lymphocytes - Represents ELISPOT. Subsets of generated Tregs (e.g., polyclonal cells, antigen-specific cells for insulin or peptide 9-23, and non-specific cells) were co-cultured with autologous T effector cells as polyclonal or antigen-specific response cells. Effector Tregs were co-cultured at the ratio given at the top of the figure. Subsequently, the co-cultures were stimulated with irradiated monocytes pre-loaded with insulin or peptide 9-23. The reading was the inhibition of IFN-gamma production by the response cells. Results are indicated by photographs of the cultures and the number of spots in specific wells. Specific details for implementing the invention

[0027] The present invention is illustrated by the following examples, which are not limitations to the invention.

[0028] Materials and Methods

[0029] An overview of the method and functional analysis of the present invention is shown in FIG. 1.

[0030] blood donor

[0031] I obtained a buffy coat from the local blood donation and treatment center in Gdansk.

[0032] B:9-23 Insulin peptides and insulin

[0033] B:9-23 insulin peptide was synthesized at Lipopharm (Gdansk, Poland) as a white powder with a purity of >90% using the HPLC method. The peptide was dissolved in deionized and autoclaved sterile water to a final concentration of 0.5 μg / μl and stored at -70°C for less than 3 months.

[0034] The insulin used in the test was commercially available (Actrapid® Penfill®, Novo Nordisk A / S) and was stored in a refrigerator at 2 to 8°C.

[0035] Cell isolation and classification

[0036] T regulatory cells and T effector cells

[0037] An overview of the preparation and classification of cells is shown in Fig. 2. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy volunteer blood donors by Ficoll-Hypaque gradient centrifugation and used fresh. Treg and Teff were freshly isolated according to the protocols described herein. Briefly, CD4 +T cells were isolated by negative selection using the EasySep Human CD4+ T Cell Enrichment Kit (Stemcell Technologies) according to the manufacturer's instructions. Subsequently, CD4+ T cells were stained with monoclonal antibodies (mAbs) specific to the following antigens: CD3, CD4, CD25, and CD127. Afterward, the cells were sorted using the FACS AriaIIu classifier (BD Biosciences, USA) to determine the Treg phenotype: CD3 + CD4 + CD25 High CD127- / Low lin - doublet - and Teffs' phenotype: CD3 + CD4 + CD25 - CD127 High lin - doublet - Isolated Treg lymphocytes and Teff cells were cultured in separate plates and incubated at 37°C in GMP-compliant culture medium X-VIVO20 (Lonza). The medium was supplemented for 24 hours with heat-inactivated human AB serum (10%), interleukin 2 (IL-2; 2,000 U / ml; Proleukin; Chiron, San Diego, CA), penicillin (100 U / ml), and streptomycin (100 mg / ml).

[0038] monocytes

[0039] Autologous CD14+ cells were isolated with a purity exceeding 95% by positive selection using the EasySep™ Human CD14 Positive Selection Kit II (StemCell Technologies) according to the manufacturer's instructions. The isolated monocytes were then cultured (10 6(cell / well), incubated at 37°C in GMP-compliant culture medium X-VIVO20 (Lonza). A pre-prepared peptide solution (25 μg / well / ml) or insulin (100 μl / well / ml) was added for 24 hours. The inventors [prescribed] B:9-23 insulin peptide (Mo9-23) or insulin (Mo INS Three conditions of monocytes were prepared, either stimulated by ) or unstimulated (Mo).

[0040] Dye labeling and cell expansion

[0041] monocytes

[0042] After 24 hours of incubation, monocytes were collected from the wells, irradiated with gamma rays (under standard conditions for blood product irradiation), counted, and a final concentration of 1x10 6 The cells were resuspended in a new medium (X-VIVO20) at a rate of cell / ml.

[0043] Treg cells and Teff cells

[0044] 24 hours after sorting, T regulatory lymphocytes were collected from the wells and washed with PBS to remove serum affecting staining. Subsequently, the cells were divided into 1 x 10⁶ 6 The cells were resuspended in PBS at a concentration of cell / ml and stained with CFSE (Cell Tracker CFSE Cell Proliferation Kit, Life Technologies) with a final CFSE concentration of 1 to 5 μM. The cells were incubated at 37°C in the dark for 20 minutes, followed by intensive washing several times with PBS and culture medium (X-VIVO20 + 10% serum + penicillin / streptomycin). T effector cells were stained in a similar manner.

[0045] Alternatively, all lymphocyte populations were stained in the above manner with violet (cell tracer violet cell proliferation kit, Life Technologies) at a final concentration of 1 to 5 μM.

[0046] Staining control was performed on a flow cytometer (Fortessa, BDBioscience).

[0047] Polyclonal stimulation

[0048] After the staining step, a portion of the cells were suspended in fresh medium (X-VIVO20, Lonza) containing 10% inactivated human serum and the antibiotic penicillin / streptomycin (Sigma Aldrich). Next, the cells were seeded into a 96-well plate (1 x 10⁶ 5 Cells / well), anti-CD3 antibodies and anti-CD28 antibodies (Treg Expansion Kit, Miltenyi Biotech) were stimulated with magnetic beads coated in a 1:1 ratio (bead:cell) and cultured for 7 days. The inventors of the Treg POLY and Teff POLY Two polyclonal cell conditions were prepared.

[0049] Antigen stimulation

[0050] After the staining step, a portion of the cells were suspended in fresh medium (X-VIVO20, Lonza) containing 10% inactivated human serum and the antibiotic penicillin / streptomycin (Sigma Aldrich). Next, the cells were seeded into a 96-well plate (1 x 10⁶ 5 Monocytes were stimulated with antigen:B:9-23 insulin peptide (Mo9-23) or insulin (MoINS) in a 1:1 (Mo:cell) ratio. Subsequently, sterile anti-CD154 (purified NA / LE mouse anti-human CD154; BD Biosciences) and anti-CD28 (purified NA / LE mouse anti-human CD28; BD Biosciences) were added to the co-culture to a final concentration of 5 µg / ml. The prepared co-culture was incubated at 37°C in 5% CO2 in culture medium (X-VIVO20 + 10% serum + penicillin / streptomycin). The inventors [researched] Treg 9-23 / INS+CD28CD154 and Teff 9-23 / INS+CD28CD154Two conditions were prepared. At the same time, the inventors prepared anti-CD28 and anti-CD154 - Treg 9-23 / INS and Teff 9-23 / INS Cells stimulated with antigen-loaded monocytes were prepared. Cells without monocytes (unstimulated, non-proliferative) were used as a negative control. Polyclonal cells (indexed POLY) were used as a positive control. Cells were cultured for 7 days.

[0051] Classification of antigen-specific cells

[0052] Cells were collected on extension day 7 and washed with fresh medium (X-VIVO). Cells were sorted using a FACS AriaIIu classifier (BD Biosciences, USA) via the SSC-A dot-plot (side scatter) versus 488 nm channel for the Cell Tracker CFSE Cell Proliferation Kit (Life Technologies) or via the SSC-A versus 450 nm channel for the Cell Tracker Violet Cell Proliferation Kit (Life Technologies). Proliferating cells (index PRO) were found to exhibit lower fluorescence intensity than negative control cells in response to antigens presented by monocytes [the cutoff for the sorting gate was assumed to be below the negative control peak fluorescence intensity, with the goal including 5% or fewer of the negative control peak events with the lowest fluorescence intensity]. Non-proliferating cells (index NON) exhibited fluorescence comparable to the cellular fluorescence from the negative control [the sorting gate was assumed to be below the negative control peak fluorescence intensity, with the goal including 80% or more of the negative control peak events]. The obtained cells were quality controlled (phenotype control, functional test for inhibiting the proliferation and production of interferon γ) or further cultured by adding magnetic microspheres coated with anti-CD3 antibody and anti-CD28 (Miltenyi Biotec) antibody in a 1:1 (cell:bead) ratio to obtain as many antigen-specific T regulatory lymphocytes as possible.

[0053] Quality Control

[0054] Phenotype verification

[0055] On extension day 7, samples of Treg and Teff were labeled with antibodies against the following antigens (Ag): CD4, CD25, CD127, CD45RA (BD Biosciences, USA), CD62L (Life Technologies, USA), and FoxP3 (eBioscience, USA) using a FoxP3 staining buffer set, and analyzed by flow cytometry (Fortessa, BD Biosciences, USA).

[0056] Proliferation inhibition test

[0057] On day 7 of expansion, an assay was performed to test the inhibitory function of interferon-γ (IFN-γ) production. Treg and Teff cells were washed with PBS buffer, purified from microspheres used for magnetic stimulation, and counted. Subsequently, the cells were resuspended in fresh culture medium containing antibiotics and human heat-inactivated human AB serum (10%). Cells under individual conditions were continued to be cultured separately for the next 2 days. During this time, IL-2 and activated microspheres were not added. After 48 hours, Teff cells were washed with PBS buffer and counted. Teff cells were stained with CFSE (Cell Trace CFSE Cell Proliferation Kit, Life Technologies, 1 μM, 15 min, 37°C) or violet (Cell Trace Violet Cell Proliferation Kit, Life Technologies, 1 μM, 15 min, 37°C) to analyze their proliferation in the presence of Treg lymphocytes. The choice of dye was determined by the previous staining of Treg lymphocytes—when Treg is stained with CFSE, Teff cells are stained purple, and vice versa.

[0058] Marked autologous Teff cells (reactive cells) were mixed with Treg cells (specific, nonspecific, and polyclonal) in the following ratios: 1:1, 1:½, 1:¼, and 1:⅛. The number of Teff cells was constant each time, while the number of Treg lymphocytes was variable. Cells were suspended in fresh culture medium containing heat-inactivated human AB serum (10%), interleukin 2 (IL-2; 1.00 U / ml), and antibiotics: penicillin (100 U / ml) and streptomycin (100 mg / ml). As a stimulant, the inventors added an antigen (Mo) in a 1:1 ratio with Teff. 9-23 Or Mo INS ) used irradiated autologous monocytes loaded with ). As a positive control, the inventors used monocytes (Mo 9-23 Or Mo INS Microspheres were used that were either Teff (no Treg cells) stimulated by ) or coated with anti-CD3 and anti-CD28 antibodies. As a negative control, the inventors used unstimulated Teff cells (refer to the reading from the cell analyzer). An additional control was unstained Treg cells cultured without Teff cells.

[0059] Cells were cultured for 6 days in culture medium (X-VIVO20 + 10% serum + p / s) at 37°C and 5% CO2. After this time, cells were harvested and analyzed using a flow cytometer (Fortessa, BD Biosciences). Unstimulated Teff cells cultured without Treg cells were used as the background and represented 100%, meaning that 100% of the cells did not divide. Stimulated Teff cells cultured without Treg cells were used as 0%, meaning that proliferation was not inhibited.

[0060] Inhibition of INF-γ production by Teff cells cultured with Treg cells

[0061] For functional tests to confirm the inhibitory effect of Treg cells on IFN-γ secretion by autologous Teff cells, Treg lymphocyte samples were collected from each culture condition. Simultaneously, samples of pre-expanded Teff lymphocytes were collected under the same conditions as the Treg cells tested as response cells in the proliferation inhibition assay. In this way, two series of tests could be performed using the following response cells: polyclonal Teff (Teff POLY ) and antigen-specific (proliferative) Teff(Teff 9-23_PRO or Teff INS_PRO ).

[0062] The cells to be tested (Treg and Teff) were washed with PBS, purified from the microspheres used for stimulation, and counted. Next, the cells were suspended in fresh medium containing heat-inactivated human AB serum (10%) and antibiotics (penicillin / streptomycin). The cells under individual conditions were cultured individually for the next 2 days. During this time, IL-2 and activated microspheres were not added.

[0063] Marked autologous Teff cells (reactive cells) were mixed with Treg cells in the following ratios: 1:1, 1:½, 1:¼, and 1:⅛. The number of Teff cells was constant each time, while the number of Treg lymphocytes was variable. Cells were suspended in fresh culture medium containing heat-inactivated human AB serum (10%), interleukin 2 (IL-2; 100 U / ml), and antibiotics: penicillin (100 U / ml) and streptomycin (100 mg / ml). As a stimulant, the inventors added an antigen (Mo) in a 1:1 ratio with Teff. 9-23 Or Mo INS ) used irradiated autologous monocytes loaded with ). As a positive control, the inventors used monocytes (Mo 9-23 Or Mo INSMicrospheres were used that were either Teff (no Treg cells) stimulated by ) or coated with anti-CD3 and anti-CD28 antibodies. As a negative control, the inventors used unstimulated Teff cells (refer to the reading from the cell analyzer). An additional control was unstained Treg cells cultured without Teff cells.

[0064] Cells were cultured in culture medium (X-VIVO20 + 10% serum + p / s) at 37°C in 5% CO2 for 6 days. After this time, the supernatant was harvested from the lower culture, and IFN-γ levels were measured by ELISA according to the manufacturer's instructions (Human IFN-Gamma OptEIA Kit II, BD Biosciences).

[0065] INF-γ - ELISpot

[0066] Co-cultures of Teff lymphocytes (reactive cells) and Treg lymphocytes were cultured on specialized ELISpot plates for 48 hours. This method allows for the accurate identification of how many cells produced the cytokine (INF-γ). After incubation, the plates were washed to remove the cells and stained according to the manufacturer's instructions (MABtech). Readings were performed using an ELISpot plate reader (Immunospot 5, CTL).

[0067] result

[0068] Effect of obtaining antigen-specific T regulatory cells stimulated with monocytes displaying antigens, anti-CD28 antibodies, and anti-CD154 antibodies

[0069] Analysis of the percentage of Tregs produced by autoantigen-presenting monocytes, i.e., antigen-specific regulatory T cells, showed that the proliferation of these cells was higher when anti-CD28 and anti-CD154 antibodies were added to the co-cultures, particularly when added to the co-cultures containing insulin (difference in t-test between antibody-free and antibody-added cultures: insulin p=0.041, peptide 9-23 p=0.044). (Fig. 3a)

[0070] By comparing the effects of the peptides used on Treg proliferation induced by autoantigen-presenting monocytes, the inventors indicate that the presented peptide 9-23 significantly and more potently increased the percentage of these cells in cultures without anti-CD28 and anti-CD154 antibodies compared to insulin (t-test p=0.032). When anti-CD28 and anti-CD154 antibodies were used in co-culture, the percentage of proliferating Treg lymphocytes was similar in both cohorts (t-test p=0.54).

[0071] Examples of point plots and analysis methods are shown in Figures 3c and 3d.

[0072] Expression of FoxP3 transcription factor by antigen-specific T regulatory lymphocytes stimulated with monocytes presenting antigens, anti-CD28 antibodies, and anti-CD154 antibodies

[0073] In all cultures throughout the entire experiment, the percentage of lymphocytes expressing FoxP3 did not drop below 90%.

[0074] The percentage of Treg cells exhibiting high expression of the FoxP3 transcription factor stimulated by autoantigen-presenting monocytes (i.e., CD3+CD4+CD25highCD127-FoxP3high phenotype) was significantly higher for the antigen-specific / proliferative population compared to the corresponding non-specific / non-proliferative Treg lymphocytes (all t-tests p<0.05) (Figs. 3b to 3d).

[0075] The percentage of Treg cells exhibiting high expression of the FoxP3 transcription factor significantly increased when anti-CD28 and anti-CD154 antibodies were added to the co-culture of both antigen-specific / proliferative Treg cells (difference in t-test with / without antibodies: insulin p=0.034 and trend only for peptide 9-23 p=0.063), as in the case of non-specific / non-proliferative Treg lymphocytes (t-test difference with / without antibodies: insulin p=0.002, peptide 9-23 p=0.042).

[0076] Clonal analysis based on the TCR repertoire of Treg lymphocytes

[0077] Analysis of clonal changes in TCR receptors in antigen-specific / proliferative Treg lymphocyte populations showed that the percentage of one to two clones in each culture increased with different TCR beta specificities each time. Nevertheless, this increase did not exceed tens of percent of all proliferative cells (Fig. 4).

[0078] Functional test - Inhibition of T-effect lymphocyte proliferation

[0079] Analysis of the immune response in the proliferation inhibition assay confirmed the inhibitory effect of all investigated Treg lymphocyte subpopulations (ANOVA, p<0.05) (Fig. 5).

[0080] The efficacy of peptides

[0081] Comparative analysis showed that when the repressed response cells in the experiment were polyclonal effector T cells (ANOVA, F=8.03 p=0.047) (Fig. 5a) and when they were effector T cells specific to the tested antigen (ANOVA, F=20.40 p=0.045) (Fig. 5b), they demonstrated statistically significantly higher Treg performance for peptide 9-23 compared to Treg specific to insulin for the test.

[0082] Polyclonal vs. specific

[0083] The effect of inhibiting proliferation was higher in tests using specific Treg compared to polyclonal Treg, but no statistical significance was reached in any test (ANOVA, p<0.05).

[0084] Nevertheless, it was found that the specific Treg component was primarily responsible for the inhibitory effect in the test. After separating specific Treg (proliferative) from non-specific Treg (non-proliferative), specific Treg inhibited much more T effector cell responses compared to non-specific Treg. This significance was associated with both the test where the responding cells were polyclonal effector T cells (significant only for peptide 9-23: ANOVA, F=8.21 p=0.028; for insulin: ANOVA, F=1.31 p=0.33) and the test where the responding cells were specific T effector lymphocytes (for peptide 9-23: ANOVA, F=186.32 p=0.005; for insulin: ANOVA, F=22.47 p=0.041).

[0085] Functional test - Inhibition of interferon secretion

[0086] Analysis of the immune response in the interferon gamma inhibition assay confirmed the inhibitory effect of all investigated Treg lymphocyte subpopulations (ANOVA, p<0.05) (Figs. 6 and 7).

[0087] The efficacy of peptides

[0088] Comparative analysis showed that when the repressed responder cells in the experiment were polyclonal T effector cells, the performance of 9-23 peptide-specific Tregs was higher compared to total insulin-specific Tregs (ANOVA, F=5.78 p=0.025) (Figs. 6a and 7). When the repressed responder cells in the experiment were T effector cells specific to the tested antigen, a similar difference was observed, but the difference did not reach statistical significance (ANOVA, F=1.86 p=0.22) (Figs. 6b and 7).

[0089] Polyclonal vs. specific

[0090] The response inhibition efficiency was higher in tests using specific Treg compared to polyclonal Treg, but no statistical significance was reached in any test (ANOVA, p<0.05).

[0091] Nevertheless, in the case of peptide 9-23 stimulation, it was found that the specific Treg component was primarily responsible for the inhibitory effect in the assay. After separating specific Tregs (proliferative) from non-specific Tregs (non-proliferative), it was found that Tregs specific to the 9-23 peptide inhibited T effector cell responses statistically significantly more than non-specific Tregs. This significance was associated with both the test in which the responding cells were polyclonal T effector cells (ANOVA, F=5.3 p=0.031) and the test in which the responding cells were T effectors specific to the 9-23 peptide (ANOVA, F=111.84 p=0.0004).

[0092] When the responder cells were insulin-specific effector T cells, stronger inhibition was observed in insulin-specific Tregs compared to non-specific Tregs. Statistical significance of the effect was observed only in some experiments and post-hoc analyses, but the overall analysis was found to be non-statistically significant (ANOVA, F=0.31 p=0.56). There was no difference between insulin-specific Tregs and non-specific Tregs regarding polyclonal responder cells (ANOVA, F=0.0004 p=0.94).

[0093] argument

[0094] The subject of the present invention is an in vitro method for obtaining antigen-specific T regulatory cells, which allows for the clinical use of such cells in the treatment of autoimmune diseases, e.g., multiple sclerosis, rheumatoid arthritis, and type 1 diabetes, and for suppressing unwanted immune responses such as transplant rejection, allergic reactions, and graft-versus-host disease (GVHD). Today, T regulatory cells used in therapy are polyclonal, meaning they recognize different antigens, so their effectiveness may be limited (Marek-Trzonkowska N 2014)(Trzonkowski P 2013)(Marek-Trzonkowska N 2013)(Marek-Trzonkowska N 2012)(Hoffmann P 2009)(Trzonkowski P 2009)(Di Ianni M 2011)(Bluestone JA 2015)(Stelmaszczyk-Emmel A 2015)(Vignali DA 2008)(Geem D 2015). Using the above method, Treg lymphocytes can be targeted to tissues expressing specific antigens and to specific autoreactive lymphocytes responsible for the inflammatory response to specific antigens. The use of antigen-specific Tregs will allow for more precise treatment and a reduction in Treg dosage. As a result, it increases the effectiveness of the treatment and reduces possible side effects.

[0095] The utility of these antigen-specific Treg lymphocytes has been demonstrated in animal models, and the first attempts to obtain such cells in humans appeared a few years ago. Initially, these included induced Treg lymphocytes and Tr1 cells, followed by natural Treg lymphocytes. In the case of Treg, the above methods are based on the use of naturally occurring antigen-presenting cells or cells appropriately prepared from cell lines that present specific antigens. Recently, attempts have also been made to genetically modify artificial lymphocytes (also known as Treg CAR lymphocytes) that are specific to a particular antigen by inserting regulatory receptors.

[0096] The potential for antigen-specific regulation of the immune response is important from a therapeutic perspective. Physiologically, the immune system recognizes and destroys dangerous external antigens while simultaneously enduring its own weaving. Nevertheless, in autoimmune diseases, such as multiple sclerosis (MS), diabetes mellitus type 1 (DM1), psoriasis, systemic lupus erythematosus (SLE), or rheumatoid arthritis (RA), these mechanisms are impaired (Senecal V 2015)(Trzonkowski P 2015)(Marek-Trzonkowska N 2012)(Pujol-Autonell I 2013)(Lima XT 2015)(Mu Q 2015)(Orent W 2015). Effector lymphocytes treat autoantigens that destroy their own tissues as foreign substances and begin to destroy their own organs. This process leads to irreversible changes. Currently, the treatment of autoimmune diseases is largely limited to pharmacological immunosuppression and the suppression of inflammatory responses. However, these therapies have proven ineffective over time. Despite initial improvements, the progression of the disease cannot be completely halted, and discontinuation is generally associated with a worsening of the condition. This treatment is also associated with a severe decline in immunity (Gupta S 2012). Consequently, patients become vulnerable to infection, and those receiving immunosuppressive drugs experience a more severe course than healthy individuals. Non-specific immunosuppression also increases the risk of developing cancer (with a higher percentage among patients receiving immunosuppressive drugs) (Andres A 2005) (Rama I 2010).

[0097] The antigen-specific regulation of immune responses is also an important issue from the perspective of transplantation. Organ transplantation is generally a life-saving procedure, but it requires the continuous use of potent immunosuppressive drugs. Discontinuation of therapy is associated with an increase in the immune response to the tissue of the transplanted organ, which leads to its destruction in the short term. As in the case of autoimmune diseases, the use of immunosuppressive drugs is associated with the occurrence of undesirable and serious side effects. Furthermore, while some of this group of drugs protect the transplanted organ from the destructive effects of the patient's immune system, they simultaneously exert toxic effects on the graft or other tissues. Examples in this study include nephrotoxicity inhibitors of calcineurin (cyclosporine and tacrolimus) used in kidney transplantation (Prokai A1 2015) or rapamycin used in pancreatic islet recipients, which impair the function of transplanted cells (Zhang N1 2006) (Berney T 2009). Issues regarding immunosuppression and the regulation of immune responses are also closely related to bone marrow transplantation. The main difference between organ transplantation and bone marrow transplantation is that in organ transplantation, the transplanted organ is protected from the destructive effects of the recipient's immune system, whereas in bone marrow transplantation, although there is no risk of rejection, the transplanted bone marrow is a source of cells that can attack the body of the recipient and lead to death (Di Ianni M 2011) (Zhao K 2015). Regardless of the origin of the immune system cells attacking the patient's body, the fight against excessive immune responses is also reduced by the use of non-specific immunosuppression. In both cases, namely allogeneic solid organ transplantation and bone marrow hematopoietic cells, allogeneic antigens are strictly defined as stimulating an immune system response, the action of which can be regulated by antigen-specific Treg lymphocytes.

[0098] In this study, the inventors decided to isolate antigen-specific T regulatory cells in vitro from all polyclonal Tregs using antigen-loaded monocytes as antigen-presenting cells (APCs). A diagram of the entire experiment presented is shown in Figure 1.

[0099] Polyclonal Treg cells with a CD3+CD4+CD25highCD127 phenotype grown with autogenous gamma-irradiated monocytes displaying a specific antigen (e.g., insulin or peptide 9-23 insulin beta chain) proliferate only when they have specificity for the antigen presented by the monocytes.

[0100] Many antigens lack affinity for Treg lymphocytes (since these cells are anaerobic), or their phenotypes may be altered during stimulation, leading to a loss of regulatory properties. Therefore, the conditions under which co-culture is performed are crucial for inducing the proliferation of specific Treg cells on the one hand, and maintaining their regulatory and inhibitory properties on the other. Both conditions were met after the addition of anti-CD28 and anti-CD154 antibodies to the co-culture, which provides the Treg cells with a missing second signal. In the presence of anti-CD28 and anti-CD154 antibodies, Treg lymphocytes specific to the presented antigens began to proliferate without losing stability, defined by the expression of the FoxP3 factor (which also increases the expression of this factor) and the activity of functional inhibition (Figs. 5 to 7).

[0101] The sorting of a pure population of antigen-specific Tregene lymphocytes was performed using a FACS cell sorter. The sorting was performed under laboratory conditions (Aria IIu sorter, BD Biosciences) or under clean laboratory conditions where the production of advanced therapeutic products is permitted (Good Manufacturing Practice conditions – GMP-compliant INFLUX sorter). Sorting was made possible thanks to the pre-staining of polyclonal Treg lymphocytes with fluorescent dyes (CFSE or violet). Treg cells that proliferate in response to present antigens, i.e., antigen-specific Tregs, begin to dilute / lose fluorescence intensity, which decreases by approximately half with each subsequent cell division. Based on this change in fluorescence, proliferating cells with low fluorescence intensity (antigen-specific Treg lymphocytes) and non-proliferating cells with high fluorescence intensity (non-specific Treg lymphocytes) can be isolated and sorted (Fig. 2). These sorted cells can be further grown and used for functional testing.

[0102] The proposed method can obtain antigen-specific Treg lymphocytes with a conserved regulatory cell phenotype identified by the expression of the FoxP3 transcription factor, and the activity in functional assays (inhibition of proliferation and inhibition of interferon γ production) is higher than the activity initiating a polyclonal T cell population.

[0103] It should be emphasized that the obtained results do not indicate high clonality of the specific cells obtained. Analysis of the TCR repertoire did not show a significant increase in the percentage of Treg lymphocytes expressing a specific class of TCR receptors (Fig. 5).

[0104] FoxP3(FoxP3 High The high expression of ) can explain the superior inhibitory properties of antigen-specific Treg lymphocytes compared to polyclonal Treg lymphocytes. FoxP3 High Because the immunomodulatory activity of cells is positively correlated with the intensity of FoxP3 factor expression, FoxP3 HighThe cell is well known to be the most inhibitory Treg cell fraction (Marek N 2011) (Ryba M 2011). The antigen specificity of FoxP3 High The fact that a high percentage of cells is induced explains the higher efficacy of therapies in which these cells are used. The cells appear to be activated only by specific antigens, and their action is limited to the tissues where said antigens are expressed. In functional tests, the inventors analyzed the effect of Treg cells on proliferation and the production of interferon γ (IFN-γ) by T effector lymphocytes. The results obtained indicate that antigen-specific Treg lymphocytes tend to inhibit the proliferation of Teff lymphocytes and the production of IFN-γ by these cells compared to polyclonal Treg lymphocytes (Figs. 5 to 7). This inhibition is observed in both polyclonal Teff lymphocytes (Figs. 5a, 6a, and 7) and antigen-specific Teff lymphocytes (Figs. 5b, 6b, and 7) in relation to the same antigens as the Treg used during the test. This system is particularly important because it relates to the in vivo situation during disease, where antigen-specific Teff lymphocytes (autoreactive lymphocytes) are primarily responsible for tissue destruction in autoimmune processes or organ rejection.

[0105] The fact that the antigen-specific Treg lymphocytes generated by the inventors can inhibit specific lymphocytes will have a significant impact on the effectiveness of the therapy.

[0106] conclusion

[0107] Antigen-specific T regulatory cells can be generated using the method of the present invention. Monocytes used in the protocol are loaded with a specific antigen. The use of a combination of anti-CD28 antibody and anti-CD154 antibody to activate the proliferation of antigen-specific Treg lymphocytes stimulates autologous monocytes released along with the antigen.

[0108] Author Contribution

[0109] DI-G, MG, and PT wrote this document. PT designed and planned the experiment. DI-G, MG, and PT performed and analyzed the experiment.

[0110] financing

[0111] This work was supported by the Polish National Research and Development Center: LIDER / 160 / L-6 / 14 / NCBR / 2015 and STRATEGMED1 / 233368 / 1 / NCBR / 2014 and the Polish Ministry of Infrastructure Program: PolTreg SA POIR.01.01.01-00-0769 / 15-01.

[0112] abbreviation

[0113] FACTT, European network activities to focus on and accelerate cell-based resistance induction therapy; Ag, antigen; APC, antigen-presenting cell; CFSE, carboxyfluorescein diacetate succinimidyl ester; COST, European Science and Technology Cooperation; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked immunospot; IFN-γ, interferon γ; INS, insulin; Mo, monocyte; NON, non-proliferative (non-specific); PBMC, peripheral blood mononuclear cell; POLY, polyclonal; p / s, penicillin-streptomycin; PRO, proliferative (antigen-specific); Treg, T regulatory cell; Teffs, T effector cell (responder cell); T1D, type 1 diabetes

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Claims

Claim 1 A method for producing antigen-specific T lymphocytes that are marked and classified with monoclonal antibodies, wherein the lymphocytes are: a) generated using autologous monocytes loaded with the antigen; b) T regulatory lymphocytes or T effector lymphocytes to be generated are suspended in PBS and intracellularly stained with a fluorescent dye; c) subsequently, the lymphocytes are cultured in a dark place; d) subsequently, the lymphocyte cells are intensively washed several times with a culture medium; e) T regulatory lymphocytes or T effector lymphocytes stained with an intracellular fluorescent dye are suspended in a culture medium containing gamma-irradiated autologous CD14+ monocytes loaded with the antigen; f) anti-CD154 antibody and anti-CD28 antibody are added to a co-culture of the T regulatory lymphocytes or T effector lymphocytes and CD14+ monocytes; g) the co-culture is incubated in a culture medium; h) after incubation, the antigen-specific T lymphocytes are classified based on the fluorescence intensity of the intracellular dye, and the fluorescence intensity is fluorescence loss A method that is a marker of antigen specificity in that it is correlated with increased proliferation. Claim 2 In claim 1, the T regulatory lymphocytes or T effector lymphocytes are 1x10 6 A method characterized by being suspended at a concentration of cell / ml PBS. Claim 3 A method according to claim 1, characterized in that the lymphocytes are stained with one of CFSE or violet blue fluorescent dyes having a final concentration of 1 to 5 μM. Claim 4 A method according to claim 1, characterized in that the lymphocytes are incubated at room temperature or 37°C for 20 minutes. Claim 5 A method according to claim 1, characterized in that the autologous monocytes are added to a co-culture at a final monocyte:lymphocyte ratio of 1:

1. Claim 6 A method according to claim 1, characterized in that the monocytes are irradiated with gamma rays. Claim 7 A method according to claim 1, characterized in that the co-culture of the monocytes and lymphocytes is cultured at a constant temperature together with an anti-CD154 antibody having a final concentration of 5 μg / ml and an anti-CD28 antibody having a final concentration of 5 μg / ml. Claim 8 A method according to claim 1, characterized in that the co-culture is cultured in a constant temperature at 37°C in 5% CO2. Claim 9 A method according to claim 1, characterized in that specificity for the antigen is evaluated in a function test in which antigen-specific T lymphocytes are more active than non-specific T lymphocytes, wherein the activity in the case of T regulatory lymphocytes is defined as the suppression of functional T effector lymphocytes, whereas the activity in the case of T effector lymphocytes is defined as increased proliferation intensity and increased production intensity of cytokines and cytotoxic factors. Claim 10 A method according to claim 9, characterized in that the antigen-specific or non-specific T lymphocytes are T lymphocytes classified based on the fluorescence intensity of an intracellular dye. Claim 11 delete

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