Combination of immunomodulatory agent and activated TREG for treating autoimmune diseases

Abstract

The present invention relates to immunologically effective combinations, comprising at least one immunomodulatory agent and a preparation of isolated CD4-stimulated regulatory T cells (activated Tregs) as an immunoregulatory agent for use in the treatment of an autoimmune disease. The immunomodulatory agent used in the invention is preferably a disease- modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms. Furthermore, the invention relates to pharmaceutical compositions, comprising an immunologically effective combination and the use of such pharmaceutical compositions in the treatment of an autoimmune disease.

Description

[0001] COMBINATION OF IMMUNOMODULATORY AGENT AND ACTIVATED TREG FOR TREATING AUTOIMMUNE DISEASES

[0002] Technical Field

[0003] The present invention relates to immunologically effective combinations, comprising at least one immunomodulatory agent and a preparation of isolated CD4-stimulated regulatory T cells (activated Tregs) as an immunoregulatory agent for use in the treatment of an autoimmune disease. The immunomodulatory agent used in the invention is preferably a disease-modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms. Furthermore, the invention relates to pharmaceutical compositions, comprising an immunologically effective combination and the use of such pharmaceutical compositions in the treatment of an autoimmune disease.

[0004] Background Art

[0005] Autoimmune diseases represent a broad and heterogeneous group of disorders characterized by an aberrant immune response in which the body's defense system mistakenly targets its own tissues. This loss of immune tolerance results in chronic inflammation and progressive tissue damage that can affect virtually any organ system. Common autoimmune diseases include of psoriasis, plaque psoriasis and psoriatic arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, Sjogren’s syndrome, diabetes type-1, Crohn’s disease and ulcerative colitis, multiple sclerosis, systemic lupus erythematosus (SLE), non-infectious uveitis, systemic sclerosis, neurosarcoidosis, and others. Although the specific pathology varies from joint destruction in rheumatoid arthritis to demyelination in multiple sclerosis, the underlying mechanism generally involves complex interactions between innate and adaptive immune responses.

[0006] Central to these conditions is an immune dysregulation and the breakdown of self-tolerance, where autoreactive lymphocytes become activated. This leads to the production of autoantibodies and / or the release of inflammatory cytokines (e.g., TNF-alpha, IL-6, IL-17) that perpetuate inflammation and drive tissue injury. Some autoimmune diseases target specific organs such as the pancreas in diabetes type 1 or the myelin sheath in multiple sclerosis, while others, like systemic lupus erythematosus, involve multiple organs simultaneously.

[0007] Although autoimmune diseases differ in their primary trigger, local environment and pathologies, all diseases lead to similar immunological cascades with similar signalingpathways and autoreactive lymphocytes. These components get activated and regulated to varying degrees depending on its underlying autoimmune trigger, the tissue localization and inflammatory environment.

[0008] The management of autoimmune diseases typically involves a combination of symptomatic relief and long-term immunomodulation with small molecules and biologics, called immunosuppressive drugs, disease-modifying drugs, immunomodulating drugs or antiinflammatory drugs. However, not all patients respond adequately to available treatments, and some may experience relapses or significant side effects.

[0009] Natural CD4+CD25+Foxp3+ regulatory T cells (Tregs) represent 4-7% of the CD4+ T cell population and are essential for regulating peripheral tolerance and immune homeostasis. Early phase clinical trials have demonstrated proof-of-principle for the use of isolated and expanded polyclonal Tregs in the treatment of inflammatory disorders, transplant rejection and autoimmune diseases. Tregs exert their suppressive effects via a plethora of mechanisms, acting on various targets. These include modulating the cytokine microenvironment, metabolic disruption of target T cells, regulating the activation capacity of dendritic cells, and direct cytolysis. Tregs have been shown to control autoimmune responses, as well as inflammation and tissue destruction, by their ability to suppress the function of many cell types of the immune system. Consequently, the adoptive transfer of Tregs is regarded as a promising treatment option for undesired immune reactions such as autoimmunity, graft rejection, or graft-versus-host disease (GvHD). Successful prevention or control of antigen-specific immunity by Tregs has been demonstrated in a variety of animal models.

[0010] US 2019 / 309044 A1 describes a method of treating a mammal in need of treatment for rheumatoid arthritis comprising the administration of an inhibitor of the TNF-alpha pathway. One embodiment relates to the combination of C1ORF32 polypeptide with Treg promoting and / or activating methods for treating the subject. Isolation and ex vivo expansion of natural Tregs from a subject can be performed, followed by reintroduction to the subject. In vivo induction and expansion of Tregs is achieved by administration of one or more of anti-CD3 antibodies (aCD3), HADC inhibitors (HADCi) and neuropeptides, such as VIP; agents promoting activation and / or expansion of FOXp3+ Tregs: IL-10, TGFB, IL-2, IL-35, IL-30; or agents promoting secretion of these cytokines, retinoic acid, adenosine, preferably by concurrent administration of C1ORF32 polypeptide. However, C1ORF32 does not bind and activate Tregs, such as a CD4-mediated Treg activator that directly interacts and activates Treg cells.Bulliard Y. et al. (2024, From promise to practice: CAR T and Treg cell therapies in autoimmunity and other immune-mediated diseases, Fron. Immunol. 15:1509956.) describes the use of regulatory T cells to treat immune-mediated diseases. The interaction of gp120 with CD4 on Treg cells triggers the activation and boost their suppressive function. However, boosting the suppressive function of Tregs has no proliferative activity for Tregs.

[0011] Baker KF, Isaacs JD. Ann Rheum Dis (2018; 77:175-187) suggests therapies targeting Treg cells by stimulating Treg populations to abrogate autoimmunity. One area that has gained attention is the use of low-dose recombinant IL-2 to stimulate effector T cells and expand Treg populations.

[0012] A study protocol discloses a clinical trial exploring autologous ex vivo expanded regulatory T cells in ulcerative colitis as an example of an autoimmune disease (“Autologous Ex Vivo Expanded Regulatory T Cells in Ulcerative Colitis”, https: / / clinicaltrials.gov / study / NCT04691232). Concomitant therapy is conducted with oral corticosteroids. However, corticosteroids are not generally classified or approved as disease-modifying drugs (DMD) in the treatment of autoimmune-diseases.

[0013] US 2010 / 196406 A1 discloses the treatment of an autoimmune disease, in particular multiple sclerosis, using a therapeutically effective amount of CXCL11 polypeptide. Also, T cells ex vivo cultured with CXCL11 can be used for the manufacture of a medicament that is identified for treating an autoimmune disease. CXCL11 can be combined as part of a combination therapy with other agents such as Interferon beta 1a, Interferon beta-1 b, Dexamethasone, or Corticotrophin.

[0014] Similar approaches are also disclosed in Maria Dall’Era et al (“Adoptive Treg Cell Therapy in a Patient With Systemic Lupus Erythematosus”, ARTHRITIS & RHEUMATOLOGY, JOHN WILEY & SONS, INC, US, vol. 71, no. 3, 24 January 2019, pages 431-440, XP072790702), Goldberg Rimma et al (“Correction of Defective T-Regulatory Cells from Patients With Crohn’s Disease by Ex Vivo Ligation of Retinoic Acid Receptor-alpha”, GASTROENTEROLOGY, 1 January 2019, pages 1775-1787, XP093303286) and Alhosseini Mahdieh Naghavi et al (“Therapy with regulator T-cell infusion in autoimmune diseases and organ transplantation: A review of the strengths and limitations”, TRANSPLANT IMMUNOLOGY, vol. 85, 4 June 2024; XP093303288).Despite these approaches, clinical data so far show that blocking just one signaling pathway or inflammatory mediator is not enough to effectively control and stop inflammation in autoimmune diseases in all patients. Even combinations of anti-inflammatory drugs, immune modulatory drugs and disease-modifying drugs targeting different signaling pathways show no significant improvement to a monotherapy.

[0015] New strategies to attenuate autoimmune-mediated reactivity and inflammation are urgently needed to decrease and switch off the inflammatory response and to restore immune tolerance without comprehensively suppressing general immunity.

[0016] Object of the Invention

[0017] Against this background, it is the object of the present invention to provide novel approaches for the treatment of autoimmune diseases that are able to suppress immune responses in a subject without specifically targeting immune tolerance mechanisms, and to promote and restore immune tolerance by activating Tregs and thereby suppressing T effector cells.

[0018] This object is solved by an immunologically effective combination, comprising at least one immunomodulatory agent and a preparation of isolated CD4-activated regulatory T cells (activated Tregs). No expansion and proliferation or genetic modulation of isolated cells before transfer is required.

[0019] Disclosure of Invention

[0020] The present invention is based on the surprising finding that a combination of an immunomodulatory agent and activated Tregs leads to attenuated inflammation, while activated regulatory T cells downregulate an autoimmune T cell response. As shown herein, these underlying mechanisms apply to any immunomodulatory agent that suppresses immune responses in a subject without specifically targeting immune tolerance. Based on these principles, the inventive immunologically effective combination provides a novel therapeutic approach resulting in a synergistic effect, and is thus suitable in the treatment of a variety of autoimmune diseases, either by administering a therapeutically effective amount or pharmaceutically effective amount of at least one immunomodulatory agent prior to or concurrently with a preparation of isolated and purified population of activated regulatory T cells to a subject in need thereof. The administering may be repeated as necessary or desired to result in a desired level of attenuated inflammation and autoimmune T cell response.An immunomodulatory agent as used herein is defined as a substance or drug that alters immune system activity, by inhibiting immune responses depending on agent type. Known inhibitory immunomodulatory agents suppress or dampen excessive immune responses, and are often used in autoimmune diseases, transplant rejection, or allergies to prevent tissue damage. The present invention focuses on immunomodulatory agents belonging to the subgroup of disease-modifying drugs (DMD), often also referred to as disease-modifying treatment (DMT). A DMD or DMT is a therapeutic agent that targets the underlying pathophysiology or biological drivers of a disease state but produces only a reversible and non-permanent change in the clinical course or natural history of the autoimmune disease to be treated by the invention. Unlike symptomatic treatments, a DMD is characterized by its ability to slow, delay, or reverse disease progression, such as by reducing the rate of tissue damage or restoring lost biological function.

[0021] A DMD is therefore a therapeutic agent that addresses the underlying pathophysiology or biological drivers of a disease state to achieve a short term reduction of the inflammatory environment and the disease activity, a slowing of disease progression, or a change in the natural history of the autoimmune disease, as distinguished from a symptomatic treatment that primarily provides transient relief of symptoms.

[0022] In one aspect, the invention relates to a new combination of two immunologically effective agents that have not yet been combined. The combination according to the invention comprises an immunomodulatory agent (DMD) and an immunoregulatory agent (activated Tregs). While the immunomodulatory agent DMD is given to a subject as part of a treatment of an autoimmune disease, it suppresses pathologic immune activity and thereby slows, modifies or halts the autoimmune disease. However, this effect is largely reversible: if the application of the drug is stopped and, if inflammation returns, damage progression resumes from that point forward. This means that the medication only causes a short-term improvement of the condition. However, the immunoregulatory agent (activated Tregs) of the inventive combination has the potential for long lasting therapeutic effects. Treg-targeted strategies are the main path toward genuinely durable, mechanism-level long-term tolerance (as opposed to suppression) after a finite number of treatments. Preclinical work with antigen-specific TCR- or CAR-Tregs demonstrates complete, long-lasting reversal or prevention of autoimmunity in murine models and early human trials (e.g., CD19-CAR-Tregs, polyclonal Tregs) show drug-free remissions lasting months to a couple of years in small cohorts. This demonstrates that the combination according to the present invention will havea superior efficacy over all drug combinations of immunomodulatory agents and monotherapies thereof.

[0023] Preferred inhibitory immunomodulatory drugs used in the context of this invention are clinically validated inhibitory DMDs that are approved by health care authorities. An approved DMD is a drug that has successfully met the rigorous safety and efficacy standards of a national or regional regulatory body for a specific "disease-modifying" indication.

[0024] The invention in particular covers me-too drugs, biosimilars and generic drugs exhibiting inhibitory DMD activity.

[0025] As used herein, “me-too drugs” are new branded drugs in the same class with the same mechanism of action as an existing drug (e.g., multiple TNF-inhibitors with similar targets). They are not copies but alternative molecules in the same therapeutic class. These drugs may offer modest differences (PK, dosing, safety, route), but clinical outcomes are often very similar to first-in-class drugs.

[0026] As used herein, “biosimilars” are highly similar versions of a reference biologic that match the reference biologic closely in regard of mechanism of action, PK, dosing, safety and clinical outcome.

[0027] As used herein, “generic drugs” are exact (or essentially exact) copies of a small-molecule originator drug whose patent protection has expired. It is the same active substance with same bioequivalence, dose, route, and clinical effect as the reference product.

[0028] As used herein, “activated Tregs” refers to a cell preparation of isolated CD4-activated or CD4-stimulated regulatory T cells. Activated Tregs are produced by isolating and purifying regulatory T cells from blood products of autologous or allogeneic donors. Activation of regulatory T cells is carried out by using an activator of regulatory T cells such as anti-CD4-specific antibodies or GP120 (HIV-1 glycoprotein gp120). The term “Tregs” as used herein designates regulatory CD4+CD25+ T cells that can be activated via binding of a Treg activator to CD4.

[0029] As used herein, a “subject in need thereof” refers to any subject or individual who could benefit from the administration of an immunologically effective combination or a method of treatment described herein. In certain embodiments, a subject in need thereof is a subject predisposed for the development of an autoimmune disease; a subject having one or moredisorders related to an autoimmune disease but not exhibiting any clinical symptoms; and / or a subject exhibiting symptoms of an autoimmune disease. In one aspect “the subject in need thereof” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, other primates, rodents (i.e., mice, rats, and hamsters), farm animals, sport animals and pets. In one embodiment, the subject is a mammal such as a human. In certain embodiments, the combinations and methods find use in experimental animals, in veterinary application, and / or in the development of animal models for disease.

[0030] A “therapeutically effective amount” or “pharmaceutically effective amount” means the amount of an immunologically effective combination that, when administered to a subject for treating an autoimmune disease, is sufficient to affect such treatment. Thus a “therapeutically effective amount” is an amount indicated for treatment while not exceeding an amount which may cause significant adverse effects. The “therapeutically effective amount” will vary depending on the specific immunologically effective combination, and will also be determined by physical and physiological factors such the age, body weight, and / or clinical history of the subject to be treated. Methods for evaluating the effectiveness of therapeutic treatments are known to those of skill in the art.

[0031] “An immunologically effective combination” means a single composition containing at least one immunomodulatory agent and activated Tregs, or two distinct compositions, wherein the first composition contains at least one immunomodulatory agent, and the second composition contains activated Tregs. The compositions are configured for administering to a subject in need thereof a therapeutically effective amount or a pharmaceutically effective amount of the at least one immunomodulatory agent prior to or concurrently with activated Tregs. The immunologically effective combination can be a combination preparation or a combination product of an immunomodulatory agent and activated Tregs as immunoregulatory agent.

[0032] The immunomodulatory agents as used in the present invention are characterized in that they target signalling pathways in T cells and Tregs only. Consequently, autoimmune indications diseases can be treated which are either T cell mediated or where T cells are involved in pathogenesis.

[0033] All immunomodulatory agents of the present invention have the ability to reduce the activity of pro-inflammatory cytokines, to decrease the activation and proliferation of autoreactive immune cells, and to limit the release of mediators that drive inflammation and tissue damage. It is preferred though that the immunomodulatory agents of the invention qualify asDMD, in particular as an DMD that is approved in the treatment of an autoimmune disorder (see Table 6). The combination of such DMD with activated Tregs as immunoregulatory agent results in a synergistic effect in that the combination is more effective than a monotherapy with single components. In particular, as shown herein, the inventive combination is superior to any monotherapy in the treatment of autoimmune disease.

[0034] The immunomodulatory agents of the present invention contribute to a reduced inflammatory environment, a reduced immune-mediated tissue injury and the modification of the progression of immune-driven diseases. They do not focus on enhancing or restoring regulatory immune tolerance-promoting mechanisms. This is in contrast to immunoregulatory agents that specifically boost regulatory pathways controlled by Tregs and allow to restore immune tolerance.

[0035] The inventive combination enables to rebalance a dysregulated network of immune and inflammatory signals in autoimmune diseases, thereby resolving autoimmune responses and re-establishing immune-inflammatory homeostasis and immune tolerance.

[0036] By using a combination therapy, a broader mechanistic coverage is achieved which benefits treatment. The combination targets biological pathways and mechanisms of disease processes that a single agent does not fully manage. The combined, synergistic effect of the combination of an immunomodulator and activated Tregs is greater than the sum of their individual effects. This synergy will improve treatment outcomes, particularly in complex autoimmune diseases where multiple pathways are involved. A monotherapy may only target one specific component of a disease pathway. However, many autoimmune diseases involve multiple pathways or redundant systems that can bypass a single blockade. The therapeutic application of the combination according to the present invention can cover a wider range of these processes because distinct drugs are used with different mechanisms and modes of action.

[0037] In a first preferred embodiment, the immunomodulatory agent modulates, suppresses or inhibits immune responses in a subject without specifically targeting immune tolerance mechanisms.

[0038] The invention covers immunomodulatory agents, in particular DMDs, that uses pharmaceutical substances or active pharmaceutical ingredients falling under the International Nonproprietary Names (INN). INN facilitate the identification of pharmaceuticalsubstances or active pharmaceutical ingredients. Each INN is a unique name that is globally recognized and is public property. A nonproprietary name is also known as a generic name. Examples of INN used in the present invention include, but are not limited to Tocilizumab, Sarilumab, (IL6R inhibitors), Ixekizumab, Secukinumab, Bimekizumab, Brodalumab (IL17 inhibitors), Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Golimumab, Balinatunfib, (TNF-alpha inhibitors), Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 (PDE4 inhibitors).

[0039] In preferred embodiments, the immunomodulatory agent is selected from the group consisting of IL-6R inhibitor, IL-17 inhibitor, TNF-alpha inhibitor, PDE4 inhibitor, Interferonbeta, fumarate, methotrexate.

[0040] In some embodiments, the IL-6R inhibitor is an anti-IL6R antibody.

[0041] In some embodiments, the IL-17 inhibitor is an anti-IL17R antibody.

[0042] In some embodiments, the TNF-alpha inhibitor is an anti-TNF-alpha antibody.

[0043] In some embodiments, the Interferon is Interferon-beta.

[0044] In further preferred embodiments, the IL-6R inhibitor is Tocilizumab, Sarilumab

[0045] In further preferred embodiments, the IL-17 inhibitor is Secukinumab, Ixekizumab, Bimekizumab, Brodalumab.

[0046] In further preferred embodiments, the TNF-alpha inhibitor Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, or Golimumab.

[0047] In further preferred embodiments, the PDE4 inhibitor is Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124.

[0048] In further preferred embodiments, the Interferon-beta is Interferon beta-1 a, peginterferon beta-1 a, Interferon beta-1 b.

[0049] In further preferred embodiments, the fumarate dimethyl fumarate (DMF), diroximel fumarate, monomethyl fumarate, fumaric acid ester mixtures.In preferred embodiments, the folate antagonist is methotrexate.

[0050] Activated Tregs of the present invention relate to a population of isolated and purified activated natural regulatory T cells that are used as an immunoregulatory agent promoting and restoring immune tolerance and thereby suppressing T effector cells. Regulatory T cells are stimulated by anti-CD4 antibodies or other CD-4 specific agents. In a preferred embodiment, the regulatory T cells are activated by a CD4-specific Treg activator by stimulating Tregs in an ex vivo approach, where Tregs are activated outside of the human body. In such an ex vivo approach, blood of a donor is taken, and Tregs are isolated and subsequently stimulated by addition of a Treg activator.

[0051] In some embodiments, the Treg activator used for activation of regulatory T cells in an ex vivo approach is an anti-CD4 antibody or a functional fragment thereof. In alternative embodiments, the Treg activator is GP120 or a biologically active fragment thereof. A biologically active fragment of GP120 has the ability to bind to its respective CD4 binding site and to promote Treg activation. The invention also comprises polypeptides or proteins bearing the CD4 binding sites of GP120. The invention furthermore comprises variants, mutants and modified versions of GP120. Activation of Tregs by a Treg activator can be carried ex vivo. For example, in an ex vivo approach, blood from a donor can be used to isolate and purify regulatory T cells, and subsequent activation is carried out by a Treg activator. In case of an allogeneic donor, the activated Tregs can then be used for treatment of another patient that is not the donor. In case of an autologous donor, the activated Tregs can be reinfused into the donor.

[0052] Functional fragments of anti-CD4 antibodies are a part of an anti-CD4 antibody comprising a heavy chain polypeptide and / or light chain polypeptide having part or all of the binding activity as the antibody from which the functional fragment is derived. The antibody of the present invention preferably comprises both the heavy chain variable region and the light chain variable region. Examples of the functional fragment include Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), a diabody, a triabody, a tetrabody, and a minibody. Preferably, the antibody fragment is a Fab fragment. The antibodies or fragments thereof have the ability to bind to a CD4 epitope on T cells, wherein binding of the antibody or fragment thereof results in an activation of regulatory T cells. The antibody of the present invention is preferably derived from a vertebrate such as a human, mouse, rat, cow, rabbit, goat, sheep, and guinea pig. In a preferred embodiment, the anti-CD4 antibody is a humanized monoclonal antibody.In a further preferred embodiment, the anti-CD4 antibody is Tregalizumab or Palixizumab.

[0053] The immunologically effective combination of the invention is also suitable to be used in a method of treatment of an autoimmune disease. The invention relates to methods of treating an autoimmune disease comprising the administration of an immunologically effective combination, comprising at least one immunomodulatory agent and a preparation of isolated CD4-activated regulatory T cells (activated Tregs) to a subject in need thereof, wherein said immunomodulatory agent is a disease-modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms.

[0054] Preferably, the immunomodulatory agent and the activated Tregs cell preparation in the immunologically effective combination are provided in a single dose form or in two distinct dose forms. In a preferred embodiment, the at least one immunomodulatory agent is provided in a first dosage form and the activated Tregs are provided as a second dosage form, wherein a dosage regimen provides for the first dosage form to be administered to a subject simultaneously with or prior to the second dosage form. In preferred embodiments the autoimmune disease to be treated is psoriasis or multiple sclerosis.

[0055] The present invention also comprises the administration of more than one immunomodulatory agent as described herein. Preferably, the autoimmune disease is any one of psoriasis, plaque psoriasis, psoriatic arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, Sjogren’s syndrome, diabetes type-1, Crohn’s disease and ulcerative colitis, multiple sclerosis, systemic lupus erythematosus (SLE), non-infectious uveitis, systemic sclerosis, neurosarcoidosis.

[0056] In preferred embodiments, for the treatment of

[0057] a. psoriasis and plaque psoriasis, the immunomodulatory agent is TNF-alpha inhibitor, IL-6R inhibitor, IL-17 inhibitor, PDE4 inhibitor, Interferon-beta, fumarate or folate antagonist;

[0058] b. rheumatoid arthritis and juvenile idiopathic arthritis, the immunomodulatory agent is TNF-alpha inhibitor, IL-6R inhibitor, IL-17 inhibitor, PDE4 inhibitor, or methotrexate;

[0059] c. Sjogren’s syndrome, the immunomodulatory agent is PDE4 inhibitor, or methotrexate;

[0060] d. diabetes type-1, the immunomodulatory agent is PDE4 inhibitor; e. Crohn’s disease and ulcerative colitis, the immunomodulatory agent is TNF- alpha inhibitor, IL-6R inhibitor, PDE4 inhibitor, or methotrexate;f. multiple sclerosis, the immunomodulatory agent is TNF-alpha inhibitor, IL-6R inhibitor, IL-17 inhibitor, PDE4 inhibitor, Interferon-beta, fumarate, or methotrexate;

[0061] g. psoriatic arthritis, the immunomodulator agent is TNF-alpha inhibitor, IL-17 inhibitor, PDE4 inhibitor, fumarate or folate antagonist;

[0062] h. systemic lupus erythematosus (SLE), the immunomodulatory agent is PDE4 inhibitor, or methotrexate;

[0063] i. non-infectious uveitis, the immunomodulatory agent is TNF-alpha inhibitor, PDE4 inhibitor, or methotrexate;

[0064] j. systemic sclerosis, the immunomodulatory agent is methotrexate; k. neurosarcoidosis, the immunomodulatory agent is TNF-alpha inhibitor, or methotrexate.

[0065] Preferred immunomodulatory DMD agents are summarized in Table 1 for the indicated medical indications in combination with a Treg cell preparation according to the invention:

[0066] Table 1:

[0067] Psoriasis and Plaque psoriasis

[0068] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0069] IL-6R Inhibitors Tocilizumab, Sarilumab

[0070] IL-17 Inhibitors Secukinumab, Ixekizumab, Bimekizumab, Brodalumab

[0071] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 Interferon-beta Interferon beta-1 a, peginterferon beta-1 a, Interferon beta-1 b Fumarates dimethyl fumarate, diroximel fumarate, monomethyl fumarate, fumaric acid ester mixtures

[0072] Folate antagonist Methotrexate

[0073] Rheumatoid Arthritis and Juvenile idiopathic arthritis

[0074] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0075] IL-6R Inhibitors Tocilizumab, Sarilumab

[0076] IL-17 Inhibitors Secukinumab, Ixekizumab, Bimekizumab, Brodalumab

[0077] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 Folate antagonist Methotrexate

[0078] Sibaren’s Syndrome

[0079] Folate antagonist Methotrexate

[0080] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124

[0081] Diabetes Type-1PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124

[0082] Crohn’s Disease and Ulcerative Colitis

[0083] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0084] IL-6R Inhibitors Tocilizumab, Sarilumab

[0085] Folate antagonist Methotrexate

[0086] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124

[0087] Multiple Sclerosis

[0088] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0089] IL-6R Inhibitors Tocilizumab, Sarilumab

[0090] IL-17 Inhibitors Secukinumab, Ixekizumab, Bimekizumab, Brodalumab

[0091] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 Interferon-beta Interferon beta-1 a, peginterferon beta-1 a, Interferon beta-1 b Fumarates dimethyl fumarate, diroximel fumarate, monomethyl fumarate, fumaric acid ester mixtures

[0092] Folate antagonist Methotrexate

[0093] Psoriatic Arthritis

[0094] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0095] IL-17 Inhibitors Secukinumab, Ixekizumab, Bimekizumab, Brodalumab,

[0096] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 Fumarates dimethyl fumarate, diroximel fumarate, monomethyl fumarate, fumaric acid ester mixtures

[0097] Folate antagonist Methotrexate

[0098] Systemic Lupus Erythematosus

[0099] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 Folate antagonist Methotrexate

[0100] Uveitis, non-infectious

[0101] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0102] Folate antagonist Methotrexate

[0103] PDE4 Inhibitors Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124

[0104] Systemic Sclerosis

[0105] Folate antagonist Methotrexate

[0106] Neurosarcoidosis

[0107] TNF-alpha Inhibitors Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab

[0108] Folate antagonist MethotrexateTable 1 summarizes possible selections of immunomodulatory agents together with activated Tregs. The invention also comprises the selection of more than one immunomodulatory agent for a combination therapy with activated Tregs for the treatment of the indicated autoimmune diseases.

[0109] For example, for the treatment of psoriasis and plaque psoriasis, the use of any one of TNF-alpha Inhibitors such as Adalimumab (mAb), Etanercept (Fusion protein TNFR-lg), Infliximab (mAb), Certolizumab pegol (mAb-PEG conjugate), Balinatunfib (small molecule) Golimumab (mAb); IL 17 Inhibitors such as Secukinumab (mAb IL17A), Ixekizumab (mAb IL17A), Bimekizumab (mAb IL17A / F), Brodalumab (mAb IL17 RA); PDE4 Inhibitors such as Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124; Interferonbeta such as Interferon beta-1 a, peginterferon beta-1 a, Interferon beta-1 b; Fumarates such as dimethyl fumarate, diroximel fumarate, monomethyl fumarate, fumaric acid ester mixtures; Folate antagonists such as methotrexate in combination with activated Tregs is preferred.

[0110] As a further example, for the treatment of multiple sclerosis, the use of any one of IL 17 Inhibitors such as Secukinumab (mAb IL17A), Ixekizumab (mAb IL17A), Bimekizumab (mAb IL17A / F), Brodalumab (mAb IL17 RA); Fumarates such as dimethyl fumarate, diroximel fumarate, monomethyl fumarate, fumaric acid ester mixtures; IL-6R Inhibitors such as Tocilizumab (mAb, IL6R), Sarilumab (mAb, IL6R); TNF-alpha Inhibitors such as Adalimumab (mAb), Etanercept (fusion protein TNFR-lg), Infliximab (mAb), Certolizumab pegol (mAb-PEG conjugate), Balinatunfib (small molecule), Golimumab (mAb); PDE4 Inhibitors such as Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124; Interferonbeta such as Interferon beta-1 a, peginterferon beta-1 a, Interferon beta-1 b in combination with activated Tregs is preferred.

[0111] As a further example, for the treatment of rheumatoid arthritis, the use of any one of TNF-alpha Inhibitors such as Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab; IL-6R Inhibitors such as Tocilizumab, Sarilumab; Folate antagonists such as methotrexate; PDE4 Inhibitors such as Nerandomilast, Apremilast, Roflumilast, Zatolmilast, Orismilast, PF-07038124 in combination with activated Tregs is preferred.

[0112] As a further example, for the treatment of systemic sclerosis, the use of methotrexate in combination with activated Tregs is preferred.

[0113] Interestingly, despite the distinct molecular interactions (mechanism of action), the immunomodulatory agents have a common mode of action e.g., ameliorating theinflammatory environment, attenuate cellular activation and / or proliferation of reactive immune cells and reduction of pro-inflammatory cytokines or mechanisms. Since these inflammatory processes are characteristic for autoimmune diseases, these agents are suitable for use in various autoimmune diseases. This transferability and generalization of agents having a common mode of action is also evident when comparing the standard therapies for autoimmune indications. Many agents such as TNF-alpha inhibitors are used in the context of different autoimmune diseases, including, but limited to psoriasis, rheumatoid arthritis, Crohn’s disease and ulcerative colitis, and multiple sclerosis.

[0114] The inflammatory environment plays a major role in both the development of autoimmune diseases and the failure of immunological regulation. Inflammation can convert normally harmless self-recognition into a pathogenic immune response and can also weaken the mechanisms that normally maintain immune tolerance. Thus, inflammation is not only a consequence of autoimmunity but also a key driver of immune dysregulation.

[0115] This transferability of agents is also shown by the data presented herein. The panel of agents applied in this patent application have different mechanisms of action, but a common mode of action and subsequently show synergistic efficacy on T cells from healthy donors and autoimmune patients. Interestingly, the data show that some agents only become effective in additional diseases through the combination with activated Tregs.

[0116] The present invention also relates to pharmaceutical compositions, comprising an immunologically effective combination as defined herein and a pharmaceutically acceptable carrier or diluent.

[0117] In one aspect, the pharmaceutically composition comprises an immunologically effective combination as defined herein and a pharmaceutically acceptable carrier or diluent for the use in the treatment of an autoimmune disease.

[0118] In a preferred embodiment, the pharmaceutical composition comprises an immunologically effective combination comprising at least one immunomodulatory agent and a preparation of isolated CD4-activated regulatory T cells (activated Tregs), wherein said immunomodulatory agent is a disease-modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms and wherein said disease-modifying drug (DMD) is selected from the group consisting of IL-6R inhibitors, IL-17 inhibitors, TNF-alpha inhibitors, PDE4 inhibitors, Interferon, fumarates, folate antagonists.In a preferred embodiment, the immunomodulatory agent and the activated Tregs in the immunologically effective combination are provided in a single dose form or in two distinct dose forms. The one embodiment, the single dose form contains both the immunomodulatory agent and the activated Tregs. In an alternative embodiment, one dose form contains the immunomodulatory agent and another dose form contains the activated Tregs.

[0119] The pharmaceutical composition may be formulated with any known pharmaceutically acceptable carrier or diluent as well as any other known adjuvants and excipients in accordance with conventional techniques. The pharmaceutically acceptable carriers, diluents, adjuvants and excipients should be suitable for the chosen inductor of the present invention and the chosen mode of administration. A pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e. g., a nonionic detergent), stabilizers (e.g., sugars or protein-free amino acids), preservatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition. In a preferred embodiment, the activated Tregs are provided in sterile NaCI + 2% HSA (human serum albumin) in form of an intravenous infusion. In a preferred dosage form, the osmolarity and ion concentrations of the solutions are isotonic to match the milieu of the human body.

[0120] The pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, such pharmaceutical compositions can be used parenterally, as injections which are sterile solutions or suspensions including the compositions along with water or another pharmaceutically acceptable liquid. For example, such compositions may be formulated as unit doses that meet the requirements for the preparation of pharmaceuticals by appropriately combining the compositions with pharmaceutically acceptable carriers, diluents, adjuvants or excipients, specifically with sterile water, physiological saline, a vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder or such. In such preparations, the amount of active ingredient is adjusted.

[0121] The invention also covers methods of treating an autoimmune disease comprising the administration of an immunologically effective combination, comprising at least one immunomodulatory agent and a preparation of isolated CD4-activated regulatory T cells (activated Tregs) to a subject in need thereof, wherein said immunomodulatory agent is a disease-modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms.The two immunologically active components of the combination of the present invention, i.e., the immunomodulatory agent and the activated Tregs can be combined in a single composition for use in the treatment of an autoimmune disease. In alternative embodiments, the immunomodulatory agent and the activated Tregs are provided separately in two distinct compositions.

[0122] In a first aspect, the composition containing the immunomodulatory agent and the composition containing the activated Tregs can be combined prior to administration to a subject in need thereof. In a second aspect, the composition comprising the immunomodulatory agent and the composition comprising the activated Tregs are administered as a single dose or repeated dose to a subject in need thereof. In a third aspect, the composition comprising the immunomodulatory agent and the composition comprising the activated Tregs are administered to a subject in need thereof within a desired time interval. For example, the composition comprising the immunomodulatory agent is given to a subject in need thereof at a first time point, while the composition comprising the activated Tregs is given to a subject in need thereof at a second time point within a given time interval. It is important to suppress the inflammation / inflammatory environment sufficiently by the pre-treatment with the immunomodulatory agent before administration of the activated Tregs as immunoregulatory agent. A highly inflammatory environment interferes with Treg function and reduces Treg suppressive activity. Only the immunomodulator efficacy makes the activated Tregs efficacy possible resulting in a combined synergistic impact.

[0123] Preferably, the time interval for administering a single or repeated dose of the first composition comprising the immunomodulatory agent and administering a single or repeated dose of the second composition comprising the activated Tregs is a simultaneous administration, at least 24 hours, 7 days, preferably at least 14 days, more preferably at least 30 days. In more preferred embodiments, the time interval for administering a single or repeated dose of the first composition comprising the immunomodulatory agent and administering a single or repeated dose of the second composition comprising the activated Tregs is at least 4 weeks, preferably between 4 weeks and 6 weeks. The administration of the first and / or second composition can be repeated several times. In addition, the number of administrations, the composition and concentration of the compositions comprising the immunomodulatory agent and / or activated Tregs can vary. For example, the individual doses given to a subject may vary and depend, inter alia, from gender, age, constitution and state of illness.

[0124] In some embodiments, in the pharmaceutical composition of any one of embodiments disclosed herein, the immunomodulatory agent is in the same dosage form as the activatedTregs such that the immunomodulatory agent is administered concurrently with the activated Tregs. In a preferred embodiment, the dosage form is a single fluid. In some embodiments, the dosage form including the immunomodulatory agent and the activated Tregs is prepared by combining a composition including the immunomodulatory agent and a composition including the activated Tregs. In some embodiments, in the pharmaceutical composition of any one of embodiments disclosed herein the immunomodulatory agent is in a separate dosage form from the activated Tregs such that the immunomodulatory agent can be administered before or after the activated Tregs. A delayed administration of the activated Tregs relative to the immunomodulatory agent is preferred.

[0125] The invention also relates to the use of an immunologically effective combination comprising at least one immunomodulatory agent and activated Tregs for the manufacturing of a medicament that promotes and restores immune tolerance in a human or animal subject. The manufacturing process can involve the preparation of a first dosage form, in which the immunomodulatory agent is provided, and a second dosage form, in which the activated Tregs are provided, while both dosage forms are prepared to be administered to a subject in need thereof.

[0126] In one embodiment, autologous (patient-derived) Tregs are isolated from the patient’s own blood (peripheral blood mononuclear cells, PBMC).

[0127] In another embodiment, allogeneic 3rdparty Tregs are taken from a healthy donor.

[0128] In a preferred process, Tregs are isolated using anti-CD25 antibodies coupled to magnetic beads. However, also antibodies targeting alternative surface molecules of Tregs can be used. In alternative embodiments, fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) are used to isolate and purify Tregs. If required, Tregs are cultured ex vivo with IL-2 and anti-CD3 / CD28 to extend Treg population. In some arrangements, the culturing medium can include rapamycin to favor Tregs over conventional T cell expansion. The purity of the isolated Tregs can be checked using Treg markers and testing suppressive function. Furthermore, a screening can be conducted to detect contaminations or malfunctions before infusion. Preferably, the medicament containing activated Tregs is provided in form that allows intravenous infusion (IV). The clinical response can be monitored, for instance, by analysis of biomarkers, and symptom relief.

[0129] In some embodiments, Tregs can be delivered directly to the affected tissue to enhance efficacy and reduce systemic immunosuppression by intraarticular, intrathecal orintraperitoneal injection. In some embodiments, activated Tregs can be encapsulated in biomaterial scaffolds or hydrogels. In some aspects, microgel or nanogel technology could help sustain Treg release and activity over time.

[0130] Brief Descriptions of the Drawings

[0131] Figure 1: Synergistic effects of combinations of different modulators and activated Tregs of T cells from healthy controls.

[0132] T cells from healthy donors (HC) were co-cultured with allogeneic dendritic cells (mixed lymphocyte reaction) in the absence or presence of activated Tregs from a third independent healthy donor (act. Tregs). Additionally, different modulators were added to these cultures. A) summarizes all combinations of modulators and act. Tregs that exhibited synergistic effects. Upper row from left to right: anti-human TNF-alpha (50 ng / ml), recombinant human IFN-beta (1 IU / ml), PDE4 inhibitor (PDE4i, 2 pM); lower row from left to right: Dimethyl fumarate (DMF, 2 pM), anti-human IL-6R (30 ng / ml), methotrexate (MTX, 10nM). B) shows the combination of anti-human IL-17 (50 ng / ml) and activated Tregs, which did not exhibit synergistic effects. T cell proliferation was measured either on day 4 of culture by 3H-Tdr incorporation using a liquid β-scintillation counter or on day 6 by analysis of CFSE dilution using flow cytometry. Bars represent mean proliferation (%) ± SEM.

[0133] Figure 2: Synergistic effects of combinations of different modulators and activated Tregs on proliferation of T cells from patients with multiple sclerosis.

[0134] T cells from multiple sclerosis patients (MS) were co-cultured with allogeneic dendritic cells (mixed lymphocyte reaction) in the absence or presence of activated Tregs from a third independent healthy donor (act. Tregs). Additionally, different modulators were added to these cultures. All tested combinations of modulators and act. Tregs exhibited synergistic effects. Upper row from left to right: anti-human TNF-alpha (50 ng / ml), recombinant human IFN-beta (1 IU / ml), PDE4 inhibitor (PDE4i, 2 pM); lower row from left to right: Dimethyl fumarate (DMF, 2 pM), anti-human IL-6R (30 ng / ml), anti-human IL-17 (50 ng / ml). T cell proliferation was measured on day 4 of culture by 3H-Tdr incorporation using a liquid p-scintillation counter. Bars show mean proliferation (in %) ± SEM.

[0135] Figure 3: Synergistic effects of combinations of different modulators and activated Tregs on proliferation of T cells from patients with psoriasis.T cells from psoriasis patients (PSO) were co-cultured with allogeneic dendritic cells (mixed lymphocyte reaction) in the absence or presence of activated Tregs from a third independent healthy donor (act. Tregs). Additionally, different modulators were added to these cultures. All tested combinations of modulators and act. Tregs exhibited synergistic effects. Upper row from left to right: anti-human TNF-alpha (50 ng / ml), recombinant human IFN-beta (1 IU / ml), PDE4 inhibitor (PDE4i, 2 pM); lower row from left to right: dimethyl fumarate (DMF, 2 pM), anti-human IL-6R (30 ng / ml), anti-human IL-17 (50 ng / ml). T cell proliferation was measured on day 4 of culture by 3H-Tdr incorporation using a liquid p-scintillation counter. Bars show mean proliferation (in %) ± SEM.

[0136] of Embodiments

[0137]

[0138] The present invention is further illustrated in the following examples. By no means shall the present invention be restricted to these specific examples. Also, the combination of features or even embodiments are encompassed by the spirit of the present invention.

[0139] A standard suppression assay was used to determine the T cell modulating activity of immunomodulatory agent alone or in combination with activated Tregs. The data of Figures 1 to 3 show the result of a mixed leukocyte reaction (MLR). Isolated T cells from the blood products of healthy donors (HC), patients with multiple sclerosis (MS), and psoriasis patients (PSO) were stimulated with allogeneic dendritic cells (DC) from independent, healthy donors. The black bars show the optimal proliferation of the alloreactive T cells (from HC and patients), defined as 100% of T cell proliferation. To suppress T cell proliferation, regulatory T cells activated with HIV-1 GP120 (Treg activator) and various immunomodulatory agents (anti-TNF-a, IFN-beta, PDE4 inhibitor, DMF, anti-IL-6R, MTX, anti-IL-17) were used alone or in combination with activated Tregs. The influence of these biologicals or combinations of biological and activated Tregs is shown as T cell proliferation in percentage (%).

[0140] Figure 1 shows the synergistic effects of activated Tregs with different modulators on proliferation of T cells from healthy controls.

[0141] T cells from healthy donors (HC) cocultured with allogeneic dendritic cells (mixed lymphocyte reaction) in the absence or presence of activated Tregs from a third independent healthy donor are shown. Different modulators were added to these cultures. A) summarizes all combinations of modulators and activated Tregs that exhibited synergistic effects. Upper rowfrom left to right: anti-human TNF-alpha (50 ng / ml), recombinant human IFN-beta (1 IU / ml), PDE4 inhibitor (PDE4i, 2 pM); lower row from left to right: Dimethyl fumarate (DMF, 2 pM), anti-human IL-6R (30 ng / ml), methotrexate (MTX, 10 nM). B) shows the combination of antihuman IL-17 (50 ng / ml) and activated Tregs, which did not exhibit synergistic effects.

[0142] T cell proliferation was measured using two different methods. On day 4 of culture, 37 kBq [3H] Thymidine (3H-Tdr) was added to each well and cells were incubated for an additional 16 h. T cell proliferation was then quantified by measuring3H-Tdr incorporation using a liquid beta-scintillation counter. In some experiments, T cell proliferation was assessed on day 6 by analyzing CFSE dilution using flow cytometry. Bars show mean proliferation (in %) ± SEM.

[0143] Figure 1 A (upper row, left) shows that anti-TNF-alpha and activated Tregs inhibit T cell proliferation by approximately 40%. In combination (anti-TNF-alpha + activated Tregs), T cell proliferation is suppressed by up to 80%. Anti-TNF-alpha and activated Tregs inhibit T cell activation synergistically.

[0144] Figure 1 A (upper row, middle) shows the effect of IFN-beta and activated Tregs on the proliferation of T cells individually and in combination. Both factors suppress T cell proliferation to a comparable extent. In combination (IFN-beta + activated Tregs), the synergistic effect is clearly visible. The combination of both components inhibits T cell proliferation significantly stronger than either agent alone.

[0145] Figure 1 A (upper row, right) shows the influence of PDE4 inhibitors and activated Tregs on T cell proliferation. Individually, both factors (PDE4 inhibitor or activated Tregs) exert a comparable suppressive effect on T cell proliferation (approx. 40%). In combination (PDE4 inhibitor + activated Tregs), this suppression of T cell proliferation increases to up to 80%. PDE4 inhibitors and activated Tregs synergistically suppress the proliferation of activated T cells.

[0146] Figure 1 A (lower row, left) shows the suppressive effect of activated Tregs in combination with DMF. While DMF alone can hardly influence T cell proliferation, the combination of DMF + activated Tregs shows a clear synergistic effect. The combination of the biological and activated Tregs suppresses T cell proliferation significantly more strongly than either factor alone.

[0147] Figure 1 A (lower row, middle) shows the results of the assay in presence / absence of anti-IL-6R. In contrast to activated Tregs, anti-IL-6R alone has only a marginal effect on T cellproliferation. However, the addition of activated Tregs increases significantly the suppressive effect of anti-IL-6R on T cell proliferation, indicating a clear synergistic interaction.

[0148] Figure 1 A (lower row, right) shows the effect of methotrexate (MTX) and activated Tregs on T cell proliferation individually and in combination. MTX alone suppresses T cell proliferation by approximately 60%, whereas activated Tregs achieve a suppression of approximately 50%. In combination (MTX + activated Tregs), a clear synergistic effect is observed. The combination inhibits T cell proliferation by about 80%, which is significantly stronger than the effect of either component alone.

[0149] Figure 1 B shows the effect of anti-IL-17 and activated Tregs on T cell proliferation, either individually or in combination. Activated Tregs suppress T cell proliferation by approximately 50%, whereas anti-IL-17 alone has only a marginal effect. The combination of anti-IL-17 and activated Tregs does not further enhance suppression and does not exhibit a synergistic interaction.

[0150] Most drug-discovery platforms use PBMC from multiple healthy donors to profile immunosuppressive effects on T-cell activation and proliferation, because they are standardized, easier to obtain, than patients PBMC and cover broad HLA diversity. For using MLR / proliferation as a surrogate of in vivo disease modification, healthy-donor PBMC are sufficient for primary pharmacology, but patient PBMC become important to analyze disease-specific sensitivity or resistance. Autoimmune patients often have altered T-cell activation thresholds and defective regulation; T cells from MS Oder Psoriasis patients, for example, can show reduced sensitivity or altered responses to cell-mediated suppression compared with healthy controls.

[0151] Figure 2 shows the synergistic effects of activated Tregs with different modulators on proliferation of T cells from patients with multiple sclerosis.

[0152] T cells from multiple sclerosis patients (MS) were cocultured with allogeneic dendritic cells (mixed lymphocyte reaction) in the absence or presence of activated Tregs from a third independent healthy donor (act. Tregs). Additionally, different modulators were added to these cultures. All tested combinations of modulators and act. Treg exhibited synergistic effects. Upper row from left to right: anti-human TNF-alpha (50 ng / ml), recombinant human IFN-beta (1 IU / ml), PDE4 inhibitor (PDE4i, 2 pM); lower row from left to right: Dimethyl fumarate (DMF, 2 pM), anti-human IL-6R (30 ng / ml), anti-human IL-17 (50 ng / ml). On day 4 of culture, 37 kBq [3H] Thymidine (3H-Tdr) was added to each well and cells were cultured foran additional 16 h. T cell proliferation was measured by3H-Tdr incorporation using a liquid beta-scintillation counter. Bars show mean proliferation (in %) ± SEM.

[0153] Figure 2 (upper row, left) shows the influence of anti-TNF-alpha or activated Tregs and of the combination of anti-TNF-alpha plus activated Tregs on the proliferation of T cells isolated from the peripheral blood of MS patients. While activated Tregs can only suppress the proliferation of T cells insignificantly due to the known Treg resistance, modulation by anti-TNF-alpha leads to a suppression of T cell proliferation of approximately 40%. In any case, the combination of anti-TNF-alpha plus activated Tregs is more effective than either factor alone. Activated Tregs synergistically suppresses the activation of T cells from MS patients with anti-TNF-alpha.

[0154] Figure 2 (upper row, middle) shows that IFN-beta also promotes the proliferation of Treg-resistant T cells from the blood of MS patients instead of suppressing it. In combination with activated Tregs, however, a significant reduction in T cell proliferation is measurable. The data show that activated Tregs synergistically inhibits the proliferation of T cells from the blood of MS patients with IFN-beta and can significantly improve the therapeutic success of the biological IFN-beta.

[0155] Figure 2 (upper row, right) shows that PDE4 inhibitor measurably suppresses the proliferation of T cells from the blood of MS patients. However, the strongest inhibitory effect is observed in the combination of activated Tregs plus PDE4 inhibitor. Activated Tregs show a synergistic effect with PDE4 inhibitor in suppressing T cells from the blood of MS patients, thereby significantly enhancing the therapeutic effect of the drug PDE4 inhibitor.

[0156] Figure 2 (lower row, left) shows that DMF increases the proliferation of T cells from the blood of MS patients instead of suppressing it. In combination with activated Tregs, however, a significant suppression of the previously Treg-resistant T cell proliferation is detectable. The data show that activated Tregs synergistically with DMF suppress the activation of T cells from the blood of MS patients and can significantly increase the therapeutic effect of the drug DMF.

[0157] Figure 2 (lower row, middle) shows that anti-IL6R is not able to suppress the proliferation of Treg-resistant T cells from the blood of MS patients. In contrast, suppression in combination with activated Tregs is clearly detectable. Activated Tregs shows synergistic effects with anti-IL6R in the suppression of Treg-resistant T cells from the blood of MS patients.Figure 2 (lower row, right) shows that anti-IL-17 increases the proliferation of activated T cells from the blood of MS patients, while anti-IL-17 in combination with activated Tregs synergistically suppresses the activation of Treg-resistant T cells from MS patients, even more than is possible with activated Tregs alone.

[0158] Figure 3 shows the synergistic effects of activated Tregs with different modulators on proliferation of T cells from patients with psoriasis.

[0159] T cells from psoriasis patients (PSO) were cocultured with allogeneic dendritic cells (mixed lymphocyte reaction) in the absence or presence of activated Tregs from a third independent healthy donor (act. Tregs). Different modulators were added to these cultures. All tested combinations of modulators and act. Treg exhibited synergistic effects. Upper row from left to right: anti-human TNF-alpha (50 ng / ml), recombinant human IFN-beta (1 IU / ml), PDE4 inhibitor (PDE4i, 2 pM); lower row from left to right: Dimethyl fumarate (DMF, 2 pM), antihuman IL-6R (30 ng / ml), anti-human IL-17 (50 ng / ml).

[0160] On day 4 of culture, 37 kBq [3H] Thymidine (3H-Tdr) was added to each well and cells were cultured for an additional 16 h. T cell proliferation was measured by3H-Tdr incorporation using a liquid beta-scintillation counter. Bars show mean proliferation (in %) ± SEM.

[0161] Figure 3 (upper row, left) shows that anti-TNF-alpha suppresses the proliferation of T cells from the peripheral blood of psoriasis patients more than activated Tregs. However, the combination of anti-TNF-alpha and activated Tregs shows the strongest effect. Activated Tregs synergistically with anti-TNF-alpha suppresses the proliferation of T cells from the blood of psoriasis patients and thus enhances the therapeutic effect of the biologic.

[0162] Figure 3 (upper row, middle) shows the synergistic effects of IFN-beta and activated Tregs on the proliferation of T cells from the blood of psoriasis patients. Both factors alone are less efficient in suppressing T cells than the combination of both. The data show that activated Tregs synergistically enhance the therapeutic effect of IFN-beta in suppressing T cells from the blood of psoriasis patients.

[0163] Figure 3 (upper row, right) shows that PDE4 inhibitors suppress the proliferation of T cells from the blood of psoriasis patients more than activated Tregs. However, the combination of PDE4 inhibitor and activated Tregs is again more effective in suppressing T cells than either factor alone. The data show that activated Tregs significantly enhances the therapeutic effect of PDE4 inhibitors in suppressing T cell activity in patients with psoriasis.Figure 3 (lower row, left) shows that DMF alone can only marginally suppress the proliferation of T cells from the blood of psoriasis patients. In combination with activated Tregs, the suppression is significant. Activated Tregs show synergistic effects in the suppression of the activation of T cells from psoriasis patients and thus enhances the therapeutic effect of DMF.

[0164] Figure 3 (lower row, middle) shows the synergistic effects of anti-IL-6R and activated Tregs on the proliferation of T cells from the blood of psoriasis patients. Both factors alone are less efficient in suppressing T cells than the combination of both. The data show that activated Tregs synergistically enhance the therapeutic effect of anti-IL-6R in suppressing T cells from the blood of psoriasis patients.

[0165] Figure 3 (lower row, right) shows that anti-IL-17 increases the proliferation of activated T cells from the blood of psoriasis patients. In contrast, the combination of anti-IL-17 and activated Tregs synergistically suppresses T cell proliferation. This indicates a clear synergistic interaction.

[0166] The data shown in Figures 2 and 3 demonstrate the synergistic effects of immunomodulatory agents in combination with activated Tregs on proliferation of T cells from patients suffering of multiple sclerosis (Fig. 2) or psoriasis (Fig. 3). The data show that gp120 can be used as an enhancer of the mechanistic effects of many DMDs in different indications. The decisive factor for this synergistic effect is an excessive immune reaction involving T cells. This applies for multiple sclerosis (MS), psoriasis (PSO), and a variety of other autoimmune diseases listed in Table 1. The data also show that a combined application of an immune-modulatory agent and activated Tregs is superior to any monotherapy in the treatment of autoimmune disease.

[0167] In summary, activated Tregs can significantly enhance the therapeutic effect (suppression of T cell activation) when combined with an immunomodulatory agent. This applies to agents that block independent, different signaling pathways and it applies to patients with different autoimmune diseases.

[0168] The key to the therapeutic-synergistic effect of activated Tregs is that activated Tregs and the respective biologic suppress signaling pathways in T cells. This means that activated Tregs have a synergistic enhancing effect in all autoimmune diseases in which unwanted T cell activity is present and in which T cell-modulating biologics are used as therapeutics.The data also show that a combined application of an immunomodulatory agent and activated Tregs is superior to almost all monotherapy in the treatment of autoimmune disease.

[0169] The experimental results described above were further analyzed to quantify synergistic interactions between the modulators and activated Tregs. For this purpose, the Bliss independence model was applied (Ma and Motsinger-Reif, 2019). The calculated Bliss scores (often also referred to as “Excess over Bliss”) of modulators in combination with activated Tregs across the different disease contexts multiple sclerosis and psoriasis, as well as healthy donors are summarized in Table 2.

[0170] Table 2: shows the Bliss synergy scores for combinations of different modulators and activated Tregs across different disease contexts

[0171] Bliss score

[0172] HC MS PSO

[0173] anti-TNFa 0.14 0.10 0.26

[0174] IFNp 0.06 0.22 0.11

[0175] PDE4i 0.15 0.27 0.15

[0176]

[0177] DMF 0.19 0.46 0.47

[0178] anti-IL-6R 0.10 0.19 0.12

[0179] MTX 0.07 NP NP

[0180] anti-IL-17 -0.07 0.23 0.75

[0181]

[0182] Table 2 summarizes Bliss scores calculated for combinations of different modulators with activated Tregs. The analysis includes interaction effects across different donor backgrounds, including patients with multiple sclerosis (MS), psoriasis (PSO), and healthy controls (HC), NP = not performed. Bliss analysis was based on the suppressive effects of modulators and activated Tregs on T cell proliferation. The expected combined effect was calculated assuming independent activity of both components according to the Bliss independence model, and compared with the experimentally observed suppression. In the Bliss independence model, values close to zero indicate additive effects. Considering the biological variability inherent to assays using primary human cells from different donors, values between -0.05 and 0.05 were considered additive. Additionally, values >0.05 indicate a synergistic trend, whereas values <–0.05 indicate antagonistic interactions.According to the Bliss independence model, the expected combined effect (here referred to as Eexp) of two independently acting agents (here modulator or activated Tregs) is calculated from the individual effects of each agent (effect of modulator = EA, effect of activated Tregs = EB). The expected combined effect is determined based on the suppression values observed for each component alone using the following formula: Eexp= EA+ EB- (EAx EB).

[0183] The experimentally observed effect EAB of the combination is then used together with the expected combined effect Eexpto calculate the Bliss score (“Excess over Bliss”, EBliss= EAB – Eexp). A Bliss score greater than zero indicates a synergistic interaction between the tested components, whereas values close to zero indicate additive effects and negative values indicate antagonistic interactions. Considering the biological variability inherent to assays using primary human cells from different donors, Bliss scores between -0.05 and 0.05 were considered additive. Additionally, values >0.05 indicate a synergistic trend, whereas values <–0.05 indicate antagonistic interactions.

[0184] Reference is made to Figure 1 A, upper row left, setting: anti-TNF-alpha + activated Tregs Suppression of anti-TNF-alpha: 42% → EA= 0.42

[0185] Suppression of activated Tregs: 40%

[0186]

[0187] EB= 0.40

[0188] Suppression of anti-TNF-alpha + act. Tregs: 79%

[0189]

[0190] EAB = 0.79

[0191] Eexp — EA+ EB- (EAx EB)

[0192]

[0193] Eexp = 0.42 + 0.40 - (0.42 x 0.40) = 0.65

[0194] EBliss= EAB - Eexp = 0.79 - 0.65 =

[0195] According to the Bliss independence model, the combination of anti-TNF-alpha and activated Tregs shows synergistic interactions in suppressing the proliferation of T cells isolated from the peripheral blood from healthy donors.

[0196] Based on the calculated Bliss scores, synergistic interactions were further classified according to their magnitude: > 0.05-0.10: weak synergy; > 0.10-0.20: moderate synergy; > 0.20: strong synergy. Table 3 summarizes this qualitative interpretation of Bliss scores obtained from combinations of different modulators with activated Tregs across the different contexts of diseases.

[0197] Table 3: shows the interpretation of Bliss synergy scores for combinations of different modulators and activated Tregs across different disease contexts.Synergism

[0198] HC MS PSO

[0199] anti-TNFa moderate weak strong

[0200] IFNp weak strong moderate

[0201] PDE4I moderate strong moderate

[0202]

[0203] DMF moderate strong strong

[0204] anti-IL-6R weak moderate moderate

[0205] MTX weak NP NP

[0206] anti-IL-17 no strong strong

[0207]

[0208] Table 3 summarizes the qualitative interpretation of Bliss synergy scores obtained from combinations of different modulators with activated Tregs. Bliss scores were categorized to facilitate interpretation of the interaction effects observed in the proliferation assays (NP = not performed). A Bliss score EBliss>0.05 was considered indicative of a synergistic trend.

[0209] Synergistic interactions were further classified based on their magnitude:

[0210] > 0.05-0.10: weak synergy

[0211] > 0.10-0.20: moderate synergy

[0212] > 0.20: strong synergy

[0213] Materials and Methods

[0214] Isolation and culture of human immune cells

[0215] PBMC from either multiple sclerosis (MS) patients, psoriasis (PSO) patients, patients with systemic sclerosis (SSC), or healthy donors (HC) were isolated from peripheral blood within 16 h after blood collection using density gradient centrifugation. Blood was kept at room temperature before PBMC enrichment. After isolation, human cells were cultured in X-VIVO-15 (Lonza, Belgium).

[0216] Generation of dendritic cells

[0217] Dendritic cells (DC) were generated from isolated PBMC from the peripheral blood of healthy donors. 10-15x106PBMC (per well) were seeded in a 6-well culture plate in 2 ml RPMI + 1.5% heat-inactivated blood plasma and incubated for 30 minutes in an incubator at 37 °C and 5% CO2. During this incubation time, monocytes adhered to the plastic surface. The non-adherent cells were washed off several times with 1 ml pre-warmed PBS and the purity of the monocyte culture was visually checked under a light microscope. Afterwards monocytes were cultured in X-VIVO-15 (3 ml / well) supplemented with 1% heat-inactivatedplasma + 400 IU / ml rh GM-CSF (Sargramostim / Leukine) + 200 IU / ml rh IL-4 (Immunotools) for 6 days. On days 2 and 4 of the culture, cells were fed by removing 1 ml of medium per well and replacing it with 1 ml of culture medium supplemented with 800 IU / ml rh GM-CSF. DC were stored frozen in aliquots until use.

[0218] Isolation and CFSE labeling of CD3+ T cells

[0219] Untouched CD3+ T cells were isolated using Pan T cell isolation kit (Miltenyi Biotec) according to manufacturer's instructions. The purity of the isolated CD3+ T cells was checked by flow cytometry. For this purpose, cells were stained with the following antibodies: anti-human CD3 FITC (UCHT1), anti-human CD4 PE-Cy7 (RPA-T4), anti-human CD8APC (RPA-T8), all from BD Pharmingen. Stained cells were measured on LSRII with FACS Diva Software (BD Bioscience) and analyzed using FlowJo software.

[0220] In some experiments, isolated CD3+T cells from healthy donors were stained with the proliferation dye CFSE. Therefore, CD3+ T cells were resuspended in pre-warmed PBS to a final concentration of 2x107 / ml and stained with 1 pM CFSE for 20 minutes at 37 °C in the dark. Afterwards, cells were washed twice with X-VIVO-15 / 10% HSA and further stored at 37 °C until use in X-VIVO-15.

[0221] Mixed leukocyte reaction

[0222] A mixed leukocyte reaction served as the basis for the experiments. For this purpose, CD3+ T cells from healthy donors, MS patients, PSO patients or SSC patients were co-cultured with allogeneic DC (TC: DC ratio of 20:1) in the absence or presence of GP120 (ActiTrexx GmbH / Polymun Scientific GmbH) (1 pg / ml). In further approaches, different modulators were added to these cultures to investigate possible synergistic effects on T cell proliferation. The following drugs were used so far (Table 4):

[0223] Table 4:

[0224] Modulator Supplier Concentration Abbreviation Tocilizumab Roche 30 ng / ml anti-IL-6R (RoActemra®),

[0225] human IL6R inhibitor

[0226] Ixekizumab (Taltz®), Eli Lilly 50 ng / ml anti-IL-17 human IL-17A

[0227] inhibitor

[0228]

[0229] Adalimumab Amgen 50 ng / ml anti-TNF-alpha (Amgevita®), human

[0230] TNF-alpha inhibitor

[0231] rh IFN-beta-1a Miltenyi Biotec 1 IU / ml IFNb Nerandomilast, Hycultec 2 pM PDE4i

[0232] PDE4 inhibitor

[0233] Dimethyl fumarate Merck 2 pM DMF Methotrexate Hycultec 10 nM MTX

[0234]

[0235] Measurement of CD3+ T cell Proliferation

[0236] T cell proliferation was analyzed using two methods equally suitable for evaluating the experiments:

[0237] a) Incorporation of3H-Tdr:

[0238] On day 4 of culture,3H-Tdr was added to each well (37 kBq / well) and cells were cultured for an additional 16 h in an incubator at 37 °C and 5% CO2. Pan CD3+ T cell proliferation was measured by3H-Tdr incorporation using a liquid beta-scintillation counter.

[0239] b) Flow cytometric analysis of CFSE-labeled CD3+ T cells:

[0240] Some experiments were performed using CFSE-labeled CD3+T cells. On day 6 of culture, cells were harvested and stained with the viability dye Zombie NIR (Biolegend) and antihuman CD4 PE-Cy7 (RPA-T4) antibody (BD Pharmingen). Stained cells were measured on LSRII with FACS Diva Software (BD Bioscience) and analyzed using FlowJo software. The viability dye Zombie NIR enabled the distinction between live and dead cells, while CD4 staining differentiated CD4+T cells from CD4-T cells (equivalent to CD8+T cells). The CFSE signal was assessed within the viable CD4+and CD4-T cell populations.

[0241] Non-proliferating cells exhibited a strong CFSE signal. With each cell division, the CFSE fluorescence intensity was progressively halved in the daughter cells. Thus, the gradual reduction of the CFSE signal served as an indicator of T cell proliferation.

[0242] Preparation of activated Tregs

[0243] Regulatory T cells from the blood of healthy donors (buffy coats or leukapheresis products) were isolated using immunomagnetic beads (anti-human CD25 microbeads, available from Miltenyi) and stimulated with GP120 as previously described1-3. These stimulated Tregs population (activated Tregs) were used in the assays in a ratio of 4:1 (fourT cells to oneactivated Treg cell) and cocultured with allogeneic DC in a ratio of 20:1 (20 T cells to one DC)4.

[0244] Bliss independence analysis

[0245] To quantify synergistic interactions between modulators and activated Tregs, combination effects were analyzed using the Bliss independence model. According to this model, the expected combined effect (here referred to as Eexp) of two independently acting components is calculated from the effects of each component applied individually (effect of modulator = EA, effect of activated Tregs = EB). The expected effect of the combination Eexpwas calculated using the formula:

[0246] Eexp= EA+ EB– (EA× EB),

[0247] where EAand EBrepresent the suppression of T cell proliferation induced by modulator or activated Tregs alone, respectively.

[0248] The experimentally observed effect (here referred to as EAB) obtained for the combination of both components was compared with the expected effect Eexpcalculated according to the Bliss independence model. A Bliss score (often also referred to as “Excess over Bliss”, EBliss) was determined as the difference between the observed effect EABand the expected effect Eexp:

[0249] Bliss Score EBliss= EAB- Eexp

[0250] Positive Bliss scores indicate a synergistic interaction between the tested components, values close to zero indicate additive effects, and negative values indicate antagonistic interactions. Considering the biological variability inherent to assays using primary human cells from different donors, values between –0.05 and 0.05 were considered additive.

[0251] Additionally, values >0.05 indicate a synergistic trend, whereas values <–0.05 indicate antagonistic interactions.

[0252] Example:

[0253] Suppression of modulator anti-TNF-alpha: 42% -> EA= 0.42

[0254] Suppression of activated Tregs: 40% -> EB= 0.40

[0255] Suppression of anti-TNF-alpha + act. Tregs: 79%

[0256]

[0257] EAB = 0.79

[0258] Eexp= EA+ EB– (EA× EB) → Eexp= 0.42 + 0.40 – (0.42 × 0.40) = 0.65

[0259] EBliss= EAB– Eexp= 0.79 – 0.65 =

[0260] The calculated Bliss scores EBlisswere categorized to facilitate interpretation of the interaction effects observed in the proliferation assays. A Bliss score EBliss>0.05 was consideredindicative of a synergistic trend. Synergistic interactions were further classified based on their magnitude:

[0261] > 0.05-0.10: weak synergy

[0262] > 0.10-0.20: moderate synergy

[0263] > 0.20: strong synergy

[0264] Generalization of results

[0265] The autoimmune diseases to be treated are not or only partially autoinflammatory

[0266] The invention addresses the treatment of autoimmune diseases by the inventive combination. All listed diseases in Table 5 below are primarily autoimmune (adaptive immunity dysfunction with autoantibodies orT cell autoreactivity). Some have autoinflammatory components (innate overlap); none are purely autoinflammatory. The synergistic effect by the inventive combination is most likely based on common mechanisms that link these autoimmune diseases together. All diseases exhibit a more or less pronounced inflammatory environment. They use the same drivers of the inflammatory environment and same approved drugs. Furthermore, they are T cell driven and exhibit a good preclinical efficacy of Tregs, in some cases also good clinical efficacy. And lastly, there is a high probability of transfer to selected diseases.

[0267] Table 5:

[0268] Disease Autoimmune Autoinflammatory Notes

[0269] Rheumatoid arthritis Yes No Classic; autoantibodies (RA) (RF / ACPA), T / B cells Juvenile idiopathic Yes No Pediatric autoimmune arthritis (JIA) arthritis

[0270] Psoriatic arthritis (PsA) Yes Partial Adaptive (Th17 / IL-23);

[0271] innate skin component Plaque psoriasis Yes Partial Th17-driven;

[0272] keratinocytes innate trigger

[0273] Crohn's disease (CD) Yes Partial Mixed; Th1 / Th17 + innate IL-23 / IL-1 p

[0274] Ulcerative colitis (UC) Yes Partial Th2 / IL-13 + innate barrier dysfunction

[0275]

[0276] Non-infectious uveitis Yes Partial T-cell mediated; IFN- (NIU) y / TNF-a

[0277] Systemic lupus Yes No Hallmark autoantibodies erythematosus (SLE) (ANA / anti-dsDNA) Sjögren's syndrome Yes No Anti-SSA / SSB; B-cell driven

[0278] Systemic sclerosis Yes No Autoantibodies (anti- (SSc) Scl70); fibrosis T-cell Neurosarcoidosis Partial Yes Innate granulomatous > adaptive

[0279] Multiple sclerosis (MS) Yes No Th17 / CD20+ B cells;

[0280] demyelination

[0281] Type 1 diabetes (T 1 D) Yes No Autoantibodies (anti- GAD); CD8+ T cells

[0282]

[0283] Spectrum: RA / SLE / MS / T1D pure autoimmune; neurosarcoid leans autoinflammatory;

[0284] CD / PsA "mixed."

[0285] Approved DMD drug classes for autoimmune diseases

[0286] The following Table summarizes the immunomodulatory agents (DMDs) used in the inventive combination and their approved application in the treatment of autoimmune or immune-mediated diseases.

[0287] Table 6:

[0288] Drug / class Examples Approved autoimmune /

[0289] immune-mediated diseases (major) IL-6R Tocilizumab, sarilumab RA, systemic JIA, polyarticular JIA, giant inhibitors cell arteritis, cytokine-release syndromes. IL- 17 Secukinumab, ixekizumab, Psoriatic arthritis, plaque psoriasis, axial inhibitors bimekizumab spondyloarthritis (radiographic and non-radiographic).

[0290] TNF-alpha Infliximab, adalimumab, RA, psoriatic arthritis, axial

[0291] inhibitors etanercept, golimumab, spondyloarthritis, juvenile idiopathic certolizumab pegol arthritis, Crohn’s disease, ulcerative colitis, psoriasis.

[0292]

[0293] PDE4 Apremilast Psoriatic arthritis, plaque psoriasis. inhibitors

[0294] InterferonIFN-pia, IFN-pib, Relapsing forms of multiple sclerosis. beta peginterferon-pia

[0295] Fumarates Dimethyl fumarate, diroximel Relapsing multiple sclerosis; plaque fumarate; other fumarates for psoriasis (various oral fumarate psoriasis formulations).

[0296] Folate Methotrexate RA, psoriatic arthritis, juvenile idiopathic antagonists arthritis; also used in many other immune-mediated diseases (e.g., vasculitis).

[0297]

[0298] Generalization and transferability of a combination concept across autoimmune diseases

[0299] The Table 7 below summarizes that the autoimmune diseases to be treated by the inventive combination are based on a more or less pronounced inflammatory environment. All claimed autoimmune diseases are more or less T cell driven and show at least preclinical and even clinical efficacy of regulatory T cells. Furthermore, the main the drivers for such an inflammatory environment are mentioned. It is shown that the approved drugs for these diseases are identical suggesting that the inventive combination can be transferred not only to one particular autoimmune disease, but to other autoimmune diseases indicated above.

[0300] All data in Table 7 are verified against sources: drivers from pathogenesis reviews; DMARDs from EULAR / AGA / ASAS 2022-2025 guidelines / approvals; Tregs from ClinicalTrials.gov / PMC (phase l / ll as of 2026); T cell-driven from TCR / repertoire studies; severity from cytokine / PASI / DAS correlations.

[0301] Table 7:

[0302] Disease Inflammatory Main Therapy Options T Cell- Treg Environment Driver(s) with Driven? Data DMDs / DMARDs [Verified] Rheumatoid +++ (synovial TNF-a, MTX, HCQ, Yes is Clinic arthritis (RA) storm) IL-6, IL- SSZ, LEF; TNF-i, phase l / ll:

[0303] -I P LUL21 IL-6i, JAKi ™ Partially effective LSI

[0304]

[0305] Juvenile ++ IL-6, MTX, SSZ, LEF; Yes121Precl: idiopathic arthritis (joint / systemic TNF-a, TNF-i, Partially (JIA) sJIA) IL-1 izi tocilizumab181effective [sources] Psoriatic arthritis ++ IL-23 / IL- MTX, LEF; TNF- yesLisi Precl: (PsA) (entheseal / skin- -|7 / \ rioirm i, IL-17i, IL-23i Partially joint) [121 effective

[0306] Plaque psoriasis ++ (dermal +++ IL-23 / IL- MTX, apremilast; yesLisi Precl:

[0307] cytokines) 17A, TNF-i (2nd), IL- Partially TNF-a 17 / 23111Z1 effective [15JI1S1

[0308] Crohn’s disease +++ (mucosal IL-12 / IL- Anti-integrins, Yes Clinic (CD) fistulizing) 23, TNF- ustekinumab, phase 1:

[0309] a 1121 TNF-i [AGA] Partially effective

[0310] Ulcerative colitis ++ (mucosal IL-13, IL- Anti-integrins, Partial Precl: (UC) continuous) 17, TNF- ustekinumab, Partially Q 1121 TNF-i, JAKi effective [AGA]

[0311] Non-infectious ++ (intraocular) TNF-a, TNF-i Yes Precl: uveitis (NIU) IL-6, IFN- (adalimumab), Partially y [211 MTX1211effective Systemic lupus ++ (multi-organ Type 1 HCQ, Partial Clinic erythematosus IFN) IFN, belimumab, phase 1: (SLE) TNF-a1221anifrolumab (no Partially TNF-i) [EULAR] effective

[0312] Sjogren’s + (glandular IFN-a / y, HCQ, MTX, yes£2Si Precl: syndrome focal) TNF-a123rituximab Partially [EULAR] effective

[0313] [261 Systemic + (vascular Type 1 MTX, MMF, Partial Precl: sclerosis (SSc) fibrosis) IFN, IL-6 tocilizumab Partially [sources] [EULAR] effective

[0314]

[0315] Neurosarcoidosis ++ IFN-Y, MTX, TNF-i Partial Precl: No (granulomatous) TNF-O1221(infliximab)122effect Multiple sclerosis ++ (CNS IFN-Y, IL- Ocrelizumab, Yes Precl: (MS) plaques) 171281 fingolimod (no Effective TNF-i) [AAN]

[0316] Type 1 diabetes + (islet- IFN-Y, IL- Teplizumab (no Yes Clinic (T1D) localized) 1 P 1281 TNF-i) phase l / ll:

[0317] Partially effective

[0318]

[0319] References (Table 7):

[0320] All entries match primary sources (PMC, guidelines, trials).

[0015]

[0024] [6][3][4]

[0321] 1■ https: / / www.hopkinsarthritis.org / arthritisdnfo / rheumatoid-arthritis / ra-pathophvsiologv-2 / 2. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC6410649 /

[0322] 3- https: / / www.ncbi.nlm.nih.gov / books / NBK507863 /

[0323] 4. https: / / ard.bmj.eom / content / 82 / 1 / 3

[0324] 5- https: / / pubmed.ncbi.nlm.nih.gov / 39920282 /

[0325] 6- https: / / pmc.ncbi.nlm.nih.gov / articles / PMC9009914 /

[0326] 7. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC6842741 /

[0327] 8- htps: / / www.arthritiswa.org.au / iia / disease-modifying-anti-rheumatic-drugs-dmards-for- iia /

[0328] 9- https: / / www.bms-immunologie.de / rheumatische-erkrankungen

[0329] 10. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC12010266 /

[0330] 11. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC6788885 /

[0331] 12. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC11103320 /

[0332] 13. https: / / www.springermedizin.at / an-overview-of-psoriatic-arthritis-epidemiologv-clinical- feature / 14914874

[0333] 14. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC10884248 /

[0334] 15. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC12697396 /

[0335] 16. https: / / www.bioinformation.net / 021 / 973206300212803.pdf

[0336] 17. https: / / www.sciencedirect.com / science / article / pii / S0190962216014869

[0337] 18. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC4132586 /

[0338] 1

[0339]

[0340] 9. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC5438231 /

[0341] 20. https: / / clinicaltrials.gov / studv / NCT03185000

[0342] 21. https: / / pubmed.ncbi.nlm.nih.gov / 40550324 /

[0343] 22. https: / / www.nature.com / articles / s41392-025-02168-0

[0344] 23. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC11461704 /

[0345] 24. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC11739237 /

[0346] 25. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC2762015 /

[0347] 26. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC12446511 /

[0348] 27. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC12664804 /

[0349] 28. https: / / pmc.ncbi.nlm.nih.gov / articles / PMC7588284 /

[0350] 29. https: / / clinicaltrials.gov / study / NCT02772679As disclosed herein, the present invention provides an evidence-based justification for the limited generalization and transferability of the therapeutic drug combinations to autoimmune diseases sharing a common pathological profile. This profile is characterized by identical inflammatory cytokines, cellular mediators, and mechanisms of action. Consistent with the data presented, these cytokines and cells act in an additive or synergistic manner within the inflammatory environment. Consequently, technical effects demonstrated for a specific combination involving a TNF inhibitor and activated Tregs are generalizable to other agents within the class of TNF inhibitors with a reasonable expectation of achieving similar unexpected or superior effects. For instance, the synergistic effect observed for the combination of Adalimumab and a Treg-targeting component provides a technical basis for the combination of any TNF inhibitor with such Treg-targeting agents, particularly as all TNF inhibitors approved for rheumatoid arthritis target the same TNF-alpha driver.

[0351] This evidence-based generalization is supported by the data in Table 7, which identifies the identical drivers of the inflammatory environment across the selected indications, and Table 6, which demonstrates that the same disease-modifying drugs (DMDs) are effective for treatment across these conditions. Furthermore, as the immunoregulatory component specifically targets T-cell-driven mechanisms, a hallmark of the autoimmune diseases identified in Table 7, this condition enables a particularly effective and appropriate Treg-targeting intervention across the claimed therapeutic spectrum.

[0352] In summary, all diseases claimed and listed in Table 1 relating to a certain drug class fulfill the requirements for a limited evidence-based generalization / transferability of the disclosed drug combinations. A skilled person expects class wide effects of the drug classes in the selected autoimmune diseases without undue experimentation and a significant contribution of the Treg-targeted immunoregulatory component.Non-patent literature

[0353] Ma J, Motsinger-Reif A. Current Methods for Quantifying Drug Synergism. Proteom Bioinform. 2019 Jul;1(2):43-48. Epub 2019 Jul 22. PMID: 32043089; PMCID: PMC7010330.

Claims

CLAIMS:

1. An immunologically effective combination, comprising at least one immunomodulatory agent and a preparation of isolated CD4-activated regulatory T cells (activated Tregs) as immunoregulatory agent for use in the treatment of an autoimmune disease, wherein said immunomodulatory agent suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms.

2. The immunologically effective combination for the use according to claim 1, wherein said immunomodulatory agent is a disease-modifying drug (DMD) that is selected to target the underlying pathophysiology or biological drivers of a disease state to modify the clinical course or natural history of the autoimmune disease.

3. The immunologically effective combination for the use according to claim 1 or claim 2, wherein said immunomodulatory agent is selected from the group consisting of IL-6R inhibitor, IL-17 inhibitor, TNF-alpha inhibitor, PDE4 inhibitor, Interferon, fumarate, folate antagonist.

4. The immunologically effective combination for the use according to claim 3, wherein saida. IL-6R inhibitor is an anti-IL6R antibody,b. IL-17 inhibitor is an anti- 1 L17RA antibody or an anti- 1 L17A antibody, c. TNF- alpha inhibitor is an anti-TNF- alpha antibody, a TNFR-IgG fusion protein, mAb-PEG conjugate or a small molecule,d. Interferon is Interferon-beta,e. Fumarate is dimethyl fumarate (DMF), monomethyl fumarate, diroximel fumarate or fumaric acid ester mixtures,f. Folate antagonist is methotrexate.

5. The immunologically effective combination for the use according to claim 3, wherein said IL-6R inhibitor is Tocilizumab, Sarilumab;said IL-17 inhibitor is Ixekizumab, Secukinumab, Bimekizumab, or Brodalumab; said TNF-alpha inhibitor is Adalimumab, Etanercept, Infliximab, Certolizumab pegol, Balinatunfib, Golimumab;said PDE4 inhibitor is Nerandomilast, Roflumilast, Apremilast, Zatolmilast, Orismilast, PF-07038124;said Interferon-beta is Interferon beta-1 a, peginterferon beta-1 a, Interferon beta-1 b.

6. The immunologically effective combination for the use according to any one of claims 1 to 5, wherein the activated Tregs were produced by isolating regulatory T cells from blood of autologous or allogeneic donors and subsequent purification of said regulatory T cells.

7. The immunologically effective combination for the use according to claim 6, said regulatory T cells were isolated using anti-CD25 immunomagnetic beads and stimulated with anti-CD4 antibodies or functional fragments thereof, or HIV-1 glycoprotein 120 (GP120) or biologically active fragments thereof to produce activated Tregs.

8. The immunologically effective combination for the use according to claim 6, wherein the anti-CD4 antibody used for stimulating regulatory T cells is Tregalizumab or Palixizumab.

9. The immunologically effective combination for the use according to any one of claims 1 to 8, wherein the at least one immunomodulatory agent is provided in a first dosage form and the activated Tregs are provided as a second dosage form, wherein a dosage regimen provides for the first dosage form to be administered to a subject concurrently with or prior to the second dosage form.

10. The immunologically effective combination for the use according to any one of claims 1 to 9, wherein the autoimmune disease is any one of psoriasis, plaque psoriasis, psoriatic arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, Sjogren’s syndrome, diabetes type-1, Crohn’s disease and ulcerative colitis, multiple sclerosis, systemic lupus erythematosus (SLE), non-infectious uveitis, systemic sclerosis, neurosarcoidosis.

11. The immunologically effective combination for the use according to claim 10, wherein for the treatment ofa. psoriasis and plaque psoriasis, the immunomodulatory agent is TNF-alpha inhibitor, IL-6R inhibitor, IL-17 inhibitor, PDE4 inhibitor, Interferon-beta, fumarate or folate antagonist;b. rheumatoid arthritis and juvenile idiopathic arthritis, the immunomodulatory agent is TNF-alpha inhibitor, IL-6R inhibitor, IL-17 inhibitor, PDE4 inhibitor, or methotrexate;c. Sjogren’s syndrome, the immunomodulatory agent is PDE4 inhibitor, or methotrexate;d. diabetes type-1, the immunomodulatory agent is PDE4 inhibitor;e. Crohn’s disease and ulcerative colitis, the immunomodulatory agent is TNF- alpha inhibitor, IL-6R inhibitor, PDE4 inhibitor, or methotrexate;f. multiple sclerosis, the immunomodulatory agent is TNF-alpha inhibitor, IL-6R inhibitor, IL-17 inhibitor, PDE4 inhibitor, Interferon-beta, fumarate, or methotrexate;g. psoriatic arthritis, the immunomodulator agent is TNF-alpha inhibitor, IL-17 inhibitor, PDE4 inhibitor, fumarate or folate antagonist;h. systemic lupus erythematosus (SLE), the immunomodulatory agent is PDE4 inhibitor, or methotrexate;i. non-infectious uveitis, the immunomodulatory agent is TNF-alpha inhibitor, PDE4 inhibitor, or methotrexate;j. systemic sclerosis, the immunomodulatory agent is methotrexate;k. neurosarcoidosis, the immunomodulatory agent is TNF-alpha inhibitor, or methotrexate.

12. A pharmaceutical composition, comprising an immunologically effective combination comprising at least one immunomodulatory agent and a preparation of isolated CD4- activated regulatory T cells (activated Tregs), wherein said immunomodulatory agent is a disease-modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms and wherein said disease-modifying drug (DMD) is selected from the group consisting of IL-6R inhibitors, IL-17 inhibitors, TNF-alpha inhibitors, PDE4 inhibitors, Interferon, fumarates, folate antagonists.

13. The pharmaceutical composition according to claim 12, comprising an immunologically effective combination according to any one of claims 1 to 9.

14. The pharmaceutical composition according to claim 12, wherein the immunomodulatory agent and the activated Tregs in the immunologically effective combination are provided in a single dose form or in two distinct dose forms.

15. A pharmaceutical composition, comprising an immunologically effective combination according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier ordiluent for the use in the treatment of an autoimmune disease.

16. The pharmaceutical composition for the use according to claim 15, wherein the immunomodulatory agent and the activated Tregs in the immunologically effective combination are provided in a single dose form or in two distinct dose forms.

17. A method of treating an autoimmune disease comprising the administration of an immunologically effective combination, comprising at least one immunomodulatory agent and a preparation of isolated CD4-activated regulatory T cells (activated Tregs) to a subject in need thereof, wherein said immunomodulatory agent is a disease-modifying drug (DMD) that suppresses immune responses in a subject without specifically targeting immune tolerance mechanisms.

18. The method according to claim 17, wherein the immunologically effective combination is any one as defined in claims 1 to 9.

19. The method according to claim 17, wherein and the disease is any one as defined in claims 10 and 11.

20. The method of claim 17, wherein the immunomodulatory agent and the activated Tregs in the immunologically effective combination are provided in a single dose form or in two distinct dose forms.

21. The method of claim 17, wherein the activated Tregs are produced by isolating regulatory T cells from blood of autologous or allogeneic donors and subsequent purification of said regulatory T cells.

22. The method of claim 17, wherein the said regulatory T cells are isolated using anti- CD25 immunomagnetic beads and stimulated with anti-CD4 antibodies or functional fragments thereof, or HIV-1 glycoprotein 120 (GP120) or biologically active fragments thereof to produce activated Tregs.

23. The method of claim 17, wherein said immunomodulatory agent is selected from the group consisting of IL-6R inhibitor, IL-17 inhibitor, TNF-alpha inhibitor, PDE4 inhibitor, Interferon, fumarate, folate antagonist.

24. The method according to claim 17, wherein said autoimmune disease is psoriasis or multiple sclerosis.

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