Use of TREG cell-derived exosomes in the treatment of psoriasis

Abstract

The invention relates to the use of treg-cell derived exosomes in the treatment of psoriasis.

Description

[0001] USE OF TREG CELL-DERIVED EXOSOMES IN THE TREATMENT OF PSORIASIS

[0002] Technical Field

[0003] The invention relates to the use of treg-cell derived exosomes in the treatment of psoriasis.

[0004] State of the Art

[0005] Psoriasis that some people are genetically predisposed to, is a chronic inflammatory and proliferative autoimmune dermatological disease that is underdiagnosed despite its prevalence and significant impact on quality of life, characterized by erythematous and scaly plaque , involves the skin and joints, often affects the scalp and nails, and is not adequately treated. Beyond skin and joint involvement, psoriasis is also associated with a number of important medical and psychiatric comorbidities, such as psoriatic arthritis, cardiovascular disease, diabetes, malignancy, depression, and anxiety, which require timely treatment to improve long-term outcomes. Psoriasis, which can be seen with many diseases and at any age, affects more than 60 million people worldwide. In 2014, the World Health Organization (WHO) defined psoriasis as a chronic, non-communicable, painful, disfiguring and disabling disease without treatment. It is divided into 2 subtypes as early onset (16-22 years) and late onset (55- 60 years) based on its genetic and immunological characteristics. Although it can be seen at an earlier age in women than in men, it is seen as equal between the genders. Its incidence is higher in the Caucasians and in high income countries. In addition, it is stated that psoriasis increases the likelihood of depression by 20% and this can extend to suicidal tendencies. In particular, the fact that the involvement is in the hands, face and genitals increases its psychological effect very much.

[0006] The progression of non-lesional skin to a fully developed psoriasis lesion or plaque involves complex and still not completely understood interactions between environmental factors and genetic predisposition. The best-defined psoriasis triggers include exposure to environmental pathogens (such as streptococcus), obesity, medications, alcohol consumption, stress, and smoking. In terms of genetic predisposition, more than 80 different genetic risk loci have been shown to predispose to psoriasis to date. With the right triggering in a genetically susceptible individual, a psoriasis plaque develops with the participation of plasmacytoid dendritic cells and type I interferons, which lead to psoriasis. During the formation of psoriasis, significant thickening of the epidermis (acanthosis), stickshaped elongation of the rete pegs and growth and expansion of the blood vessels in the dermal papilla are observed. Various immune cell subsets, including IL-17 secreting group 3 natural lymphoid and Thl7 and Tcl7 cells, IFN-y secreting Thl and Tel cells, and neutrophils, are attracted to the skin along with activated macrophages and dendritic cells. With the inclusion of the lymph nodes that clear out the skin, the initial immune response creates a self-sustaining cycle of inflammation that maintains disease activity. During treatment-related remission, resolved psoriasis plaques contain memory cells located in the tissue, typically CD8+IL- 17-positive or CD8+IFN-y-positive cells. When treatment is stopped or interrupted, these cells reactivate, causing skin inflammation to reappear in the areas of previous involvement.

[0007] The severity of the disease may help guide management. Various topical treatments are safe and effective for mild to moderate disease. More severe disease may require systemic treatment, including phototherapy, acitretin, methotrexate, cyclosporine, or biological therapy.

[0008] In the pathogenesis of psoriasis, keratinocytes (KCs) produce antimicrobial peptides (AMPs) such as P-defensins, SI 00 proteins, and cathelici din.. A disintegrin and metalloprotease domain (ADAMTSL5) containing LL37 and thrombospondin type 1 motif-like are considered autoantigens. LL37-DNA complexes stimulate plasmacytoid dendritic cells (pDCs) and pDCs secrete interferon alpha (IFNa). LL37-RNA complexes stimulate myeloid dendritic cells (mDCs) and mDCs produce tumor necrosis factor alpha (TNFa), interleukin (IL)-23, and IL- 12. IL-23 stimulates Thl7 cells and Thl7 cells produce IL-17 and IL-22 through the JAK- STAT pathway. Langerhans cells (LCs) also secrete IL23 and stimulate Thl7 cell (Figure 1). The aryl hydrocarbon receptor (AhR) in cutaneous vascular endothelial cells (VECs) reduces neutrophil uptake.

[0009] Other medical conditions of the patient should be considered when developing a treatment approach to psoriasis. Because psoriasis progresses with many diseases. Psoriatic arthritis occurs in 10-40% of patients. Recently, psoriasis has been associated with hypertension, obesity, type 2 diabetes, dyslipidemia (sometimes collectively called metabolic syndrome), and cardiovascular disease. Other conditions that are more common in psoriasis patients than the general population include chronic obstructive pulmonary disease, asthma, chronic kidney disease, hepatobiliary cancer, and inflammatory bowel disease. The International Federation of Psoriasis Associations has updated the diagnosis of psoriasis, which is mild, moderate, and severe, to be suitable for topical treatment or systemic treatment. Topical and systemic treatments are applied according to the severity of the disease in psoriasis.

[0010] Topical treatment: If psoriasis is confined to a small area (less than 3-5% of the body surface), the mainstay of treatment is administered to the target with means such as corticosteroids, vitamin D3 analogues, calcineurin inhibitors, keratolytics, and combination topical agents cream, ointment, foam, or gel. Targeted phototherapy is also used. Crude coal tar and dithranol (anthralin) are used in daily treatment centers and dermatology departments with inpatient beds, but are replaced by modem, more cosmetically acceptable topical drugs, if not more effective. A significant disadvantage of topical treatments is poor adherence to treatment.

[0011] Phytotherapy: Ultraviolet radiation is locally immunosuppressive. It directly affects Langerhans cells, inhibits epidermal hyperproliferation and angiogenesis, and causes selective reduction of cutaneous T cells through apoptosis. Different phototherapy methods include narrowband (311-313 nm) ultraviolet B radiation, broadband (280-320 nm) ultraviolet B radiation, targeted phototherapy, and oral psoralen ultraviolet A (photochemotherapy). It is now used less frequently in some parts of the world due to the cumulative risk of skin cancer.

[0012] Oral systemic therapies: Before the advent of biological drugs, oral systemic therapies had been the primary treatment for moderate-to-severe plaque psoriasis for decades. The mechanisms of action, efficacy, and safety profiles of these oral agents differ considerably. Because these small molecules interact with ubiquitous intracellular targets, their actions can be relatively broad compared to biological ones. Oral agents commonly used in psoriasis include methotrexate, cyclosporine, acitretin, fumarates, and apremilast. An oral inhibitor of TYK2 is in late phase development for psoriasis. Methotrexate has been used for more than 50 years to treat psoriasis and psoriatic arthritis. Side effects of methotrexate include bone marrow suppression, nausea, vomiting, hair loss, teratogenicity, and pulmonary toxicity. Cyclosporine is a systemic calcineurin inhibitor used in the short-term treatment of patients with severe plaque psoriasis or in crisis, and as bridging therapy to long-term treatments, such as biological drugs or other oral drugs. Its action is fast, and its effectiveness is solid. It should not be used for more than 1 year due to the risk of irreversible nephrotoxicity. Other side effects of cyclosporine include hypertension, increased risk of infection, nausea, hirsutism, gingival hyperplasia, drug-drug interactions, and electrolyte disorders. Acitretin is a systematic synthetic retinoid that can be used to treat severe plaque psoriasis. It normalizes keratinocyte proliferation and exerts immunomodulatory effects to reduce proinfl ammatory cytokines such as IL-6 and IFN-y. It is sometimes used to treat pustular forms of psoriasis. Fumaric acid esters are small molecules that prevent the maturation of dendritic cells, induce T cell apoptosis, and prevent leukocyte extravasation. Typically, the dose is gradually increased due to significant side effects, such as flushing and diarrhea, seen in 40% of patients. Apremilast is a phosphodiesterase-4 inhibitor licensed for the treatment of moderate to severe psoriasis and psoriatic arthritis. It exerts immunomodulatory effects to reduce proinflammatory cytokines (such as TNFa, IL-2 and IL-12) and increase anti-inflammatory cytokines (such as IL-10). Adverse effects may include nausea, diarrhea, and weight loss.

[0013] Biologicals: They are biological drugs that are mostly recombinant monoclonal antibodies or receptor fusion proteins and target completely human, humanized or human-mouse chimeric and specific inflammatory mediators. Except for infliximab, all biological drugs used in psoriasis are administered by subcutaneous injection. There are 11 biological treatments in four different classes (anti-TNFa, anti-IL-17, anti-IL-12p40 or IL-23p40 and anti-IL-23pl9) currently in use for the treatment of moderate to severe psoriasis. Four anti-TNFa agents are currently used for psoriasis: adalimumab, certolizumab pegol, etanercept and infliximab, etanercept is a fusion protein with the Fc end of IgGl of tumor necrosis factor receptor 2 (TNFRSF1B). Certolizumab pegol is a poly ethylene-gly col-conjugated monoclonal antibody fragment. Pegylation reduces its immunogenicity and extends its half-life, and certolizumab, unlike other biological agents, does not cross the placental barrier. Adalimumab is a completely human antibody and infliximab is a chimeric antibody. Golimumab, a fifth anti- TNFa agent, is currently approved for the treatment of psoriatic arthritis, but not for psoriasis. Three anti-IL-17 agents have been approved: secukinumab, ixekizumab and brodalumab. Both secukinumab and ixekizumab specifically target IL-17A, and brodalumab inhibits IL- 17A, IL-17F, and two other members of the IL-17 cytokine family (IL-17C, IL-17E, or IL-25) by targeting IL- 17 receptor A unit (IL-17RA). Bimekizumab, which targets both IL-17A and IL-17F, is in phase 3 of the clinical study for psoriasis. There are currently four agents targeting IL-23 in clinical use for psoriasis: ustekinumab, which blocks the common p40 subunit of IL-12, and guselkumab, risankizumab, and tildrakizumab, which target the pl9 subunit of IL-23. Mirikizumab, a fourth anti-IL-23pl9 biological, is currently in phase 3 clinical studies (NCT03482011 and NCT03535194). Retinoic acid receptor-associated orphan nuclear receptor gamma t (RORyt or RORc2) is a key transcription factor for Thl7 cell differentiation. Inhibition of RORyt activity is thought to be a promising strategy for the treatment of psoriasis. A phase 2a clinical study of the RORyt inhibitor VTP-43742 was conducted in psoriatic patients. A significant decrease was observed in the severity index (PASI) score of the psoriasis area compared to the placebo. IL-23 plays a key role in maintaining and replicating Thl7 and cytotoxic T cells type 17 (Tcl7) responses. This inhibition of IL-17 responses is thought to be the main mechanism behind the therapeutic efficacy of these drugs. Targeting of highly specific inflammatory mediators by biological agents may enable immune responses to overcome blockade leading to worsening disease with a shift in their clinical and immunological properties. The best feature of paradoxical reactions is the recent onset or worsening of psoriasis during anti-TNFa treatment or anti-IL-6 treatment. These reactions are more common in women and often present as palmoplantar pustulosis. Another phenotypic shift is the transformation of psoriasis into atopic dermatitis with morphology, the presence of itching, and eosinophilia. This shift is seen in all biologicals, but it appears prominent in anti-IL-17 and anti-IL23pl9 classes. Although many biologies are highly effective, not all patients respond in the same way. Some people may have no response at all (primary treatment failure) or, much more commonly, may have an initial response that subsequently disappears within months to years (secondary failure). The duration of treatment discontinuation is variable between different biologic drugs, and dramatically, the risk of treatment failure increases as the number of biologies a patient has previously received increases. The underlying causes of primary and secondary failure are unclear. The development of low-cost biosimilars of biologicals has made access to this class of drugs more affordable. Many biosimilar versions of infliximab, etanercept, and adalimumab are available, and these can provide patients in low- and middle-income countries with the opportunity to receive treatment, as well as providing cost savings in high- income countries. At the grade level, biological treatments anti-IL17, anti-IL12 / 23, anti-IL23 and anti-TNF alpha were found to be significantly more effective than small molecules and non-biological systemic agents in reaching PASI 90. When we compared drug-level treatments, infliximab, ixekizumab, secukinumab, brodalumab, risankizumab and guselkumab were significantly more effective than ustekinumab and three anti-TNF alpha agents (adalimumab, certolizumab and etanercept) in reaching PASI 90. Ustekinumab and adalimumab were significantly more effective than etanercept in reaching PASI 90; ustekinumab was more effective than certolizumab and the clinical efficacy of ustekinumab and adalimumab was found to be similar. No significant difference was found between tofacitinib or apremilast and three non-biological drugs: fumaric acid esters (FAEs), cyclosporine and methotrexate, by meta-analysis also showed that infliximab, ixekizumab, risankizumab, bimekizumab, secukinumab, guselkumab and brodalumab performed better than other drugs in reaching PASI 90 compared to placebo. The clinical efficacy of these drugs was found to be similar except for ixekizumab, which had a higher chance of reaching PASI 90 compared to secukinumab, guselkumab and brodalumab.

[0014] JAK inhibitor therapy: JAK inhibitor blocks the JAK-STAT signaling pathway in Thl7 cells. (Ponesimod, a selective S1P1 agonist, prevents lymphocytes from exiting lymph nodes and reduces the number of peripheral lymphocytes and the transfer of lymphocytes to peripheral tissues. KD025, ROCK2 inhibitor reduces IL-17 secretion in Thl7 cells. The AhR agonist Tapinarof reduces IL-17 and IL-22in Thl7 cells.

[0015] Extracellular vesicles consist of microparticles and exosomes, they are secreted by almost all cell types. Microparticles are secreted by cells directly through the membrane budding, while exosomes are secreted through the endosomal transport pathway. As critical messengers for intercellular communication, exosomes transmit bioactive cargoes to the recipient cells and are involved in various physiopathological processes such as embryonic development, tissue repair and regeneration, material metabolism and immunoregulation. In addition, exosomes play a critical role in regulating tumorigenesis and tumor progression. A more in-depth investigation of disease-state exosomes can contribute to a better understanding of pathogenic mechanisms and help to develop innovative diagnostic or treatment strategies.

[0016] The life process of exosomes involves the production, secretion, transport, and interaction of exosomes with target cells, which are under the control of a number of complex regulatory molecules (Figure 2). A deeper understanding of the life course of exosomes helps to identify the functions of exosomes and to develop strategies for the specific regulation of exosomes. The formation mechanism of exosomes consists of the stages of initiation, endocytosis, formation of multivesicular bodies (MVBs) and secretion. Intracellular trade of MVBs is mediated by Rab GTPases. The fusion of MVBs with the plasma membrane is facilitated by SNAREs. There are three types of interactions between exosomes and cells: (1) membrane proteins on exosomes and target cells bind directly and then trigger the intracellular signaling chain; (2) exosomes carry their contents to the target cells by fusing with the cell membrane; and (3) exosomes are ingested by the cells and broken down by the lysosomes to release the signaling molecules.

[0017] Several studies have investigated the biological effects of MSC-EVs, including their specific cargo and immunomodulatory properties. There is evidence that MSC-EVs contain growth factors and cytokines, including hepatocyte growth factor (HGF), TGFP, interleukin-6 (IL-6) and IL- 10, and thus contribute to the mechanisms of immunoregulation. In contrast, the content of Treg-EVs has not been so extensively researched. Like the original Treg cells, Treg-EVs have been reported to express CD73, CD39, and CD25. Both CD73 and CD39 are involved in the adenosinergic pathway, which plays a key role in modulating immune responses. Meanwhile, CD25 is estimated to promote T-effector apoptosis; however, these assumptions require further study.

[0018] Extracellular vesicles (EVs) are produced by various cells and are present in most biological fluids. They play an important role in cell-cell signaling, immune response and tumor metastasis and also have theranostic potential. They provide many functional biomolecules, including DNA, microRNAs (miRNA), messenger RNA (mRNA), long non-coding RNA (IncRNA), lipids, and proteins, thus affecting different physiological processes in the target cells. Decreased immunogenicity compared to liposomes or viral vectors and the ability to overcome physiological barriers such as the blood-brain barrier make them an attractive and innovative option as diagnostic biomarkers and therapeutic carriers. Mesenchymal stem / stromal cells (MSCs) and regulatory T cells (Tregs) are two types of cells that can produce functional EVs.

[0019] MSC-derived EVs (MSC-EVs) are the most researched. The potential of MSCs for selfrenewal and multi-generational differentiation contributes to the unique therapeutic properties of their paracrine activities. There is also evidence that the conditioned environments of MSCs show similar therapeutic effects to transplanted cells, which means that cytokines, chemokines, and most importantly, EVs stand out as possible key factors determining MSCs. Therefore, MSC-EVs are extensively researched and used to treat a wide range of diseases, including cardiovascular, neurological, immunological and kidney pathologies. It is thought that immune regulation may become a unique tool in the treatment of many pathological conditions from inflammatory and autoimmune diseases to cancer.

[0020] Regulatory T-cells (Treg cells) represent a basic T-cell with the ability to develop selftolerance and balance excessive inflammation. These cells have a central role in preventing autoimmunity and reducing the proinflammatory responses of myeloid cells, T cells and B cells. Most of the emerging clinical data show that Treg therapy shows promise in providing an alternative to existing pharmacological immunosuppressive treatments. While MSCs and their EVs exhibit some immunomodulatory properties, immune cells and, most importantly, regulatory T cells (Tregs) may be a possible source of EV with stronger and specific effects. Tregs are a subpopulation of CD4 + T lymphocytes that, unlike CD8 + cytotoxic T lymphocytes, play a crucial role in establishing immunological self-tolerance. Although the mechanism of its regulatory effects has not been fully elucidated, Tregs have been reported to produce anti-inflammatory factors, including interleukin- 10 (IL- 10), transforming growth factor beta 1 (TGF-P) and IL-13, thus reducing the inflammatory response and inflammatory damage. Accordingly, numerous studies have shown that type 1 diabetes, multiple sclerosis, myasthenia gravis, rheumatoid arthritis and other autoimmune diseases are caused by Treg deficiencies. Meanwhile, Tregs have been reported to promote tumor formation by reducing the anticancer activity of immune cells. Thus, a reduction in Treg activity may facilitate anticancer immune responses in vivo, and targeting Tregs may increase tumor treatment efficacy. In contrast to the extensively studied MSC-EVs, Treg-EV-based studies are still in their infancy. However, Treg-EVs have begun to gain more attention due to their immunosuppressive effects, such as prolonging survival in animal allograft models. To date, there have been 257 clinical trials evaluating the effects of Treg in the treatment of different conditions (www.clinicaltrials.gov), and there are no clinical trials evaluating the effects of Treg-EV. However, both Tregs and their EVs hold great promise in increasing the effectiveness of autoimmune disease treatments or improving the condition of transplant patients.

[0021] Autoimmune conditions are characterized by an abnormal immune response that causes the destruction of healthy cells. They include, but are not limited to, psoriasis, multiple sclerosis (MS), type 1 diabetes (T1D), rheumatoid arthritis (RA), and inflammatory bowel disease (IBD). Allograft rejection also contains an autoimmune component. MSC-EVs are being studied in autoimmune diseases such as psoriasis, multiple sclerosis, type 1 diabetes (UMB- derived MSC-EVs have entered phase II / III clinical trials for the treatment of T1D (NCT02138331)) and rheumatoid arthritis.. Treg-EVs have begun to be studied in allograft rejection and inflammatory bowel disease models, and it is now a very new and untouched field. Regulatory T-cell exosomes can be used in autoimmune diseases, organ transplantation and rejection, immunotherapy and cancer therapy, and inflammatory diseases. Studies are ongoing in these areas.

[0022] Descriptions of the Figures

[0023] Figure 1 : Pathogenesis of Psoriasis. iDC: inflammatory dendritic cell; TNF: tumor necrosis factor; JAK: Janus kinase; STAT: signal transducer and activator of transcription; Neu: neutrophil; HLA: human leukocyte antigen.

[0024] Figure 2: The life course of exosomes. Figure 3: Flow plots showing our strategy used when sorting and the amount of purity during sorting. A) Surface staining of all lymph nodes of 1 FOXP3YFP reporter mouse before and after the sorting is shown. It is a representative plot (of staining performed on a single day). B) Percentages of CD4+ cells and Treg cells were shown after sorting from lymph nodes taken from a single 1 FOXP3YFP reporter mouse.

[0025] Figure 4: Flow plots showing our strategy used when sorting and the amount of purity during sorting. A) Surface staining of the lymphocytes taken from the spleen of 1 FOXP3YFP reporter mouse before and after the sorting is shown. It is a representative plot (of staining performed on a single day). B) Percentages of CD4+ cells and Treg cells were shown after sorting from lymph nodes taken from a single 1 FOXP3YFP reporter mouse.

[0026] Figure 5: A demonstration of the percentages of differentiation by flow cytometry at the end of the 5th day of ex-vivo Treg differentiation from CD4+ FOXP3-cells obtained from mouse lymph nodes. Unstim.; condition in which CD4+FOXP3 -cells are cultured only with IL-2 (50 ng / ml). Stim; Condition in which TGF-P (5ng / ml) and IL-2 (50 ng / ml) were differentiated for 5 days in 96 well plates coated with 2ug / ml CD3 / lug / ml CD28 combination of non-Treg (CD4+FOXP3-) T cells.

[0027] Figure 6: A demonstration of the percentages of differentiation by flow cytometry at the end of the 5th day of ex-vivo Treg differentiation from CD4+ FOXP3-cells obtained from mouse spleen tissue. Unstim.; condition in which CD4+FOXP3 -cells are cultured only with IL-2 (50 ng / ml). Stim; Condition in which TGF-P (5ng / ml) and IL-2 (50 ng / ml) were differentiated for 5 days in 96 well plates coated with 2ug / ml CD3 / lug / ml CD28 combination of non-Treg (CD4+FOXP3-) T cells.

[0028] Figure 7: a) Size of Treg exosomes produced from mouse spleen cells analyzed by SEM b): Size of Treg exosomes produced from mouse lymph node cells analyzed by SEM (Scale bar 200 nm)

[0029] Figure 8: Size of Treg exosomes produced from mouse spleen cells analyzed by NTA

[0030] Figure 9: Size of Treg exosomes produced from mouse lymph node cells analyzed by NTA

[0031] Figure 10: Creation of an animal model of psoriasis

[0032] Figure 11 : a) Intradermal and topical application decreased disease scores according to PASI scores, b) Weight loss in mice was observed due to weight gain. Exosome application did not affect weight change.

[0033] Figure 12: Representation of LN CD4 / CD8 T cells with flow cytometry graphs

[0034] Figure 13: Immune responses in the lymph node were compared

[0035] Figure 14: Immune responses in the spleen tissue were compared Figure 15: The myeloid lineage in the lymph node tissue was examined

[0036] Figure 16: The myeloid lineage was examined

[0037] Figure 17: Intracellular staining of T cell cytokine production capacity in LN tissue was examined.

[0038] Figure 18: Intracellular staining of T cell cytokine production capacity in LN and spleen tissue was examined.

[0039] Figure 19: Intracellular staining of T cell cytokine production capacity in LN and spleen tissue was examined.

[0040] Figure 20: Intracellular staining of T cell cytokine production capacity in spleen tissue was examined.

[0041] Figure 21 : Examination of lymph tissue FOXP3+ cells

[0042] Figure 22: Examination of FOXP3+ cells in spleen tissue

[0043] Figure 23:The expression of inflammation-related cytokines in the samples taken from the skin was examined with real time qPCR.

[0044] Figure 24: Inflammatory cytokine protein levels in the serum were examined by ELISA.

[0045] Figure 25: Inflammatory cytokine protein levels in the tissue with skin lesions were examined by ELISA.

[0046] Brief Description of the Invention

[0047] There is no complete cure for psoriasis. The drugs used are classified as oral systemic, topical, or biological drugs. Side effects and secondary failures of long-term use of drugs cause psychological stress for patients.

[0048] Treatments with exosomes instead of cellular treatment with Treg provide a method that will prevent the development of allogeneic reactions, can be stored long-term, and provide general use without being specific to the person.

[0049] Detailed Description of the Invention

[0050] The critical role of Treg-EVs in Treg-mediated suppression of immune cells, demonstrated in recent in vitro and in vivo studies, opens the possibility of using Treg-EV-based therapies for conditions characterized by immune system over-reactivity. However, successful use of Treg- EV has been reported not only in immune-related disorders and transplantation tolerance models, but also in models of scar formation in myocardial infarction and wound healing. Treg-EV is a very important prediction, suggesting that the range of possible applications is wider than treating extreme immune reactions. The immunosuppressive effect of Treg-EVs has been reported to be beneficial for conditions characterized by immune system overreaction (cytokine storm accompanied by ARDS, autoimmune diseases, allograft rejection).

[0051] In addition to the fact that the treatment method of the invention will be applied as a biological treatment, another advantage is that very effective results have been obtained in the topical application and the possible side effects will not affect the whole body, and no side effects have been observed in animal models in the experiment.

[0052] The invention showed that topical application to the skin, where psoriasis was observed in mice with a psoriasis model of exosomes obtained from the Treg cell, reversed the symptoms of psoriasis.

[0053] CD4+ Foxp3YFP+ Treg cells were isolated from lymph nodes (cervical, axillary, brachial and inguinal) and spleens of Foxp3YFP reporter mice.

[0054] Obtaining Lymphocytes from the Lymph Node

[0055] After the sacrificed mice were fixed to a flat surface, all lymph nodes that could be removed by making an incision from the abdomen were taken into Eppendorf tubes containing antibiotic PBS solution or medium, and the Eppendorf tubes were kept on ice. Then, the lymph nodes were made into a single cell with the help of BD falcon Cell Strainer (70 pm) and these cells were counted with the help of the thoma slide. Cells were sorted according to FACSAria III in the GENKOK Flow laboratory. Therefore, the necessary steps for device calibration and sterilization were performed in order. 96 well plates coated with CD3(2ug / ml) CD28(lug / ml) for 2 hours were used for Treg differentiation.

[0056] Obtaining Lymphocytes from the Spleen

[0057] After the sacrificed mice were fixed to a flat surface, the spleen was removed by making an incision from the abdomen and taken into Eppendorf tubes containing antibiotic PBS solution or medium, and the Eppendorf tubes were kept on ice. Then, the spleens were divided into single cells with the help of a cell disintegrator (Cell Strainer-70 pm in diameter). The supernatant was removed by centrifuging. 1 ml of Red Cell Lysis solution was added and waited for 5 minutes at room temperature. 10 ml of PBS was added and centrifuged at 400 g for 5 minutes and the supernatant was removed. 1 ml of PBS was added to the remaining cells and then these cells were counted with the help of the thoma slide. Then, CD4-Pe-cy7 staining was performed on the cell pellet. After the lymphocyte was obtained, the cells were kept on ice and sterile media and materials were used at all stages of the experiment. Treg Cells are Obtained in Enough Quantity and Number to Suffice for Experiments (Regarding the Number of Cells):

[0058] Regarding Sorted Treg and non-Treg cells:

[0059] In Figure 3 and Figure 4 below, the purity percentages of the samples read before and after the sorting of CD4+ and F0XP3+ cells obtained from LN and spleen cells, respectively, are presented. There is no problem in non-Treg (CD4+FOXP3-) and Treg (FOXP3+) T cell sorting and cell production. As can be seen in the sorting report in Table 2, 3-4 million non- Treg CD4+ T cells are always obtained from the Lymph node (LN) for experiments. The cells are distributed to the wells as at least triplicate.

[0060] For Treg differentiation, 100,000 non-Treg (CD4+FOXP3-) T cells are always planted and differentiation with TGF-P (5ng / ml) and IL-2 (50 ng / ml) is performed for 5 days in 96 well plates coated with 2ug / ml CD3 / lug / ml CD28 combination. Cell culture information is summarized in Table 1. The percentages of differentiation by flow cytometry after differentiation of Treg cells, which we performed ex vivo from both LN and spleen non-Treg (CD4+FOXP3-) T cells, are given in Figure 5 and Figure 6, respectively. As seen in Figure 5, the percentage of Tregs that differ from lymph node cells by culture for 5 days is 61.6, while the percentage of Tregs that differ from spleen cells in Figure 6 is 68.8.

[0061] Table 1: Cell Culture Information Table 2: Cell sorting number information

[0062] Exosome Isolation from the Spleen and Lymph Node

[0063] CD4+FOXP3+ cells we took from the lymph node and spleen were expanded with IL-2 (1000U) for 5 days in 96 well plates coated with 2ug / ml CD3 / lug / ml CD28 combination. In addition, CD4+ FOXP3-cells obtained from the lymph node and spleen were converted into de novo-induced Treg cells for 5 days as mentioned above. On the 5th day, the cells whose differentiation percentages were controlled in flow cytometry were taken to the RPMI complete medium without FBS. After 12 hours, the cells were centrifuged at 300 ref for 3 minutes and supernatants were collected. Secretomes were used in exosome isolation.

[0064] Exosome Isolation Method

[0065] The ExoQuick-TC (#EXOTC50A-1, System Biosciences, USA) kit produced by System Bio Sciences was used as the exosome isolation method. For exosome production, mouse spleen and mouse lymph node CD4+FOXP3+ cells in the culture were incubated with serum-free medium for 18 hours, 12 hours and 6 hours, and the incubation period was decided as 12 hours. As a result of the optimization studies performed with trypan blue, the cells in the culture were kept in a serum -free environment for 12 hours and then the exosome was isolated using their secretomes. For this procedure, the protocol written in the kit procedure was applied. According to the protocol; 3 ml of cell culture medium was collected from a 1x25 cm2 flask. The medium was centrifuged for 15 minutes at 3000x g, and the cells and cell wastes were precipitated.

[0066] The supernatant was taken into a new tube and 4 ml of exosome isolation solution was added for 20 ml of medium, 1 / 5 of the supernatant volume. It was thoroughly vortexed to obtain a homogeneous mixture. Then, it was increased to +4°C and incubated for 24 hours. After incubation, the samples were centrifuged at +4°C for 30 minutes at 1500x g. The supernatant formed after centrifugation was discarded and the pellet was resuspended with 500 ul PBS and increased to -80°C.

[0067] Characterization of Exosomes

[0068] Characterization with SEM

[0069] SEM (Scanning Electron Microscope-Scanning Electron Microscope Zeiss Gemini 500) device was used to analyze the diameter of the exosomes. For this, 30 pl of exosome suspension was dropped on a clean slide and spread with the help of a tip. The sample was allowed to dry at room temperature in a fume hood and then displayed in SEM.

[0070] Characterization with NTA

[0071] The size of Treg exosomes produced from the mouse spleen and lymph node was determined by a Nanoparticle Monitoring Analysis system (NTA, Malvern Instrument Nanosight NS300, UK). The samples were diluted in PBS to a final volume of 1 ml. Measurement concentrations were found by pre-testing the ideal particle value per frame (20-100 particles / frame). Adjustments were made according to the manufacturer's software manual (NanoSight NS300 User Manual, MAN0541-01-TR-00, 2017). The passage of 1 ml samples through the device was detected and the measurement was completed. The results were determined as mean diameter and concentration.

[0072] Creation of an animal model of psoriasis

[0073] The psoriasis model was determined as the model that was accepted to occur in the presence of sharply limited erythematous, easily poured squames and covered plaques in animals. It was created in the C57BL / 6 mouse model using Imiquimod.

[0074] • All mice were shaved on their backs in a rectangular shape with a diameter of 2x2 cm.

[0075] • On days 0-1-2-3-4-5-6, 5% Aldar cream 250 mg was divided into 4 equal parts and applied to the shaved area of each mouse. In addition, he was shaved in the naive group and vaseline cream was applied every day.

[0076] • On days 1-2-3-5-6, the exosome topical group was applied to the back area like a cream, and the intradermal exosome group was given by intradermal injection.

[0077] • Lesion scoring and weight weighing were performed every day.

[0078] • On the 7th day, all were photographed before the slaughter. On days 0-1-2-3-4-5-6, 5% Aldar cream 250 mg was divided into 4 equal parts and applied to the shaved area of each mouse. In addition, vaseline cream was applied every day in the naive group.

[0079] PASI scoring was performed every day before the application. PASI values were recorded in 3 parts: erythema, squamous and induration. Each parameter was evaluated over 4. The total value was calculated over 12 for each day. Psoriasis model was created, and the prophylactic effect of polyclonal Treg-derived exosomes was tested in both intradermal and topical application. Spleen sizes increased due to Aldara application.

[0080] After the mice were sacrificed, a swap was taken from the tissue for microbiome analysis. The treated skin part was divided into 4 equal parts. 2 pieces were appropriately separated for immunohistochemical analysis. Spleen and lymph nodes were removed for analysis in flow cytometry. Blood was taken for the legend plexus, and 1 part of the skin was homogenized with PBS for this procedure. In addition, the last part of the skin was taken into Trisol for expression analysis and subjected to homogenization.

[0081] Our invention has shown that topical application of exosomes obtained from Treg cells to the skin of mice modeled for psoriasis reverses psoriasis symptoms.

[0082] 1- Lymph Node and Spleen Isolation from Mice i. 37 C57BL6 male mice aged 5-10 weeks were used. ii. The animal's limbs were opened and fixed in place, as shown in the figure. Taking care not to pierce the deep tissue, the skin was cut longitudinally from the anus to the jaw. iii. The skin was opened with forceps and fixed to provide easy access to the lymph nodes, as shown in the figure. iv. Accessible lymph nodes were collected from the locations shown in the figure. And it was transferred to a collection container. v. The spleen was taken from the place shown in the figure. It was transferred to a collection container.

[0083] The cells obtained from the collected lymph nodes and spleens were divided equally and made ready for use for flow cytometry. During staining, the real numbers of the cells were calculated by adding a commercial bead (counting bead).

[0084] 2 -Biolegend Rbc Lysis Buffer (lOx)

[0085] (Cat No: 420302) It was purchased as a 1 Ox solution and used by diluting to IX in deionized water.

[0086] Purification of the spleen from red blood cells: i. The spleen of the mouse was homogenized with the help of a filter and taken into a cell suspension. ii. The cells were precipitated by centrifugation (350 x g); the supernatant was discarded. iii. The pellet was resuspended in 1 ml of IX Lysis buffer. iv. It was incubated on ice for 4-5 minutes by stirring occasionally. v. The reaction was stopped by diluting the lysis buffer with 1.5 ml IX PBS. vi. The cells (350 x g) were centrifuged, and the supernatant was discarded. vii. The pellet was resuspended in the appropriate buffer.

[0087] Surface Painting Protocol

[0088] Approximately 1x106 cells were blocked with Fc-block for 5 minutes in a 100 pl staining buffer (supplemented with PBS, 2% FBS). The antibody cocktail was then added and incubated for 30 minutes on ice in the dark. The cells were then washed twice with a stain buffer by rotating at 400g for 3 minutes. The cells were resuspended with 0.2ml 4% paraformaldehyde-PBS fixative. It was incubated at room temperature for 10 minutes. The cells were washed twice with a stain buffer by rotating at 400g for 3 minutes.

[0089] Intracellular cytokine staining (ICC) protocol-BD Cytofix / Cytoperm Plus Protocol

[0090] (Fixation / Permeabilization Kit With BD Golgi Stop)

[0091] (Cat. No: 554715)

[0092] LN and Spleens were isolated and counted. The cells were centrifuged, and the supernatants were discarded. The cells were resuspended in the appropriate buffer.

[0093] A. Stimulating Cells

[0094] The cells were stimulated with PMA-Ionomycin-Golgistop. The stimulation was incubated at 37° C for 4 hours.

[0095] B. Stain Cell Surface Antigens

[0096] The cells were stained by adding 20 pl of stain buffer with an appropriate amount of fluorochrome-conjugated monoclonal antibody specific to the surface antigen (30 minutes, 4°C, ice).

[0097] The cells were washed by treating the pellet with Stain Buffer twice and centrifuged.

[0098] Fixing and permeabilizing cells

[0099] Cells were then fixed and stored to continue intracellular staining. The cells were then thoroughly resuspended and 100 pl per well was added for micro-well plates of the fixation / permeabilization solution for 20 minutes at 4°C in the dark.

[0100] The cells were washed twice in 1 * BD Perm / W ash™ buffer and pellet.

[0101] Staining for Intracellular Cytokines

[0102] The cells fixed / permeable in 20 pl IX BD Perm / Wash™ buffer containing anti-cytokine antibody or appropriate negative control were thoroughly resuspended.

[0103] It was incubated at 4°C in the dark for 30 minutes.

[0104] Before flow cytometry analysis, the cells were washed twice with Stain Buffer.

[0105] Transcription Factor (FOXP3) Staining Protocol

[0106] (Cat. No: 424401)

[0107] Cell surface painting was done (if necessary!). After washing, the supernatant was discarded.

[0108] 200 pl IX fixative was added to each well and incubated at room temperature in the dark for 45-60 minutes.

[0109] The plate was centrifuged at 400 xg for 5 minutes at room temperature, and the supernatant was discarded.

[0110] 200 pl IX Perm Buffer was added to each well and the plate was centrifuged at 400 xg for 5 minutes at room temperature, and the supernatant was discarded. This step was repeated two times.

[0111] An appropriate amount of fluorochrome-conjugated antibody diluted in IX Perm buffer was added to each well and incubated in the dark at room temperature for at least 30 minutes.

[0112] 200 pl IX Perm Buffer was added to each well and the plate was centrifuged at 400xg for 5 minutes at room temperature, and the supernatant was discarded. After washing, the supernatant was discarded. The stain was resuspended in the cells in the buffer.

[0113] All steps were performed at room temperature.

Claims

CLAIMS1. A therapeutic composition for use in treatment of psoriasis, characterized in comprising exosomes derived from regulatory T cells.

2. The therapeutic composition characterized in comprising exosomes derived from regulatory T cells.

3. The therapeutic composition according to claim 2, wherein the composition is intradermal.

4. The therapeutic composition according to claim 2, wherein the composition is topical.

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