Adiponectin, alone or in combination with in vitro light separation substitution (ECP), for treatment of immune-related adverse events of immune checkpoint inhibitors

Abstract

The present invention relates to a substance adiponectin and / or an adiponectin receptor agonist (ARA) for use in the treatment of immune checkpoint blocking (ICB)-induced immune-related adverse events (irAE) in a subject, wherein the subject preferably accepts checkpoint blocking therapy as a cancer therapy. The present invention also relates to a combination medicament comprising adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer for combined use to treat immune checkpoint blocking (ICB) induced immune related adverse events (irAE) in a subject wherein the treatment further comprises in vitro irradiation of a blood sample of the subject with ultraviolet A (UVA), preferably with in vitro photoseparation displacement (ECP) therapy.

Description

Technical Field

[0001] The present invention relates to a substance adiponectin and / or adiponectin receptor agonist (ARA) for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, wherein the subject preferably receives checkpoint blockade therapy as cancer therapy. The present invention also relates to a combination drug comprising adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer for use in combination to treat immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, wherein the treatment further comprises irradiating a blood sample of the subject with ultraviolet A (UVA) in vitro, preferably using extracorporeal photopheresis (ECP) therapy. Background Art

[0002] Immune checkpoint blockade (ICB) has been shown to have clinical benefits in the treatment of various cancers. Immune checkpoint blockade enhances anti-tumor immunity by blocking negative regulators of immunity such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), or programmed cell death ligand-1 (PD-L1). Immune-mediated organ tissue damage caused by ICB therapy is classified as immune-related adverse events (irAEs). irAEs can affect any organ system, with the most common involving the gastrointestinal tract, lungs, endocrine glands, skin, and liver.

[0003] Treatment of irAEs involves discontinuation of ICB therapy and / or administration of glucocorticoids, depending on the severity of the irAE. These measures carry the risk of missing ICB therapy and immunosuppression that may reduce antitumor immune responses. To date, no randomized trials have examined the effects of glucocorticoids on antitumor immunity in patients treated with ICBs, and retrospective data are controversial.

[0004] In addition, glucocorticoids cause serious side effects, including hyperglycemia, fluid retention, psychologic disturbances, iatrogenic adrenal insufficiency (if glucocorticoids are tapered too rapidly), and infectious complications.

[0005] In addition, second-line therapies such as TNF antagonists may interfere with antitumor effects, as TNF has been shown to be essential for antitumor effects in hematological malignancies (Schmaltz et al., 2003), and TNF blockade for autoimmunity has been associated with a numerically increased risk of lymphoproliferative cancers and non-melanoma skin cancers. A retrospective study analyzing 790 patients with advanced melanoma who received ICB reported a 13.5% incidence of serious infections in the subgroup of patients who received glucocorticoids or infliximab (TNF antagonists). Recent clinical studies have shown that TNF blockade, as well as immunotherapy, is associated with a 50% nonresponse to ICB.

[0006] In summary, patients who develop severe irAEs (grades 3 and 4) after ICB are currently treated with complete and prolonged interruption of immunotherapy and administration of glucocorticoids. Both interventions may negatively impact the anti-tumor immune response. There are currently no prospective randomized trials supporting the use of glucocorticoids or second-line therapies such as the frequently used mycophenolate mofetil (MMF), TNF antagonists, or cyclosporine A. There is a lack of new approaches to effectively combat irAEs.

[0007] Therefore, novel therapeutic approaches that interfere with irAEs without reducing antitumor effects and causing few systemic side effects are an unmet medical need. Summary of the Invention

[0008] In light of the prior art, the technical problem underlying the present invention is to provide an improved therapeutic strategy for treating irAEs, especially during ICB therapy, which does not interfere with the ICB-mediated antitumor effects and causes fewer side effects than prior art glucocorticoid treatment.

[0009] This problem is solved by the features disclosed herein (including features of the independent and dependent claims). The inventors have found that adiponectin, alone or in combination with adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer, can be successfully used to treat immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in subjects in the context of extracorporeal photopheresis (ECP) therapy.

[0010] The inventors have previously successfully applied extracorporeal photopheresis (ECP) to treat ICB-induced colitis in patients with metastatic melanoma (Apostolova et al., 2020). When analyzing the mechanism by which ECP alleviates post-ICB colitis, the inventors found that ECP induces the expression of adiponectin in the intestine, which leads to the release of arginase-1 (Arg-1) from myeloid cells and a decrease in T cell activation and regulatory KLRG1. + BTLA + CD47 + TOX 低 T cell expansion. Adiponectin production is induced by apoptotic cells phagocytosed by macrophages in the intestine. The inventors found that ECP reduced clinical and histological signs of ICB-induced colitis in mice, as well as the in vivo expansion of pathogenic luciferase-transgenic T cells that infiltrated irAE target organs. Anti-tumor immunity in melanoma-bearing mice treated with anti-PD1 remained intact during ECP treatment, while corticosteroids reduced anti-melanoma immunity. Consistent with the findings in mice, ECP induced adiponectin in the blood and intestinal tissues of patients with irAEs.

[0011] The inventors identified ECP as a novel immunomodulatory therapy that controls ICB-induced irAEs such as colitis by activating the adiponectin / Arg-1 axis and acting directly on T cells and macrophages. Using a gene loss-of-function approach (adiponectin KO, arginase-1 KO mice) and pharmacological intervention, the inventors were able to demonstrate a functional role for the adiponectin / Arg-1 axis in the protective effects of ECP.

[0012] The inventors found that administration of adiponectin and / or adiponectin receptor agonist (ARA) enhanced the activity of ECP in treating ICB-induced irAEs, which was observed, for example, as reduced intestinal inflammation and less weight loss during / after ICB treatment.

[0013] Therefore, in a first aspect, the present invention relates to a combination drug comprising the substance adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer, for combined use to treat immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, wherein the treatment (also) comprises ex vivo irradiation of a blood sample of the subject with ultraviolet A (UVA), preferably using extracorporeal photopheresis (ECP) therapy.

[0014] In a preferred embodiment, the photosensitizer is administered ex vivo to the subject's blood sample prior to in vitro UVA irradiation of the subject's blood sample.In a preferred embodiment, the subject is receiving or has received checkpoint blockade therapy, for example as a cancer therapy.

[0015] Unexpectedly, the inventors further found that administration of adiponectin and / or adiponectin receptor agonist (ARA) alone can also reduce and prevent ICB-induced irAEs.

[0016] Therefore, the present invention relates to adiponectin and / or adiponectin receptor agonist (ARA) for treating or preventing immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject. In a preferred embodiment, the subject is receiving or has received checkpoint blockade therapy, for example, as a cancer therapy.

[0017] ICB checkpoint blockade therapy typically includes the administration of at least one antibody, preferably at least two antibodies, preferably selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3). In embodiments of adiponectin and / or ARA used according to the present invention, adiponectin and / or ARA are administered at least one hour before, during, and / or after administration of checkpoint blockade therapy.

[0018] In embodiments of adiponectin and / or ARA for use according to the invention, adiponectin and / or ARA is administered at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes, or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, or 48 hours, or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, 28 days, 30 days, or at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20 weeks, 24 weeks, or 52 weeks after administration of checkpoint blockade therapy. In embodiments of adiponectin and / or ARA for use according to the invention, adiponectin and / or ARA is administered at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes, or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, or 48 hours, or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, 28 days, 30 days, or at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20 weeks, 24 weeks, or 52 weeks prior to administration of checkpoint blockade therapy. In embodiments of adiponectin and / or ARA for use in accordance with the present invention, adiponectin and / or ARA is administered at least once daily for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, 28 days, 30 days, or at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20 weeks, 24 weeks, or 52 weeks during administration of checkpoint blockade therapy.

[0019] Adiponectin and / or ARA is administered at least once daily, preferably for at least 2 weeks to 5 weeks during the administration of the checkpoint blockade therapy, more preferably for at least 3 weeks during the administration of the checkpoint blockade therapy.

[0020] In embodiments of adiponectin and / or ARA for use according to the present invention, the subject has cancer.

[0021] In embodiments of adiponectin and / or ARA for use in accordance with the present invention, the cancer is selected from the group comprising malignant melanoma, breast cancer, or another cancer treatable by checkpoint blockade therapy, such as any cancer disclosed herein.

[0022] In embodiments of adiponectin and / or ARA for use according to the invention, the checkpoint blockade therapy comprises administration of at least one antibody, preferably at least two antibodies.

[0023] In embodiments of adiponectin and / or ARA for use according to the present invention, the at least one antibody is selected from the group comprising cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3).

[0024] In embodiments of adiponectin and / or ARA used according to the present invention, the irAE may involve any organ, preferably the gastrointestinal tract, lungs, endocrine glands, skin and / or liver.

[0025] In embodiments of adiponectin and / or ARA for use in accordance with the present invention, the irAE comprises ICB-induced colitis.

[0026] In embodiments of adiponectin and / or ARA for use in accordance with the present invention, administration of adiponectin and / or ARA reduces ICB-induced colitis.

[0027] In embodiments of adiponectin and / or ARA for use in accordance with the present invention, administration of adiponectin and / or ARA does not interfere with anti-tumor responses induced by checkpoint blockade therapy, preferably anti-PD1 treatment, due (at least in part) to tissue-specific adiponectin induction.

[0028] In embodiments of adiponectin and / or ARA used according to the present invention, the anti-tumor immune response induced by anti-PD-1 mediated by T cells in the tumor microenvironment is not impaired by the administration of adiponectin and / or ARA, wherein the intact anti-tumor immune response is preferably demonstrated by recall immune experiments.

[0029] In another aspect, the present invention relates to a combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, the combination drug comprising at least two of the following components: a. adiponectin and / or adiponectin receptor agonist (ARA) and b. a photosensitizer,

[0030] And wherein the treatment further comprises treating the subject's blood sample with ultraviolet A (UVA) extracorporeal irradiation, preferably extracorporeal photopheresis (ECP).

[0031] In a preferred embodiment, the photosensitizer is administered ex vivo to the blood sample prior to treating the blood sample with ultraviolet A (UVA) in vitro irradiation, preferably extracorporeal photopheresis (ECP).

[0032] In an embodiment, a combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject comprises the following components:

[0033] a. Adiponectin and / or adiponectin receptor agonist (ARA),

[0034] b. Photosensitizers and

[0035] c. a blood sample of the subject and / or lymphocytes, preferably T cells, extracted therefrom,

[0036] Wherein the blood sample and / or lymphocytes extracted therefrom have been subjected to treatment with a photosensitizer ex vivo and subsequently subjected to ultraviolet A (UVA) irradiation in vitro, preferably extracorporeal photopheresis (ECP).

[0037] In other words, in an embodiment, the combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject comprises the substance adiponectin and / or an adiponectin receptor agonist (ARA), a photosensitizer, and a blood sample and / or lymphocytes (preferably T cells) contained in or extracted from the blood sample, wherein the blood sample and / or lymphocytes have been subjected to an in vitro treatment comprising (i) administering a photosensitizer to the blood sample and / or lymphocytes, and subsequently (ii) in vitro irradiation with ultraviolet A (UVA), preferably in vitro photopheresis (ECP).

[0038] In an embodiment, the combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject comprises:

[0039] a. Adiponectin and / or adiponectin receptor agonist (ARA),

[0040] b. Photosensitizers and

[0041] c. Leukocytes, preferably (immunoregulatory) T cells, obtained by a method comprising the following steps:

[0042] (i) providing a sample obtained from an isolated blood sample of the subject,

[0043] (ii) adding the photosensitizer to the sample, and

[0044] (iii) subjecting the sample to UVA irradiation, preferably ECP treatment.

[0045] As shown in the examples, the positive effects and efficacy of the administration of samples treated with ECP according to the present invention may be at least partially due to the regulation of the functions of leukocytes (particularly T cells) contained in the blood sample, wherein the regulation is the result of irradiation in the presence of a photosensitizer. Therefore, the present invention also encompasses leukocytes, preferably lymphocytes, more preferably T cells, and even more preferably immunomodulatory / immunomodulatory T cells, as a combination drug with adiponectin and / or adiponectin receptor agonists (ARAs), for use in combination to treat and / or prevent immune-related adverse events (irAEs) in subjects receiving checkpoint inhibitor therapy. Preferably, immunomodulatory T cells are produced by subjecting blood, buffy coat, lymphocytes, and / or MNC cells from a human, preferably a subject who has received checkpoint inhibitor therapy and suspected or has experienced irAE to the ECP method as described herein, and are subsequently used to treat a subject who has received checkpoint inhibitor therapy and suspected or has experienced irAE, the subject being preferably a blood and / or cell donor.

[0046] In a preferred embodiment, prior to treatment with the photosensitizer and UVA irradiation, the subject's blood sample is subjected to in vitro centrifugation so that only the buffy coat (preferably comprising lymphocytes and optionally other white blood cells and platelets) is subjected to in vitro treatment with the photosensitizer and UVA irradiation and is preferably then returned to the subject.

[0047] In preferred embodiments, the subject is receiving or has received checkpoint blockade therapy.

[0048] The present invention also relates to a combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, wherein the combination drug comprises:

[0049] (i) adiponectin and / or adiponectin receptor agonist (ARA),

[0050] (ii) photosensitizer and

[0051] (iii) Closed extracorporeal photopheresis (ECP) system,

[0052] The closed ECP system forms a closed circuit with the subject's vascular system, preferably the venous system.

[0053] In a preferred embodiment, a blood sample is received from a vein of a subject via a venous catheter in a closed ECP system, wherein after the buffy coat is separated from the residual blood by centrifugation within the ECP system, the residual blood is then reintroduced into the subject's vein, while the buffy coat is simultaneously mixed or combined with a photosensitizer and subjected to UVA irradiation within the ECP system, and finally, the treated and irradiated buffy coat and / or lymphocytes derived therefrom are reinfused into the subject's vein via the venous catheter.

[0054] In preferred embodiments, the buffy coat comprises T cells. In preferred embodiments, the re-infused buffy coat comprises immunoregulatory T cells.

[0055] In an embodiment of the present invention, the photosensitizer is a psoralen agent, preferably 8-methoxypsoralen. Preferably, the "ECP method" or "ECP treatment" of a blood sample, a fraction thereof, or cells derived therefrom herein comprises treating or mixing the blood sample, a fraction thereof, or cells derived therefrom with a photosensitizer prior to UVA irradiation thereof. In a preferred embodiment of the present invention, the photosensitizer is administered ex vivo / in vitro to a blood sample or fraction thereof of a subject.

[0056] Aspects of the present invention are based on the discovery that if a patient who has received checkpoint inhibitor therapy (e.g., as a cancer treatment) develops an irAE, it can be effectively treated by administering adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer followed by in vitro irradiation of the subject's blood sample with ultraviolet A (UVA), preferably using ECP. In a preferred embodiment, ECP essentially refers to a method comprising adding a photosensitizer in vitro to a blood sample or blood-derived sample of an irAE patient and subjecting the sample to in vitro UVA irradiation. The study found that implementation of this combined treatment approach reduced the incidence and severity of irAEs in subjects without compromising the anti-cancer effects of ICB therapy.

[0057] In an embodiment of the substances adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer for combined use according to the present invention, the subject is receiving or has received checkpoint blockade therapy.

[0058] In another aspect, the present invention relates to a combination drug for treating and / or preventing immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, the combination drug comprising adiponectin and / or an adiponectin receptor agonist (ARA) and lymphocytes obtained from a method comprising the following steps:

[0059] - providing a sample obtained from an isolated blood sample of the subject,

[0060] - adding a photosensitizer to the sample, and

[0061] - subjecting the sample to UVA irradiation,

[0062] Preferably, the subject is receiving or has received checkpoint blockade therapy, for example as a cancer therapy. In a preferred embodiment, the lymphocytes include (immunomodulatory) KLRG1 + BTLA + CD47 + TOX低 T cells. Therefore, in a preferred embodiment, the combination drug for treating and / or preventing immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject comprises adiponectin and / or an adiponectin receptor agonist (ARA) and (immunomodulatory) T cells obtained from a method comprising the following steps:

[0063] - providing a sample obtained from an isolated blood sample of a subject,

[0064] - adding a photosensitizer to the sample, and

[0065] - subjecting the sample to UVA irradiation,

[0066] Wherein preferably, the subject is receiving or has received checkpoint blockade therapy, for example, as a cancer therapy. This aspect of the present invention is based on the following observations: the T cells contained in the blood-derived sample subjected to irradiation after the addition of a photosensitizer adopt an immunoregulatory phenotype, which is particularly advantageous when the resulting cells are combined with the administration of adiponectin and / or adiponectin receptor agonist (ARA) for the treatment and / or prevention of irAE in the subject. In a preferred embodiment, the blood sample for producing the immunoregulatory T cells of the present invention is from a subject who has received checkpoint inhibitor therapy and suspects the occurrence or occurrence of symptoms of immune-related adverse events (irAEs). This embodiment is particularly advantageous because the cells are autologous for the patient and adverse events do not occur when allogeneic cells are transplanted.

[0067] Preferably, the immunoregulatory T cells used for such treatment or prevention are produced by adding a photosensitizer to a sample from a human subject's blood sample and then subjecting the sample to UVA irradiation. Adding a photosensitizer to a blood-derived sample and subjecting the sample to UVA irradiation preferably produces or induces (forms) immunoregulatory T cells in the sample.

[0068] In a preferred embodiment, the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation. In the context of the present invention, irradiation is preferably performed by an extracorporeal photopheresis (ECP) system. Any suitable system or irradiation device known to the skilled person can be used to perform irradiation of the blood sample.

[0069] The ECP treatment or ECP method according to the present invention relates to a method comprising the following steps:

[0070] - provide a sample obtained from a blood sample of a subject who has received checkpoint inhibitor therapy and is suspected of or has developed symptoms of an immune-related adverse event (irAE),

[0071] - adding a photosensitizer to the sample, and

[0072] - Subjecting the sample to UVA irradiation, preferably extracorporeal photopheresis (ECP) therapy.

[0073] In a preferred embodiment of the combination drug according to the present invention, adiponectin and / or ARA are administered before, during and / or after ECP treatment of a sample of a subject, wherein the ECP treatment comprises treating a patient's blood sample with a photosensitizer in vitro (ex vivo) and subjecting the sample to in vitro (ex vivo) UVA irradiation, and wherein the combination drug preferably further comprises returning the ECP-treated blood sample, its fraction and / or lymphocytes derived therefrom to the subject. In a preferred embodiment, prior to ECP treatment, the subject's blood sample is subjected to in vitro centrifugation so that only the buffy coat (preferably comprising lymphocytes and optionally other white blood cells and platelets) is subjected to in vitro treatment with a photosensitizer and UVA irradiation, and is preferably then returned to the subject.

[0074] Combined ECP treatment of a blood sample from a subject, such as a blood sample containing, in particular, mononuclear cells (MNCs), results in the generation of immunoregulatory T cells in the sample. In this context, "generating" immunoregulatory T cells should be understood as inducing or inducing the formation of such cells in the sample. In other words, the cells contained in the sample differentiate into or adopt the phenotype of immunoregulatory T cells.

[0075] It was found that the sample produced by the ECP treatment according to the present invention, in particular the induced immunoregulatory T cells contained in the sample, can be used to treat irAE patients. It can be demonstrated that administering such a sample or the cells contained in such a sample treated by the method of the present invention to a subject who has received a checkpoint inhibitor therapy in combination with adiponectin / ARA is effective in preventing the occurrence of irAEs or even in treating established irAEs in the subject. When compared to treating a subject with only one drug, a combination drug comprising adiponectin and / or ARA and a subject's ECP-treated blood sample or lymphocytes extracted therefrom even exhibits a synergistic effect in preventing or treating ICB-induced irAEs.

[0076] Completely unexpectedly, therapies comprising administration of adiponectin and / or ARA and using samples or cells produced by the ECP methods of the present invention are effective even in patients with irAEs who are refractory to other immunosuppressive therapies (such as steroids or anti-TNF antibodies) and who continue to exhibit irAE symptoms or still have irAEs after cessation of checkpoint inhibitor treatment.

[0077] Importantly, ECP treatment according to the present invention can be performed on a sample from the same subject who has received checkpoint inhibitor therapy. Thus, cells or samples produced by the methods of the present invention can be administered to the same subject who is the blood donor, and the resulting sample can represent an autologous cell therapy. In other embodiments, ECP treatment according to the present invention can be performed on a sample from another subject who is a compatible donor to the subject receiving ICB therapy, and the resulting sample can represent an allogeneic cell therapy.

[0078] As used herein, the term "a subject who is receiving or has received checkpoint inhibitor therapy" includes subjects who are currently receiving checkpoint inhibitor therapy, or subjects who have received checkpoint inhibitor therapy but have stopped (e.g., after the onset of irAE symptoms) or completed it.

[0079] In an embodiment, the ECP treatment according to the present invention is carried out in vitro or ex vivo. As used herein, the terms in vivo and ex vivo are used synonymously. In the context of the present invention, these terms relate to methods carried out on a blood-derived sample taken from the human body, wherein the method of the present invention is carried out on a blood-derived sample outside the human body. For this purpose, the blood of the donor subject is extracted from the body, which means extracted from the physiological circulatory system. As used herein, a "separated blood sample" is a blood sample (meaning a certain volume of blood) that is moved from the donor's circulatory system to a certain position outside the human body. This separated blood sample can be subjected to or introduced into an extracorporeal photopheresis (ECP) system or a blood separation system to perform at least some steps of the method of the present invention. Wherein, this system can be an online system that is fluidically connected to the subject's blood circulatory system. In an alternative embodiment, the method can be carried out off-line, wherein the blood-derived sample subjected to irradiation is disconnected from the donor subject's circulatory system.

[0080] Thus, in embodiments, the ECP treatment of the invention is an in vitro method. In embodiments of the methods of the invention, the blood sample is an isolated blood sample. In embodiments, the method is an in vitro method and the blood sample is an isolated blood sample.

[0081] As used herein, the term "blood sample" includes all kinds of blood-derived samples, including blood cell samples, such as MNC samples generated from blood.

[0082] In an embodiment of the present invention, the subject (recipient of the blood donor and / or cell) exhibits irAE symptoms or suffers from irAE. In another embodiment, the subject has received immune checkpoint inhibitor therapy (ICB), but the checkpoint inhibitor therapy is completed or stops after the symptoms and / or manifestations of irAE appear in the subject. In embodiments, the symptoms and / or manifestations of irAE appear in the subject after the checkpoint inhibitor therapy stops. In embodiments, the symptoms and / or manifestations of irAE are maintained after the checkpoint inhibitor therapy stops.

[0083] In an embodiment of the present invention, in the case of a subject who has received immune checkpoint inhibitor therapy (ICB) and suspected of having or having irAE symptoms as a blood donor, the provision and / or separation of a blood sample may occur at any time point described in the above embodiments. For example, sample separation may be performed before or after the onset of irAE symptoms. In addition, sample separation may be performed during checkpoint inhibitor therapy or after stopping the therapy.

[0084] In a preferred embodiment of the present invention, a sample resulting from ECP treatment according to the present invention or T cells used according to the present invention are administered to a subject when undergoing checkpoint inhibitor therapy.

[0085] In a preferred embodiment, sample separation occurs during ongoing checkpoint inhibitor therapy, or before or after the onset of irAE symptoms. By administering samples / cells to the subject during ongoing checkpoint inhibitor therapy, the sample or cells produced by the method of the embodiment can be used prophylactically or therapeutically. Therefore, in a preferred embodiment of the present invention, the subject who has received checkpoint inhibitor therapy can receive immunoregulatory T cells, which are preferably autologous and have been produced by irradiating a blood-derived sample according to the methods described herein, while undergoing checkpoint inhibitor therapy. Such embodiments are particularly advantageous because when a patient receives the cells of the present invention and / or the cells produced by the methods of the present invention, checkpoint inhibitor therapy, such as anti-cancer checkpoint inhibitor therapy, can be maintained.

[0086] Therefore, in such embodiments, checkpoint inhibitor therapy can be administered to the subject for a longer period of time, because the administration of irradiated cells prevents the occurrence of irAE or improves irAE, so that checkpoint inhibitor therapy can continue. Administration of such cells produced by the method of the present invention, particularly the immunomodulatory T cells of the present invention, does not interfere with the subject's anti-cancer response to checkpoint inhibitor therapy. Compared with known treatment / preventive measures for irAE, particularly the administration of immunosuppressive drugs (such as corticosteroids), this is an important advantage. ECP does not significantly change the anti-cancer effect of checkpoint inhibitor therapy, while the simultaneous administration of glucocorticoids (a specific class of corticosteroids), particularly prednisolone, leads to worse results, indicating that the effectiveness of checkpoint inhibitor treatment is reduced.

[0087] Furthermore, in embodiments where a (human) blood-derived sample that has undergone a method of adding a photosensitizer to the sample and irradiating the sample is used to treat and / or prevent irAEs in a subject who has received checkpoint inhibitor therapy, regardless of the source of the blood-derived sample (autologous or allogeneic), administration of the sample or cells produced by this method can be performed, for example, before or after the onset of irAE symptoms, and / or during checkpoint inhibitor therapy or after cessation of checkpoint inhibitor therapy.

[0088] In embodiments, the subject (blood donor and / or recipient of the cells) has cancer, such as malignant melanoma or another cancer treatable by checkpoint inhibitor therapy.

[0089] In embodiments, the subject is receiving immunosuppressive drugs, such as steroids, corticosteroids, cyclosporine, and / or anti-TNF antibodies (eg, infliximab), and / or is refractory to immunosuppressive drugs.

[0090] In embodiments of the present invention, irAE (of blood donor and / or cell recipient) comprises symptoms of autoimmune disease and / or is caused by autoimmune reaction.In preferred embodiments, irAE comprises or is autoimmune colitis.

[0091] In embodiments, the irAE comprises at least one irAE selected from the group consisting of autoimmune colitis, autoimmune hepatitis, autoimmune thyroiditis, and autoimmune dermatitis.

[0092] In embodiments, the irAE comprises at least one irAE selected from the group consisting of immune checkpoint inhibitor-associated colitis, immune checkpoint inhibitor-associated hepatitis, immune checkpoint inhibitor-associated thyroiditis, and immune checkpoint inhibitor-associated dermatitis.

[0093] Administration of cells produced by the methods described herein, such as immunoregulatory T cells, has a therapeutic effect on patients with irAEs who have been administered to treat irAE symptoms and who are refractory to immunosuppressive drugs. For such patients, there is no effective treatment available to improve irAE symptoms, and therefore the present invention represents a completely unexpected possibility for treating irAEs in these patients.

[0094] In embodiments, checkpoint inhibitor therapy comprises administering at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody.

[0095] In an embodiment, the immunoregulatory T cells used to treat and / or prevent irAEs in a subject who has received checkpoint inhibitor therapy are autologous to the subject. In alternative embodiments, the T cells can be allogeneic to the subject.

[0096] In embodiments, a combination drug comprising adiponectin and / or adiponectin receptor agonist (ARA) and lymphocytes (preferably (immunomodulatory) T cells) used according to the present invention is administered when irAE symptoms occur. In embodiments, after irAE symptoms occur, cells are administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks or 50 weeks, preferably at least 1-3 weeks, more preferably at least 2 weeks, and ... After AE symptoms occur, adiponectin and / or adiponectin receptor agonist (ARA) are applied for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks or 50 weeks, preferably 1-5 weeks, more preferably at least 3 weeks. In embodiments, a combination drug comprising adiponectin and / or adiponectin receptor agonist (ARA) and immunomodulatory T cells is applied during or after check point inhibitor therapy is stopped or completed.

[0097] In an embodiment of the combination drug, lymphocytes (preferably (immunoregulatory) T cells) are administered to the subject at least once, preferably at least twice, preferably on consecutive days. Thus, the subject may receive more than one dose of lymphocytes / T cells, wherein preferably no more than one dose is administered per day. In another embodiment, the subject receives at least 2, 3, 4 or 5 doses of lymphocytes / T cells of the present invention. In an embodiment, the lymphocytes / immunoregulatory T cells of the present invention are administered to the subject at least every 8 weeks, preferably every 2-4 weeks. In an embodiment, adiponectin and / or adiponectin receptor agonist (ARA) are administered before, during and / or after the administration of lymphocytes / immunoregulatory T cells. In an embodiment, adiponectin is administered at least once, more preferably once every two days, and at least once a day.

[0098] In an embodiment of the combined medicament or substance for combined use of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer according to the present invention, adiponectin and / or ARA and the photosensitizer are administered at least 30 minutes after administration of the checkpoint blockade therapy.

[0099] In an embodiment of the combined medicament or substance for combined use of adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer according to the present invention, the subject has been diagnosed with and / or is suffering from cancer.

[0100] In an embodiment of the combination according to the present invention or a substance for combined use comprising adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the photosensitizer is a psoralen agent, preferably 8-methoxypsoralen. In an embodiment of the combination according to the present invention or a substance for combined use comprising adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the wavelength of the UVA irradiation is a wavelength of 3200 angstroms to 4000 angstroms.

[0101] In an embodiment of the combined medicament or the substance for combined use of adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer according to the present invention, the subject suffers from cancer.

[0102] In an embodiment of the combined medicament or substance for (combination) use according to the invention of adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer, the cancer is selected from the group comprising malignant melanoma, breast cancer or another cancer treatable by checkpoint blockade therapy.

[0103] In an embodiment of the combined medicament or substance for (combined) use according to the invention of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the checkpoint blockade therapy comprises the administration of at least one antibody, preferably at least two antibodies.

[0104] In an embodiment of the combined medicament or substance for (combination) use according to the present invention of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the at least one antibody is selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3).

[0105] In embodiments of the combination drug according to the present invention or substances for (combination) use of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the irAE may involve any organ, preferably the gastrointestinal tract, lungs, endocrine glands, skin and / or liver.

[0106] In an embodiment of the combination medicament or substance for (combination) use according to the present invention of adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer, the irAE comprises ICB-induced colitis.

[0107] In an embodiment of the combination medicament or the substances for (combination) use of adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer according to the invention, the combination medicament reduces ICB-induced colitis.

[0108] In an embodiment of the combination according to the invention or the substances adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer for (combination) use, the combination induces apoptosis of leukocytes, which are phagocytosed by intestinal macrophages, thereby causing adiponectin production in the inflamed intestine.

[0109] In an embodiment of the combination medicament or substance for (combination) use of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer according to the present invention, the combination medicament induces adiponectin, arginase-1 (Arg1) and tolerogenic KLRG1 in the inflamed intestine of the subject. + BTLA + CD47 + TOX 低 Increase of T cells but not induction of adiponectin, arginase-1 (Arg1), and tolerogenic KLRG1 in cancer tissues + BTLA + CD47 + TOX 低 Increase in T cells.

[0110] In an embodiment of the combination according to the invention or the substances for (combined) use of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the combination does not interfere with the anti-tumor response induced by anti-PD1 therapy due to tissue-specific adiponectin induction.

[0111] In an embodiment of the combination drug according to the present invention or the substance for (combination) use of adiponectin and / or adiponectin receptor agonist (ARA) and a photosensitizer, the anti-PD-1 induced anti-tumor immune response mediated by T cells in the tumor microenvironment is not impaired by the combination drug, wherein the intact anti-tumor immune response is demonstrated by a recall immunoassay experiment.

[0112] In another aspect, the present invention relates to adiponectin and / or an adiponectin receptor agonist (ARA) for use in treating a tumor patient or a subject suffering from cancer, wherein the patient / subject has experienced immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB), wherein the treatment comprises the combined administration of adiponectin and / or ARA and anti-tumor immunotherapy, wherein the immunotherapy comprises the administration of immunoregulatory T cells and / or blood samples.

[0113] In an embodiment of the invention for treating adiponectin and / or ARA in a tumor patient, immunoregulatory T cells and / or a blood sample are obtained from a method comprising the following steps:

[0114] - providing a sample derived from an isolated blood sample of said patient,

[0115] - adding a photosensitizer to the sample, and subjecting the sample to irradiation.

[0116] In an embodiment of the invention for use in treating adiponectin and / or ARA in a tumor patient, the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation.

[0117] In another aspect, the present invention relates to a method of treating a subject who has received checkpoint inhibitor therapy and is suspected of experiencing or has experienced symptoms of an immune-related adverse event (irAE), the method comprising administering adiponectin and / or an adiponectin receptor agonist (ARA) to the subject at least once before, during, or after subjecting the subject to extracorporeal photopheresis (ECP) therapy (such as blood irradiation therapy by an ECP system).

[0118] In embodiments, extracorporeal photopheresis (ECP) therapy comprises administering a photosensitizer to a blood sample or fraction thereof from a subject prior to administering UVA irradiation to the blood sample or fraction thereof.

[0119] Each optional or preferred feature of the present invention disclosed or described in the context of one aspect of the invention is also disclosed herein in the context of other aspects of the invention described herein.

[0120] All features disclosed in the context of the ECP methods for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy of the present invention are also related to and disclosed herein in the context of the in vitro methods of the present invention, the treatment methods of the present invention, and lymphocytes and / or T cells for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy of the present invention, and vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] The present invention is further described by the following figures. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention provided to better illustrate the disclosure described herein.

[0122] Figures 1a to 1m It was shown that ECP treatment alleviated ICI-enhanced colitis in vivo through adiponectin induction.

[0123] Specifically, Figure 1a Figure 2 shows a graph depicting body weight normalized to body weight on day 0 of the experiment. Body weight was measured daily. Groups included untreated mice (UT), animals with DSS-induced colitis treated with anti-PD1 or anti-PD1 plus ECP. Data represent the mean of the combined body weights of all animals in a given group. Arrows indicate when ECP was performed (days 3 and 6). Data from 3-6 experiments were combined.

[0124] Figure 1b Shown is a scatter bar graph showing the mean (± sem) colon length of untreated animals, mice with DSS / anti-PD1-induced colitis, and mice with DSS / anti-PD1-induced colitis and treated with ECP, as indicated. Pooled values ​​from two experiments are shown. P values ​​were calculated using one-way ANOVA.

[0125] Figure 1c Representative colons isolated from mice in the indicated groups are shown. Colons were isolated on day 8 of the experiment.

[0126] Figure 1d Representative histological images of colon samples from mice with DSS-induced colitis treated with anti-PD1 or anti-PD1 plus ECP are shown. Arrows point to neutrophils in the intestinal wall.

[0127] Figure 1e and Figure 1fGraphs depicting neutrophil counts in untreated (UT) mice or mice with DSS-induced colitis treated with anti-PD1 or anti-PD1 plus ECP ( Figure 1e ) and lymphocytes ( Figure 1f ) Histopathological grade of infiltration into the colon (0 = absent, 1 = mild, 2 = extensive). Each data point represents the value of a single mouse. The experiment was performed twice and the results were combined (mean ± sem). P values ​​were calculated using the Kruskal-Wallis test.

[0128] Figure 1g Relative gene expression is shown, depicted as a microarray heat map ("Z-score intensity") of RNA isolated from colitis tissue of mice treated with DSS and anti-PD1 (n=4) or DSS and anti-PD1 plus ECP (n=5). The tiled display shows the most significant differentially regulated genes determined according to a linear model-based approach. The scale represents the row-wise scaling level of gene expression, with red being the highest and blue being the lowest. The arrow points to Adipoq, the gene most upregulated in the ECP group. The right column represents the log2 fold change between anti-PD1 plus ECP and anti-PD1. Asterisks highlight significant regulation.

[0129] Figure 1h Figure 2 shows Adipoq gene expression in the colon of mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. Gene expression data were measured by qPCR and normalized to Hprt expression and presented as fold change relative to anti-PD1 treatment. Results from three separate experiments were combined (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0130] Figure 1i Shown is a scatter plot showing the mean (± sem) of adiponectin in serum samples from mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. Each data point represents a single mouse. Results from nine experiments were combined. p values ​​were calculated using an unpaired Student's t-test.

[0131] Figure 1j Shown are scatter bar graphs depicting colon length of mice with DSS / anti-PD1 induced colitis and treated with ECP as indicated. WT: wild type receptor, Adipoq - / - : Adiponectin receptor-deficient. ECP donors for both groups were WT. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using an unpaired Student's t-test.

[0132] Figure 1kShown are graphs showing the body weight of mice treated with DSS, anti-PD1 and ECP, normalized to the body weight on day 0 of the experiment. WT: wild type receptor, Adipoq - / - : Adiponectin-deficient receptors. ECP donors in both groups were WT. Arrows indicate the time point of ECP transplantation. Data represent the mean of the combined body weights of all animals in the indicated group. Results from two separate experiments were combined.

[0133] Figure 11 and Figure 1m Shown are scatter plots depicting neutrophil counts in mice with DSS-induced colitis treated with anti-PD1 plus ECP ( Figure 11 ) and lymphocytes ( Figure 1m ) Scattered bar graph of histopathological grades of infiltration into the colon. WT: wild-type receptor, Adipoq - / - : Adiponectin receptor-deficient. ECP donors for both groups were WT. Results from two separate experiments were combined (mean ± sem) and significance was calculated using the Mann-Whitney test.

[0134] Figures 2a to 2u It was shown that engulfment of apoptotic leukocytes resulted in the expression of adiponectin in phagocytes.

[0135] Specifically, Figure 2a CD45.2 is shown + Representative flow cytometry images of CellTrace Violet (CTV) in macrophages. ECP-treated CTV was stained for CD45.1 + Splenocytes exposed to CD45.2 + Bone marrow-derived macrophages were co-cultured for 48 hours.

[0136] Figure 2b Shown Figure 2a The cell trace shown is violet-positive CD45.2 + Quantification of macrophages. Data from multiple experiments were combined (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0137] Figure 2c BMDM (CD11b + ), DAPI, and ECP-treated splenocytes. Splenocytes were exposed to BMDM for 24 hours. Scale bar: 20 μm. Arrows indicate cells engulfing ECP-treated splenocytes.

[0138] Figure 2dQuantification of macrophages in splenocytes treated with ECP is shown, expressed as the percentage of single cells. Data from two experiments were combined. P values ​​were calculated using the Wilcoxon test.

[0139] Figure 2e to Figure 2h CD45.2 is shown + Phosphorylated STAT6 in BMDM ( Figure 2e )、Arg1( Figure 2f )、CD206( Figure 2g )、CD301( Figure 2h ) Scatter bar graph of protein levels of CD45.1 in BMDM left untreated or treated with ECP + Splenocytes were co-cultured for 48 hours. Data from four experiments were combined (mean ± sem) and presented as fold change in MFI compared to the medium control. Significance was calculated using the Wilcoxon test. Figure 2i Shown is a scatter bar graph depicting CD45.2 + Adipoq gene expression in BMDM (PrimeFlow RNA assay) treated with ECP and CD45.1 + Splenocytes were co-cultured for 48 hours. Data from two experiments were combined (mean ± sem) and depicted as fold change in MFI compared to the medium control. Significance was calculated using the Wilcoxon test.

[0140] Figure 2j Shown are representative histograms of Adipoq RNA (PrimeFlow, gene expression analysis) in BMDM alone or in BMDM co-cultured with ECP-treated splenocytes for 48 hours.

[0141] Figure 2k Representative fluorescent images of colitis tissue from melanoma-bearing mice with DSS / anti-PD1-induced colitis are shown. Mice received VivoTrack680-stained ECP splenocytes or were left untreated (control). Images were taken 12 hours after transplantation.

[0142] Figure 2l Representative fluorescence images of subcutaneous melanoma tissue from tumor-bearing mice with DSS / anti-PD1-induced colitis are shown. Mice received VivoTrack680-stained ECP splenocytes or were left untreated (control). Images were taken 12 hours after transplantation.

[0143] Figure 2m Shown are graphs depicting the ECP-treated cells at 6 and 12 hours post-transfer from ( Figure 2k and Figure 2lQuantification of tissue fluorescence signal (mean radiant efficiency) of the tissue samples shown in ). Significance was calculated using the Kruskal-Wallis test.

[0144] Figure 2n Shown is a scatter plot of CD45.2 isolated from the colonic lamina propria or subcutaneous melanoma tissues of tumor-bearing mice with DSS / anti-PD1-induced colitis. + VT680 + CD45.1 cells in mice treated with ECP + VT680 + Splenocytes. Results from two separate experiments were combined (mean ± sem) and p values ​​were calculated using one-way ANOVA (*** is p < 0.0001).

[0145] Figure 2o shows that CD45.1 + VT680 + CD45.2 isolated from the colonic lamina propria of tumor-bearing mice with DSS / anti-PD1-induced colitis 12 hours after splenocyte transfer + CD11b + VT680 + Arginase-1 protein levels in cells. Results from two separate experiments were combined (mean ± sem) and p values ​​were calculated using the Wilcoxon test.

[0146] Figure 2p Shown Figure 2o Representative histograms of Arg-1 for samples described in .

[0147] Figure 2q to Figure 2u Scatter bar graphs showing the expression of CD45.1 + CD45.2 splenocytes co-cultured for 48 hours + Phosphorylated STAT6 in BMDM ( Figure 2q )、Arg1( Figure 2r )、CD206( Figure 2s ) and CD301( Figure 2t ) protein level or Adipoq gene expression ( Figure 2u BMDM were treated with vehicle or the phagocytosis inhibitors Endosidin 9 or PitStop 2. Experiments were performed three times, and data were combined (mean ± sem). Results are shown as the fold change in MFI compared to vehicle. Significance was calculated using the Friedman test.

[0148] Figures 2EA to 2EKshowed that the immunosuppressive effect of ECP was reduced after depletion of intestinal phagocytes.

[0149] Specifically, Figure 2EA shows that CD11b + f / 4 / 80 + Macrophages are depicted as a scatterplot of the percentage of all immune cells in the colon wall of mice treated with DSS / anti-PD1 / ECP. Mice received vehicle (Encapsome) or phagocyte depletion treatment (Clodrosome). Data from two separate experiments were combined (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0150] Figure 2EB Shown are body weights normalized to the body weight on day 0 of the experiment. All mice received DSS / anti-PD1 / ECP treatment. Mice were treated with vehicle (Encapsome) or phagocyte depletion treatment (Clodrosome). Arrows indicate Encapsome and Clodrosome treatment or ECP transplantation. Data represent the mean of the combined body weights of all animals in the specified group. Data from two experiments were combined.

[0151] Figure 2EC Shown is a scatter plot showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis. Mice received ECP in combination with vehicle or phagocyte depletion (Clodrosome). Experiments were performed twice and p values ​​were calculated using an unpaired Student's t-test.

[0152] Figure 2ED and Figure 2EE Neutrophils ( Figure 2ED ) and lymphocytes ( Figure 2EE ) Histopathological scoring of colonic infiltration. Scoring was performed on day 8 of the experiment. Each data point represents the value of a single mouse. Results from two experiments were combined (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0153] Figure 2EF A scatter bar graph is shown, which shows Ly6G + Neutrophils are shown to be all CD11b in the colon wall of mice treated with DSS / anti-PD1 / ECP and vehicle or clodrosome depletion + The percentage of cells was calculated using the unpaired Student's t-test.

[0154] Figure 2EG Shown are representative flow cytometry images showing neutrophils in the colon wall of mice treated with vehicle or clodrosome-depleting DSS / anti-PD1 / ECP.

[0155] Figure 2EH Shown is a scatter plot showing serum levels of adiponectin in mice treated with DSS / anti-PD1 / ECP and vehicle or phagocyte-depleting Clodrosomes. Data from two separate experiments were combined (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0156] Figure 2EI and Figure 2EJ Adipoq ( Figure 2EI ) or Arg1( Figure 2EJ ) gene expression. Gene expression was measured by qPCR and normalized to Hprt expression and shown as fold change relative to vehicle treatment. Data from two separate experiments were combined (mean ± sem) and significance was calculated using an unpaired Student's t-test.

[0157] Figure 2EK Shown is a scatter plot of CD45.2 isolated from the lamina propria of colon tissue of tumor-bearing mice with DSS / anti-PD1-induced colitis. + VT680 + cells (mean ± sem). Mice received 2 × 10 6 ECP-processed VT680 + MODE-K cells or 2 × 10 6 ECP-treated CD45.1 + VT680 + Splenocytes. p values ​​were calculated using the Mann-Whitney test.

[0158] Figures 3a to 31 showed that scRNA-seq-based analysis of colonic immune cells revealed major differences in myeloid cells after ECP treatment.

[0159] Specifically, Figure 3a A bar graph showing the frequency of intestinal immune cells based on their expression profiles is shown. Cells were isolated from the colonic lamina propria of mice treated with DSS / anti-PD1 alone or in combination with ECP. Two mice were analyzed per group.

[0160] Figure 3b Shown is a UMAP visualization of immune cells isolated from the colonic lamina propria of mice treated with DSS / anti-PD-1 alone or in combination with ECP. Distinct cell types were identified by Louvain cluster analysis and marker gene expression. Neutrophils and macrophages are highlighted. Two mice were analyzed per group.

[0161] Figure 3c and Figure 3d PPARγ ( Figure 3c ) and STAT6( Figure 3d ) UMAP visualization of transcription factor activity. Figure 3b Clusters are described in [ ]. Macrophage populations were labeled. Transcription factor activity was calculated based on changes in expression of downstream genes. Two mice were analyzed per group.

[0162] Figure 3e Shown Figure 3b Visualization of PPARγ and STAT6 transcription factor activity in the macrophage clusters shown. Macrophages from mice treated with DSS / anti-PD1 alone or in combination with ECP are shown. Numbers indicate calculated activity scores, while the size of the circles indicates the number of cells with detected activity. Two mice were analyzed per group. Figure 3f Figures showing body weight normalized to day 0 are shown. Body weight was measured daily. Mice received DSS / anti-PD1 treatment in combination with ECP and were treated with vehicle, PPARγ inhibitor (GW9662) or STAT6 inhibitor (AS1517499). Arrows indicate when treated with vehicle, PPARγ inhibitor or STAT6 inhibitor, or when mice received ECP transplantation. Each data point represents the mean of the combined body weights of all animals in the specified group. Data from two experiments were combined.

[0163] Figure 3g Shown is a scatter plot showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis and receiving ECP transplantation. Mice were treated with vehicle, PPARγ inhibitor, or STAT6 inhibitor as indicated. Data from both experiments were combined and significance was calculated using standard one-way ANOVA.

[0164] Figure 3h and Figure 3i Neutrophils in mice receiving DSS / anti-PD1 treatment in combination with ECP are shown ( Figure 3h ) and lymphocytes ( Figure 3i) Histopathological scoring of colonic infiltration. Mice were treated with vehicle, PPARγ inhibitor, or STAT6 inhibitor as indicated. Scoring was performed on day 8. Two individual experiments were combined (mean ± sem). P values ​​were calculated by Kruskal-Wallis test.

[0165] Figure 3j Shown is a graph depicting adiponectin serum levels in mice that received DSS / anti-PD1 treatment in combination with ECP and were additionally treated with vehicle, a PPARγ inhibitor, or a STAT6 inhibitor, as indicated. Data from both experiments were combined (mean ± sem) and significance was calculated by the Kruskal-Wallis test.

[0166] Figure 3k and Figure 3l Adipoq ( Figure 3k ) or Arg1( Figure 3l ) gene expression graph, as shown. Gene expression was measured by qPCR and normalized to Hprt expression and shown as fold change relative to vehicle treatment. Data from two separate experiments were combined (mean ± sem) and significance was calculated using standard one-way ANOVA or Kruskal-Wallis test.

[0167] Figures 3EA to 3EE showed that adiponectin expression was reduced in vitro after STAT6 and PPARγ blockade.

[0168] Specifically, Figures 3EA to 3ED Scatter bar graphs showing the expression of CD45.1 + CD45.2 splenocytes co-cultured for 48 hours + Arg1( Figure 3EA )、CD206( Figure 3EB ) and CD301( Figure 3EC ) protein level or Adipoq gene expression ( Figure 3ED BMDM were treated with vehicle, a STAT6 inhibitor (AS1517499), or a PPARγ inhibitor (T0070907). Data from three separate experiments were combined (mean ± sem). Results are shown as the fold change in MFI compared to vehicle. Significance was calculated using the Friedman test.

[0169] Figure 3EEViolin plots depicting differential gene expression in colonic macrophage clusters identified by scRNA-Seq analysis were shown. Cells isolated from DSS / anti-PD1 colitis mice treated with or without ECP were analyzed. Significance is indicated by p < 0.01 (*), p < 0.001 (**), and p < 0.0001 (***).

[0170] Figures 4a to 4p showed that adiponectin and adiponectin receptor agonist monotherapy and in combination with ECP reduced intestinal inflammation.

[0171] Specifically, Figure 4a A graph showing body weight normalized to the initial body weight on day 0 is shown. Body weight was measured daily. Each group included animals with DSS / anti-PD1-induced colitis and treated with vehicle or adiponectin receptor agonist (AdipoR agonist; ARA). Data represent the mean of the combined body weights of all animals in a given group. Arrows indicate when treatment with vehicle or AdipoR agonist was performed. Data from two experiments were combined.

[0172] Figure 4b Shown is a scatter plot showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis treated with vehicle or AdipoR agonists, as indicated. Data from two experiments were combined. P values ​​were calculated using an unpaired Student's t-test.

[0173] Figure 4c and Figure 4d Shown are scatter plots depicting neutrophil counts in mice with DSS / anti-PD1-induced colitis treated with vehicle or AdipoR agonists ( Figure 4c ) and lymphocytes ( Figure 4d ) Histopathological grade of infiltration into the colon, as shown. Results from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Mann-Whitney test.

[0174] Figure 4e A graph showing body weight normalized to initial body weight on day 0 is shown. Each group included animals with DSS / anti-PD1-induced colitis and treated with vehicle or recombinant adiponectin. Data represent the mean of the combined body weights of all animals in a given group. Arrows indicate when treatment with vehicle or adiponectin occurred. Data from two experiments were combined.

[0175] Figure 4fShown is a scatter plot showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis and treated with vehicle or recombinant adiponectin, as indicated. Data from two experiments were combined. P values ​​were calculated using an unpaired Student's t-test.

[0176] Figure 4g and Figure 4h Shown are scatter plots depicting neutrophil counts ( Figure 4g ) and lymphocytes ( Figure 4h ) Histopathological grade of infiltration into the colon, as shown. Results from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Mann-Whitney test.

[0177] Figure 4i A graph showing body weight normalized to initial body weight on day 0 is shown. Each group included animals with DSS / anti-PD1-induced colitis treated with ECP in combination with vehicle or an AdipoR agonist. Data represent the mean of the combined body weights of all animals in a given group. Arrows indicate when treatment was with vehicle or an AdipoR agonist and ECP. Data from two experiments were combined.

[0178] Figure 4j Shown are scatter bar graphs showing the mean (± sem) of mouse colon length. Each group included animals with DSS / anti-PD1-induced colitis treated with ECP in combination with vehicle or AdipoR agonist. Pooled values ​​from two experiments are shown. P values ​​were calculated using an unpaired Student's t-test.

[0179] Figure 4k and Figure 4l Shown are scatter plots depicting neutrophil counts in mice with DSS / anti-PD1-induced colitis treated with ECP in combination with vehicle or AdipoR agonists ( Figure 4k ) and lymphocytes ( Figure 4l ) Histopathological grade of infiltration into the colon. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using the Mann-Whitney test.

[0180] Figure 4mFigure 2 shows body weight normalized to initial body weight on day 0. Each group included animals with DSS / anti-PD1-induced colitis treated with ECP in combination with vehicle or recombinant adiponectin. Data represent the mean of the combined body weights of all animals from a given group. Arrows indicate when treated with vehicle or adiponectin (grey) and ECP (black). Data from two experiments were combined.

[0181] Figure 4n Shown are scatter bar graphs showing the mean (± sem) of mouse colon length. Each group included animals with DSS / anti-PD1-induced colitis treated with ECP in combination with vehicle or recombinant adiponectin. Pooled values ​​from two experiments are shown. P values ​​were calculated using an unpaired Student's t-test.

[0182] Figure 4o and Figure 4p Shown are scatter plots depicting neutrophil counts ( Figure 4o ) and lymphocytes ( Figure 4p ) Histopathological grade of infiltration into the colon. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using the Mann-Whitney test.

[0183] Figures 5a to 5o showed that ECP caused Arg1 upregulation in myeloid cells.

[0184] Specifically, Figures 5a to 5c Shown are scatter plots showing Adipoq ( Figure 5a )、Adipor1( Figure 5b ) and Adipor2( Figure 5c ) Mean (± SEM) of gene expression. Shown are the pooled values ​​of at least two separate experiments (n = 4-12 per cell type). Each data point represents one biological replicate. P values ​​were calculated using standard one-way ANOVA.

[0185] Figure 5d and Figure 5e Shown are scatter plots showing the effects of exposure to vehicle or increasing concentrations of recombinant adiponectin ( Figure 5d ) or AdipoR agonists ( Figure 5e )F4 / 80 + MFI of Arg1 in macrophages. The experiment was performed twice and the mean (± sem) is shown. Each data point shows biological replicates (n = 4). P values ​​were calculated using standard one-way ANOVA.

[0186] Figure 5f Graph showing relative Adipoq expression (y-axis) and Arg1 expression (x-axis) as a percentage of Hprt in BMDM. Pooled values ​​from four experiments (n=21) are shown. Correlation coefficients and p-values ​​were calculated using regression analysis.

[0187] Figure 5g Opt-SNE plot showing CD45.2 in the colonic lamina propria from mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP + Recipient cells (ECP donor: CD45.1). Immune cell analysis by CyTOF. Colors correspond to FlowSOM-guided cell population clustering, identified by signature marker expression.

[0188] Figure 5h An overlay of the opt-SNE plot shown in Figure 5G is shown. The plot shows intestinal CD45.2 from mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. + Receptor immune cells.

[0189] Figure 5i Opt-SNE plot showing CD45.2 isolated from the colonic lamina propria of mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP + Arginase-1 expression in myeloid clusters of recipient cells.

[0190] Figure 5j Shown is a scatter plot depicting Arg1 gene expression in the colon of mice with DSS-induced colitis and treated with anti-PD1 alone or with anti-PD1 plus ECP. Gene expression data were measured by qPCR and normalized to Hprt expression and presented as fold change relative to anti-PD1 treatment. Results from three separate experiments were combined (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0191] Figure 5k A scatter bar graph is shown showing the difference between the expression of WT C57BL / 6 and Adipoq - / - mArg1 expression in mouse-derived M2-polarized BMDM, expressed as a percentage of mHprt expression. Results from three experiments were combined (mean ± sem). p values ​​were calculated using an unpaired Student's t-test.

[0192] Figure 5l Shown are the WT or Adipoq- / - M2 polarization F4 / 80 in mice + Arg1 protein levels in BMDM. Data from four experiments were pooled (mean ± sem) and shown as fold change in MFI compared to WT. Significance was calculated using an unpaired Student's t-test.

[0193] Figure 5m Shown are the WT or Adipoq - / - M2 polarization F4 / 80 in mice + Representative histograms of Arg1 protein in BMDMs.

[0194] Figure 5n and Figure 5o Scatter bar graphs showing the differences in the expression of WT or Adipoq - / - M2 polarization F4 / 80 in mice + CD206 ( FIG. 5N ) and CD301 ( FIG. 5O ) protein levels on BMDM are presented as MFI fold changes compared to WT. Data from four experiments were combined (mean ± sem). P values ​​were calculated using unpaired Student's t-test and Mann-Whitney test.

[0195] Figures 6a to 6m showed that Arg1 deficiency in the receptor compartment reduces the protective ECP effect.

[0196] Specifically, Figure 6a A graph showing relative Adipoq expression (y-axis) as a percentage of Hprt and relative Arg1 expression (x-axis) as a percentage of Hprt in colon tissue of mice with DSS / anti-PD1-induced colitis is shown. Pooled values ​​from three experiments (n=13) are shown. Correlation coefficients and p-values ​​were calculated using regression analysis. No correlation was found.

[0197] Figure 6b Figure 2 shows a graph of relative Adipoq expression (y-axis) and relative Arg1 expression (x-axis) expressed as a percentage of Hprt in colon tissue of mice with DSS / anti-PD1-induced colitis and treated with ECP. Pooled values ​​from three experiments (n=13) are shown. Correlation coefficients and p-values ​​were calculated using regression analysis.

[0198] Figure 6cA graph showing body weight normalized to initial body weight on day 0 is shown. Each group included animals with DSS / anti-PD1-induced colitis treated with ECP and vehicle or the Arg1 inhibitor CB1158. Data represent the mean combined body weight of all animals in the designated group. Arrows indicate ECP transplantation and treatment with vehicle or Arg1 inhibitor. Data from two experiments were combined.

[0199] Figure 6d Shown is a scatter plot showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis treated with ECP and vehicle or Arg1 inhibitor, as indicated. Pooled values ​​from two experiments are shown. p values ​​were calculated using an unpaired Student's t-test.

[0200] Figure 6e and Figure 6f Shown are scatter plots depicting neutrophil counts ( Figure 6e ) and lymphocytes ( Figure 6f ) Histopathological grade of infiltration into the colon. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using the Mann-Whitney test.

[0201] Figure 6g Graphs showing body weight normalized to initial body weight on day 0 are shown. Groups included animals with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. When indicated, ECP-treated splenocytes were derived from WT or Arg1 - / - Donor mice. Arrows indicate ECP transplantation. Data represent the mean combined body weight of all animals in the indicated group. Data from two experiments were combined.

[0202] Figure 6h Shown are scatter dot bar graphs showing the mean (± sem) of colon lengths of mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. Where indicated, ECP-treated splenocytes were derived from WT or Arg1 - / - Donor mice. Pooled values ​​from two experiments are shown. p values ​​were calculated using the Kruskal-Wallis test.

[0203] Figure 6i and Figure 6j Shown are scatter plots depicting neutrophil counts in mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP ( Figure 6i) and lymphocytes ( Figure 6j ) Histopathological grade of infiltration into the colon. When indicated, ECP-treated splenocytes were derived from WT or Arg1 - / - Donor mice. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using the Kruskal-Wallis test.

[0204] Figure 6k and Figure 6l Shown are scatter plots showing neutrophil counts in mice with DSS-induced colitis treated with anti-PD1 alone or in combination with ECP ( Figure 6k ) and lymphocytes ( Figure 6l ) Histopathological grade of infiltration into the colon. When indicated, recipient mice were WT or Arg1 - / - ECP donor mice were WT. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using the Kruskal-Wallis test.

[0205] Figure 6m Representative H&E images of colon samples from mice with DSS-induced colitis treated with anti-PD1 alone or in combination with ECP are shown. Where indicated, recipient mice were WT or Arg1 - / - . Figure 6m Arrows pointing to immune cell infiltrates in the colon wall and to pseudomembranes on the colonic mucosa are also included.

[0206] Figures 7a to 71 showed that ECP and adiponectin reduced the activation of pathogenic T cells.

[0207] Specifically, Figure 7a and Figure 7b Shown are pan T cells treated with vehicle or recombinant adiponectin in the presence of anti-CD3 / CD28 activation beads. The graph shows CD4 + ( Figure 7a ) and CD8 + ( Figure 7b ) CD25 expression on T cells. T cells were adoptively transferred into recipients with DSS / anti-PD1-induced colitis. Data from both experiments were combined. Significance was calculated using the Wilcoxon test.

[0208] Figure 7c Shown are the changes in CD4 + and CD8 + Representative histograms of CD25 expression on T cells.

[0209] Figure 7d and Figure 7e Shown are scatter plots showing neutrophil counts in mice with DSS / anti-PD1-induced colitis that received adoptive transfer of anti-CD3 / CD28 activated T cells treated with vehicle or adiponectin for 72 hours ( Figure 7d ) and lymphocytes ( Figure 7e ) Histopathological grade of infiltration into the colon. Results from two separate experiments were combined (mean ± sem). p values ​​were calculated using the Mann-Whitney test.

[0210] Figures 7f to 7i Violin plots showing splenic CD4 + or CD8 + TOX in T cells ( Figure 7f )、BLTA( Figure 7g )、KLRG1( Figure 7h ) and CD47( Figure 7i ) protein levels. + Gating was performed on receptor cells. Cells were isolated from mice with DSS-induced colitis and treated with anti-PD1 alone or in combination with ECP. Data are from one experiment (n=5).

[0211] Figure 7j and Figure 7k Shown are scatter plots showing neutrophil counts in secondary recipients with DSS / anti-PD1-induced colitis who received adoptive transfer of splenic pan T cells from primary donors ( Figure 7j ) and lymphocytes ( Figure 7k ) Histopathological grade of colon infiltration. Donors had DSS-induced colitis and were treated with anti-PD1 or anti-PD1 plus ECP. Results from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Mann-Whitney test.

[0212] Figure 7l Violin plots are shown showing the expression of selected genes in the colon effector CD8 T cell cluster identified by scRNA Seq analysis. Cells isolated from DSS / anti-PD1 colitis mice treated with or without ECP were analyzed. Significance is indicated by p < 0.01 (*), p < 0.001 (**), and p < 0.0001 (***).

[0213] Figures 8a to 8v These results suggest that a protective ECP effect against irAE colitis allows for an antitumor immune response.

[0214] Specifically, Figures 8a to 8i Shown are scatter plots showing CD4 T cells isolated from spleens of mice treated with vehicle, prednisolone, or ECP. + cell( Figure 8a ), CD8 + cell( Figure 8b ), CD19 + cell( Figure 8c ), CD11b + cell( Figure 8d ), NK1.1 + cell( Figure 8e ), CD4 + TNF + cell( Figure 8f ), CD4 + INFγ + cell( Figure 8g ), CD8 + TNF + cell( Figure 8h ) and CD8 + INFγ + cell( Figure 8i The mean (± sem) of the absolute counts of ) are shown in the figure. Mice received vehicle or prednisolone treatment daily for 50 days or weekly from CD45.1 + Donor ECP transplantation, lasting eight weeks. + Pre-gating was performed on recipient cells. Each data point represents the value of a single mouse. Significance was calculated using the Kruskal-Wallis test or ordinary one-way analysis of variance.

[0215] Figure 8j Survival analysis of C57BL / 6 mice (C57BL / 6 background) transplanted intravenously with B16.F10 melanoma cells is shown. All mice were treated with anti-PD1 and received ECP transplantation or steroid treatment as indicated. Data from three separate experiments were combined. Significance was calculated by Mantel-Cox test.

[0216] Figure 8k Intravenous transplantation of BRAF V600E - Survival analysis of C57BL / 6 mice harboring mutant 4434 melanoma cells (C57BL / 6 background). All mice were treated with anti-PD-1 and received ECP transplantation or steroid treatment as indicated. Data from two separate experiments were combined. Significance was calculated by the Mantel-Cox test.

[0217] Figure 8l B16.F10 is shown luc / GFPRepresentative bioluminescent images of mice on day 23 after melanoma metastasis. All mice also received DSS to induce colitis and were treated with isotype control, anti-PD-1, or anti-PD-1 in combination with ECP or steroids, as indicated. For BLI, the following parameters remained constant for all groups: amount of injected luciferin, type of luciferin, exposure time, and distance from the camera.

[0218] Figure 8m Shown from Figure 8l Quantification of bioluminescent signals from mice shown and described in . The fold change in total flux compared to the isotype control is shown. Data from two separate experiments were combined (mean ± sem). P values ​​were calculated using standard one-way ANOVA.

[0219] Figure 8n and Figure 8o Shows the display Figure 8l Pmel( Figure 8n ) or Dct( Figure 8o ) gene expression. Gene expression was measured by qPCR and normalized to Hprt expression and shown as fold change relative to isotype control treatment. Data from two separate experiments (mean ± sem) were combined and significance was calculated using the Kruskal-Wallis test.

[0220] Figure 8p A scatter plot bar graph showing the number of OT-1 T cells per mg of tumor tissue is shown. Mice bearing subcutaneous B16.F10-OVA tumors received adoptive transfer of OT-1 T cells and were treated with anti-PD-1 alone or in combination with ECP or steroids. Tumors were digested to isolate TILs on day 16 after tumor cell transplantation. Data from two separate experiments were combined (mean ± SEM). P values ​​were calculated using standard one-way analysis of variance.

[0221] Figure 8q Shown is a scatter plot showing OVA-specific CD8 + The number of T cells / mg tumor tissue. dim Mice bearing tumors were treated with anti-PD-1 alone or in combination with ECP or steroids. Tumors were digested on day 16 after tumor cell implantation to isolate TILs. Data from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Kruskal-Wallis test.

[0222] Figure 8rSurvival analysis of C57BL / 6 mice (C57BL / 6 background) transplanted intravenously with B16.F10 melanoma cells is shown. Recipients received sublethally irradiated prior to tumor cell transplantation. If eligible, mice received adoptive transfer of T cells from mice bearing B16.F10 melanoma and treated with anti-PD1+ or anti-PD1+ steroids. Data from two separate experiments were combined. Significance was calculated using the Mantel-Cox test.

[0223] Figure 8s and Figure 8t The results show that Adipoq ( Figure 8s ) or Arg1( Figure 8t ) Gene expression in mice that received OT-1 T cells and were treated with anti-PD-1 alone or in combination with ECP. Gene expression was measured by qPCR and normalized to Hprt expression and is shown as fold change relative to anti-PD-1 treatment. Data from three separate experiments were combined (mean ± sem) and significance was calculated using an unpaired Student's t-test or a Mann-Whitney test.

[0224] Figure 8u and Figure 8v Shown are scatter plots of subcutaneously grown B16.F10-OVA tumors that received OT-I T cell transfer ( Figure 8u ) or MC38-OVA with subcutaneous growth dim Tumor ( Figure 8v Adiponectin serum concentrations in mice treated with anti-PD1 alone or in combination with ECP. Data from three separate experiments were combined (mean ± sem) and significance was calculated using unpaired Student's t-test or Mann-Whitney test. Figures 9a to 9j The clinical activity of ECP in patients with irAEs was shown.

[0225] Specifically, Figure 9aThe remission assessment of immune-related adverse events (irAEs) during / after ECP treatment is shown. The figure shows the absolute number of patients with complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD) at weeks 6 and 12 in n=14 patients enrolled in a prospective clinical Phase Ib / II trial. irAEs were graded according to CTC-AE V5.0. One patient was ineligible (ne) for a remission assessment at week 12 due to withdrawal from the study. CR was defined as complete resolution of the irAE, PR was defined as a decrease in the initial CTC-AE grade of at least one organ, SD was defined as an unchanged CTC-AE grade, and PD was defined as an increase in the initial CTC-AE grade. All patients with irAEs had an inadequate response to high-dose glucocorticoid therapy for at least 72 hours before the start of ECP therapy. The overall response rate (ORR) at weeks 6 and 12 after the start of ECP treatment is shown above each bar.

[0226] Figure 9b Figure 2 shows the time course of ALT levels in patients with ICI-induced hepatitis during ECP treatment. Relative blood ALT levels of patients enrolled in a prospective clinical phase Ib / II trial were expressed as a percentage of baseline ALT levels before the start of ECP therapy as a surrogate parameter for the severity of irAE hepatitis. Each line represents a single patient. ALT levels were assessed at weekly time points until week 12 of ECP treatment and then at extended follow-up intervals. One patient (*line) had a very mild elevation in ALT levels (CTC-AE I) before the start of ECP in addition to his severe colitis. ALT levels did not decrease much during ECP therapy as they were close to normal.

[0227] Figure 9c Figure 2 shows the temporal evolution of ICI-induced colitis during ECP therapy. Colitis severity in patients enrolled in the prospective Phase Ib / II clinical trial was graded using the CTC-AE v5.0 scale, based on diarrhea frequency at weekly time points until week 12 of ECP therapy, followed by extended follow-up intervals. Each color represents a single patient.

[0228] Figure 9d The IrAE response assessments for colitis, hepatitis, and dermatitis at week 12 of ECP treatment are shown. The absolute number of patients with complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) as defined above is shown. The ORR is shown above each bar. One patient was not evaluated (ne) due to withdrawal from the study before week 12.

[0229] Figure 9eRepresentative endoscopic images of a patient with ICI-induced colitis before and after ECP therapy are shown. Images were captured before the start of ECP therapy (Pre-ECP) while the patient was immunosuppressed with high-dose methylprednisolone, and after 22 ECP treatments (Post-ECP) when immunosuppression had fully subsided. The patient was included in a real-world patient cohort, and written informed consent was obtained for the publication of the endoscopic images.

[0230] Figure 9f Representative histological images of the colonic mucosa of a patient with colitis before and after ECP (week 6) are shown. Arrows indicate crypt abscesses.

[0231] Figure 9g Graphs showing the severity of histopathological colitis before and after at least 8 ECP treatments are shown. Each data point represents a single patient (n=7). p values ​​were calculated using a two-sided Student's paired t-test.

[0232] Figure 9h A graph representing the relative reduction in corticosteroid dose after 12 weeks of ECP therapy (week 12) compared to the first day of ECP treatment in patients with irAEs enrolled in a prospective clinical phase Ib / II trial is shown. Each point represents a single patient. One patient was ineligible for calculation of the reduction in corticosteroid dose due to withdrawal from the study before week 12. The p value was calculated using a one-sample Wilcoxon signed rank test.

[0233] Figure 9i Adiponectin concentrations in patient serum before and 2 weeks after ECP treatment are shown. Each data point represents a single patient. P values ​​were calculated using a two-sided Student's paired t-test.

[0234] Figure 9j Figure 2 shows ADIPOQ gene expression in colitis tissue from patients with irAE-colitis before and after ECP therapy. Gene expression was measured by qPCR, normalized to TBP expression, and shown as fold change relative to "pre-ECP." Each dot represents a single patient. P values ​​were calculated using a one-sample Wilcoxon signed-rank test.

[0235] Figures 9EA to 9ED The clinical activity of ECP in patients with irAEs in a real-world patient cohort is shown.

[0236] Specifically, Figure 9EAThe irAE remission assessment in a retrospective real-world patient cohort after ECP treatment is shown. The relative amounts of irAEs (colitis, arthralgia, hepatitis, dermatitis, mucositis) that responded best to ECP therapy are shown in a bar graph. In the retrospective analysis, grading was performed according to CTC-AE V.5.0. The percentages of patients with complete remission (CR) (defined as complete resolution of the irAE), partial remission (PR) (defined as a decrease in the initial irAE grade in at least one organ), and stable disease (SD) (defined as unchanged irAE grade) are shown above each bar.

[0237] Figure 9EB The reduction of immunosuppressive dose in patients with irAE during ECP treatment is shown. The relative change in immunosuppressive dose before the start of ECP therapy (blue) and after the end (red) is expressed as a percentage from baseline. One patient's systemic immunosuppression was completely reduced before the start of ECP treatment, and the patient was still receiving ECP treatment at the time of analysis (not shown). Each data point represents a single patient. P values ​​were calculated using the Wilcoxon test. Figure 9EC and Figure 9ED Scatter bar graphs showing that immunodeficient Rag2 - / - yc - / - Mouse neutrophils ( Figure 9EC ) and lymphocytes ( Figure 9ED ) Histopathological grade of colon infiltration. Mice received ECP-treated human PBMCs or steroids if eligible. Analysis was performed on day 15 after the first PBMC transfer. Results from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Kruskal-Wallis test.

[0238] Figure S1a Figure S1p shows ECP treatment and ICI colitis models.

[0239] Specifically, Figure S1a The experimental setup of DSS / anti-PD1-induced colitis treated with ECP is shown. DSS was administered for 3 days, and mice were treated with ICI on days 0, 3, and 6. ECP-treated splenocytes from donor mice were transplanted on days 3 and 6. Unless otherwise indicated, serum and tissue samples were harvested on day 8 after the start of DSS treatment.

[0240] Figures S1b to S1c show graphs depicting the histopathological grade (0 = absent, 1 = mild, 2 = abundant) of neutrophil (Figure S1b) and lymphocyte (Figure S1c) infiltration into the colon of mice with DSS-induced colitis and treated with isotype control or anti-PD1. Each data point represents the value of a single mouse. The experiment was performed twice, and the results were combined (mean ± sem). P values ​​were calculated using the Mann-Whitney test.

[0241] Figure S1d Figure 2 shows a graph showing colon length (y-axis) and body weight as a percentage of initial body weight (x-axis) of mice treated with DSS / anti-PD1 and DSS / anti-PD1 / ECP. Colon length and body weight were measured on day 8 after the start of DSS treatment. Pooled values ​​from five experiments (n=25) are shown. Correlation coefficients were calculated using regression analysis.

[0242] Figure S1e A graph showing body weight normalized to the initial body weight on day 0 is shown. Body weight was measured daily. Groups included animals with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 and untreated donor splenocytes. Each data point represents the mean of the combined body weights of all animals in a given group. Arrows indicate splenocyte transfer. Data from two experiments were combined.

[0243] Figure S1f Shown are scatter bar graphs showing the mean (±sem) of colon length in mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 and untreated donor splenocytes. P values ​​were calculated using an unpaired Student's t-test.

[0244] Figures S1g and S1h show graphs depicting the histopathological grading of neutrophil (Figure S1g) and lymphocyte (Figure S1h) infiltration into the colon of mice with DSS-induced colitis treated with anti-PD1 alone or in combination with untreated donor splenocytes. Each data point represents the value of a single mouse. The experiment was performed twice, and the results were combined (mean ± sem). P values ​​were calculated using the Mann-Whitney test.

[0245] Figure S1i A graph showing body weight normalized to the initial body weight on day 0 is shown. Body weight was measured daily. Each group included animals with DSS / anti-PD1-induced colitis and treated with ECP or steroids. Each data point represents the mean combined body weight of all animals in the designated group. Arrows indicate ECP (black) and steroid (grey) treatment. Data from two experiments were combined.

[0246] Figure S1jShown is a scatter plot showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis and treated with ECP or steroids. Data from two experiments were combined. P values ​​were calculated using an unpaired Student's t-test.

[0247] Figures S1k and S11 show graphs depicting histopathological grading of neutrophil (Figure S1k) and lymphocyte (Figure S11) infiltration into the colon of mice with DSS / anti-PD1-induced colitis and treated with ECP or steroids. The experiment was performed twice and the results were combined (mean ± sem). P values ​​were calculated using the Mann-Whitney test.

[0248] Figure S1m A graph is shown depicting body weight normalized to the body weight on day 0 of the experiment. Body weight was measured daily. Groups included patients with DSS-induced colitis treated with anti-PD1, anti-PD1 plus 10 7 ECP-treated splenocytes or anti-PD1 plus 10 7 Figure 5. ECP-treated PBMC-treated mice. Data represent the mean of the combined body weights of all animals in the indicated group. Arrows indicate when ECP was performed (days 3 and 6).

[0249] Figure S1n Shown are scatter bar graphs showing the mean colon length (± sem) of mice with DSS / anti-PD1-induced colitis, mice with DSS / anti-PD1-induced colitis treated with ECP-treated splenocytes, and mice with DSS / anti-PD1-induced colitis treated with ECP-treated PBMCs, as indicated. P values ​​were calculated using one-way ANOVA.

[0250] Figures S1o and S1p show histopathological scores for neutrophil (Figure S1o) and lymphocyte (Figure S1p) infiltration into the colon of mice with DSS / anti-PD1-induced colitis and mice with DSS / anti-PD1-induced colitis treated with ECP-splenocytes or ECP-PBMCs. Scoring was performed on day 8 of the experiment. Each data point represents the value of a single mouse (mean ± sem). P values ​​were calculated by Kruskal-Wallis test.

[0251] Figures S2a to S2e These results suggest that ECP treatment is effective in anti-CTLA4-induced colitis.

[0252] Specifically, Figure S2aA graph showing body weight normalized to initial body weight on day 0 is shown. Body weight was measured daily. Each group included animals with DSS-induced colitis treated with an isotype control, anti-CTLA4, or anti-CTLA4 plus ECP. Each data point represents the mean combined body weight of all animals in the designated group. Arrows indicate ECP treatment. Data from two experiments were combined.

[0253] Figure S2b Shown is a scatter bar graph showing the mean (± sem) of colon length in mice with DSS-induced colitis treated with isotype control, anti-CTLA4, or anti-CTLA4 plus ECP. Data from two experiments were combined. P values ​​were calculated using standard one-way ANOVA.

[0254] Figures S2c and S2d show graphs depicting histopathological grading of neutrophil (Figure S2c) and lymphocyte (Figure S2d) infiltration into the colon of mice treated with DSS-induced colitis and treated with isotype control, anti-CTLA4, or anti-CTLA4 plus ECP. The experiment was performed twice, and the results were combined (mean ± sem). P values ​​were calculated by Kruskal-Wallis test.

[0255] Figure S2e Shown is a scatter bar graph showing the mean (± sem) of adiponectin serum concentrations in mice with DSS-induced colitis treated with anti-CTLA4 alone or in combination with ECP. Data from two experiments were combined and p values ​​were calculated using an unpaired Student's t-test.

[0256] Figure S3a Figure S3h shows that adiponectin deficiency in donor leukocytes does not reduce ECP efficacy.

[0257] Specifically, Figure S3a A graph showing body weight normalized to initial body weight on day 0 is shown. Body weight was measured daily. Groups included Adipoq mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. - / - animal.

[0258] Animals were adiponectin deficient in both the donor and recipient compartments (Adipoq - / - ). Each data point represents the mean combined body weight of all animals in a given group. Arrows indicate ECP treatment. Data from two experiments were combined.

[0259] Figure S3b Shown is a scatter plot of Adipoq mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. - / -Mean (± sem) of mouse colon length. Animals were adiponectin-deficient in both the donor and recipient compartments. Data from two experiments were combined and p values ​​were calculated using an unpaired Student's t-test.

[0260] Figures S3c and S3d show graphs depicting Adipoq mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. - / - Histopathological grading of neutrophil (Figure S3c) and lymphocyte (Figure S3d) infiltration into the colon of mice. Animals were adiponectin-deficient in both the donor and recipient compartments. Experiments were performed twice, and the results were combined (mean ± sem). P values ​​were calculated by Mann-Whitney test or unpaired Student's t-test.

[0261] Figure S3e A graph showing body weight normalized to initial body weight on day 0 is shown. Body weight was measured daily. Groups included WT animals with DSS / anti-PD1-induced colitis treated with ECP. Mice received either Adipoq from a WT donor or - / - ECP cells from donors as indicated. Each data point represents the mean of the combined body weights of all animals in the designated group. Arrows indicate ECP treatment. Data from two experiments were combined.

[0262] Figure S3f Shown is a scatter plot showing the mean colon length (±sem) of WT mice with DSS / anti-PD1 induced colitis treated with ECP. - / - ECP cells from donors, as shown. Data from the two experiments were combined and p values ​​were calculated using an unpaired Student's t-test. Figures S3g and S3h show graphs depicting the histopathological grading of neutrophil (Figure S3g) and lymphocyte (Figure S3h) infiltration into the colon of WT mice with DSS / anti-PD1-induced colitis and treated with ECP. Mice received either ECP from a WT donor or Adipoq - / - The experiments were performed twice, and the results were combined (mean ± sem), and the p-values ​​were calculated by Mann-Whitney test.

[0263] Figures S4a to S4n These results showed that ECP induced rapid apoptosis of leukocytes in vitro.

[0264] Figure S4aShown is a scatter bar graph showing the relative viability (% of the 0 h time point) of mouse splenocytes that were left untreated (UT) or treated with UVA, 8-MOP, or ECP, as indicated. Cell viability was monitored using a luminescence-based viability assay. Data from three experiments were combined (mean ± sem).

[0265] Figure S4b Figure 1 shows the percentage of dead cells in all analyzed mouse splenocytes at different time points after treatment as analyzed by flow cytometry. Splenocytes were kept untreated (UT) or treated with UVA, 8-MOP, or ECP as shown. P values ​​were calculated using one-way ANOVA.

[0266] Figure S4c Representative Western blot images of p53 and vinculin protein levels in splenocytes from mice left untreated or treated with ECP are shown. Analyses were performed at various time points after treatment. Vinculin was used as a loading control.

[0267] Figure S4d Shown is a scatterplot bar graph showing the fold change in p53 protein levels in ECP-treated mouse splenocytes relative to UT splenocytes at various time points after treatment. P53 was normalized to vinculin, used as a loading control. Each data point represents the value from a single mouse. P values ​​were calculated using two-way ANOVA. Figure S4e A graph depicting the fold change in caspase 3 / 7 activity in ECP-treated murine splenocytes relative to UT splenocytes at various time points after treatment is shown. Caspase 3 and caspase 7 activity were measured using a luminescence-based assay. Each data point represents the value from a single mouse. P values ​​were calculated using a two-way ANOVA.

[0268] Figures S4f to S4n Shown are mouse splenocytes that were left untreated (UT) or treated with UVA, 8-MOP, or ECP as indicated. Proteins were isolated 24 hours after treatment and analyzed by Western blotting; β-actin was used as a loading control. The scattered bar graphs show protein levels of total protein or cleavage products. PARP ( Figure S4f , Figure S4g and Figure S4h), caspase 9 ( Figure S4i and Figure S4j ), caspase 3 ( Figure S4k and Figure S4l ) and caspase 8 ( Figure S4m and Figure S4n ) abundance.

[0269] Figure S5a and Figure S5bThese results indicate that ECP-treated murine splenocytes undergo cell death in vivo.

[0270] Specifically, Figure S5a Shown are the results of multiple time points after transfer in the presence of C57BL / 6 luc After spleen cell transplantation from donor mice, Rag2 - / - γc - / - Total flux in the receptor (photons / s). C57BL / 6 luc Donor mice were transplanted with B16.F10 melanoma and treated with anti-PD-1. Splenocytes were isolated on day 9 and kept untreated (UT) or treated with ECP before transplantation.

[0271] Figure S5b shows the results of the experiment with C57BL / 6 luc After transplantation of spleen cells from donor mice, Rag2 - / - γc - / - Quantification of BLI signals from ex vivo imaging of different organs isolated from recipients. Analysis was performed on day 30 after transplantation. Donor mice were treated as described in (a). Data from two experiments were combined (mean ± sem). P values ​​were calculated using two-way ANOVA. Figures S6a to S6h These results indicate that ECP-treated splenocytes are phagocytosed by antigen-presenting cells.

[0272] Specifically, Figure S6a Calreticulin protein levels on live splenocytes that were untreated (UT) or exposed to UVA, 8-MOP, or ECP at different time points after treatment are shown. Data are shown as fold change (mean ± sem) relative to the MFI of UT cells. P values ​​were calculated using the Kruskal-Wallis test.

[0273] Figure S6b Shown are representative histograms of calreticulin on the surface of splenocytes that were untreated (UT) or exposed to ECP at 6 and 24 hours post-treatment.

[0274] Figure S6c Shown is the expression of CD45.2 + Representative flow cytometry images of CellTrace Violet (CTV) in BMDCs. ECP-treated CTV was stained for CD45.1 + Splenocytes exposed to CD45.2 + BMDCs were maintained for 48 hours.

[0275] Figure S6d Shows CTV + CD45.2 + Quantification of the percentage of BMDCs in all BMDCs. ECP-treated CTV was stained for CD45.1+ Splenocytes exposed to CD45.2 + BMDC were maintained for 48 hours. BMDC were used as controls only. Data from multiple experiments were combined (mean ± sem). P values ​​were calculated using the Wilcoxon test.

[0276] Figure S6e Shown is the effect of CD45.2 + Analysis of SIINFEKL-H2kb on BMDMs treated with ECP-treated CTV loaded with SIINFEKL ovalbumin peptide + CD45.1 + Splenocytes were co-cultured for 48 hours. - CD45.2 + MFI fold change compared to BMDM. BMDM that engulfed ECP-treated cells became CTV positive. Data from two experiments were combined (mean ± sem). P values ​​were calculated using the Wilcoxon test. Figure S6f A scatter plot is shown showing the difference between the + Co-culture of BMDM with CD45.1 + ECP-treated splenocytes. Macrophages were treated with vehicle or the phagocytosis inhibitors Endosidin 9 or PitStop 2. Live CD45.1 + Splenocytes represent cells that were not phagocytosed. Data from two experiments were combined (mean ± sem). Significance was calculated using one-way analysis of variance.

[0277] Figure S6g and Figure S6h Figure 2 shows Adgre 1 expression in colitis tissues of DSS / anti-PD1 / ECP-treated mice receiving vehicle or phagocyte-depleted clodrosomes. Figure S6g ) or Cd68( Figure S6h ) Gene expression graph. Gene expression was measured by qPCR, normalized to Hprt expression, and shown as fold change relative to vehicle treatment. Data from two separate experiments were combined (mean ± sem) and significance was calculated using unpaired Student's t-test or Wilcoxon test.

[0278] Figures S7a to S7c showed that splenic immune cells have higher integrin expression compared with epithelial cells.

[0279] Specifically, Figure S7aRelative gene expression of molecules involved in transendothelial migration is shown. RNA was isolated from FACS-sorted splenocytes from untreated mice or from the intestinal epithelial cell line MODE-K (n=5 per group). Gene expression was measured by qPCR and normalized to Hprt expression. The scale represents the row-scaled level of gene expression. Figure S7b Figure 2 shows a graph showing the percentage of dead MODE-K epithelial cells as a percentage of all analyzed cells analyzed by flow cytometry 24 hours after treatment. Cells were either left untreated or treated with ECP, and data from two experiments are shown (mean ± sem). P values ​​were calculated using Student's t-test.

[0280] Figure S7c Calreticulin protein levels on live MODE-K epithelial cells that were untreated or exposed to ECP are shown. Calreticulin was measured 24 hours after treatment. Data from two experiments are shown as fold change (mean ± sem) relative to the MFI of untreated cells. P values ​​were calculated using the Wilcoxon test.

[0281] Figure S8a Figure S8l showed that ECP-treated epithelial cells had no protective effect against ICI-induced colitis.

[0282] Specifically, Figure S8a and Figure S8e Body weights normalized to the initial body weight on day 0 are shown. Body weights were measured daily. FACS-sorted spleen myeloid cells ( Figure S8a ) or T cells ( Figure S8e ) were transplanted into mice with DSS / anti-PD1-induced colitis. Each data point represents the mean combined body weight of all animals in the designated group. Arrows indicate ECP transplants.

[0283] Figure S8b and Figure S8f Shown is a scatter plot of FASC-sorted spleen myeloid cells ( Figure S8b ) or T cells ( Figure S8f )-treated mice (±sem). P values ​​were calculated using unpaired Student's t-test.

[0284] Figures S8c and S8d show graphs depicting histopathological grades (mean ± sem) of neutrophil (Figure S8c) and lymphocyte (Figure S8d) infiltration into the colon of mice with DSS / anti-PD-1-induced colitis and treated with ECP-exposed, FASC-sorted myeloid cells. P values ​​were calculated by Mann-Whitney test or unpaired Student's t-test.

[0285] Figures S8g and S8h show graphs depicting histopathological grades (mean ± sem) of neutrophil (Figure S8g) and lymphocyte (Figure S8h) infiltration into the colon of mice with DSS / anti-PD-1-induced colitis and treated with ECP-exposed, FASC-sorted T cells. P values ​​were calculated by Mann-Whitney test.

[0286] Figure S8i Body weights were normalized to the initial body weight on day 0. Body weights were measured daily. Each group included spleen cells (2×10 6 ) or MODE-K epithelial cells treated with anti-PD1 plus ECP (2×10 6 ) treated mice. Each data point represents the mean combined body weight of all animals in the designated group. Arrows indicate ECP transplantation.

[0287] Figure S8j Shown is a scatter plot of the effect of anti-PD1, anti-PD1 plus ECP on splenocytes (2×10 6 ) or anti-PD1 plus ECP-exposed epithelial cells (2×10 6 Mean colon length (± SEM) of mice treated with ). P values ​​were calculated using one-way ANOVA.

[0288] Figures S8k and S8l show graphs depicting the results of 3-D-type splenic leukemia (2 × 10 6 ) or epithelial cells (2×10 6 ) Histopathological grades of neutrophil (Figure S8k) and lymphocyte (Figure S8l) infiltration into the colon of mice treated with the combination (mean ± sem). P values ​​were calculated using the Kruskal-Wallis test.

[0289] Figure S9a and Figure S9b These results showed that M2 macrophage-related transcription factors were upregulated after ECP.

[0290] Specifically, Figure S9a Shown is a visualization of KLF4 and PPARα transcription factor activity in colonic macrophage clusters identified by scRNA-seq. Macrophages from mice treated with DSS / anti-PD-1 alone or in combination with ECP are shown. Numbers indicate calculated activity scores, while circle size indicates the number of cells with detected activity. Two mice were analyzed per group.

[0291] Figure S9b Violin plots depicting differential gene expression of colonic neutrophil clusters identified by scRNA-Seq analysis are shown. Cells isolated from DSS / anti-PD1 colitis mice treated with ECP or without ECP were analyzed. Significance is indicated by p < 0.01 (*), p < 0.001 (**), and p < 0.0001 (***).

[0292] Figures S10a to S10d showed that AdipoRon reduced anti-CTLA-4-induced colitis.

[0293] Specifically, Figure S10a Body weights normalized to the initial body weight on day 0 are shown. Body weights were measured daily. Mice with DSS / anti-CTLA4-induced colitis were treated with vehicle or the adiponectin receptor agonist AdipoRon. Each data point represents the mean combined body weight of all animals in the designated group. Arrows indicate AdipoRon treatment. Data from two experiments were combined.

[0294] Figure S10b Shown are scatter bar graphs showing mean colon length (±sem) of mice with DSS / anti-CTLA4-induced colitis treated with vehicle or AdipoRon. Data from two experiments were combined and p values ​​were calculated using an unpaired Student's t-test.

[0295] Figure S10c and Figure S10d Figures are shown depicting neutrophil counts in mice with DSS / anti-CTLA4-induced colitis treated with vehicle or AdipoRon. Figure S10c ) and lymphocytes ( Figure S10d ) Histopathological grading of colon infiltration. The experiment was performed twice, and the results were combined (mean ± sem), and p values ​​were calculated by Mann-Whitney test.

[0296] Figures S11a to S11c showed that ECP induces CD45.2 in the spleen of mice with DSS / anti-PD1-induced colitis + Cell changes.

[0297] Specifically, Figure S11a Shown is an overlay of opt-SNE plots showing CD45.2 in spleens of mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP + Recipient cells (ECP donor: CD45.1). Immune cells were analyzed by CyTOF.

[0298] Figure S11b Shown are separate opt-SNE plots showing CD45.2 in mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. + Recipient cells (ECP donor: CD45.1). Immune cell analysis by CyTOF. Colors correspond to FlowSOM-guided cell population clustering, identified by signature marker expression.

[0299] Figure S11c Violin plots showing splenic CD45.2 from mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP are shown. + CD11b + Arg1 protein levels in recipient cells were analyzed by CyTOF and compared using an unpaired Student's t-test.

[0300] Figures S12a to S12e showed that ECP rendered recipient myeloid cells tolerogenic in colitis mice.

[0301] Specifically, Figure S12a Shown is a heat map showing the splenic receptor CD45.2 isolated from mice with DSS-induced colitis and treated with anti-PD1 alone or in combination with ECP + CD11b + Protein levels of tolerogenic markers on myeloid cells.

[0302] The scale represents the row-wise zoomed level of marker expression, with red being highest and blue being lowest. Analysis was performed by CyTOF.

[0303] Figures S12b to S12e Violin plots showing the expression of the splenic receptor CD45.2 isolated from mice with DSS-induced colitis and treated with anti-PD1 alone or in combination with ECP are shown. + CD11b + CD39 on myeloid cells ( Figure S12b )、PD-L1( Figure S12c )、Tim3( Figure S12d ) and B7-H4( Figure S12e ) Protein levels were analyzed by CyTOF and analyzed using an unpaired Student's t-test.

[0304] Figure S13a and Figure S13b showed that the expression of intestinal stem cell genes was increased in mice with colitis after receiving ECP.

[0305] Specifically, Figure S13a and Figure S13bFigures are shown showing the expression of Ascl2 ( Figure S12a ) or Axin2( Figure S13b ) gene expression. Gene expression was measured by qPCR and normalized to Hprt expression and is shown as fold change relative to anti-PD1 treatment. Data from three separate experiments were combined (mean ± sem) and significance was calculated using an unpaired Student's t-test.

[0306] Figures S14a to S14q showed that ECP increased tolerogenic T cells in ICI-induced colitis.

[0307] Specifically, Figures S14a to S14c Shown are pan T cells treated with vehicle or recombinant adiponectin in the presence of anti-CD3 / CD28 activating beads. + ( Figure S14a ) and CD8 + ( Figure S14b )T cells as a percentage of all T cells, and FoxP3 + T reg ( Figure S14c ) accounts for CD4 + The percentage of T cells was calculated. Data from two experiments were combined (mean ± sem). The significance was calculated using paired Student's t-test.

[0308] Figures S14d to S14f The expression of BTLA ( Figure S14d )、CD47( Figure S14e ) or KLRG1( Figure S14f ) of spleen CD8 + Absolute counts of T cells. Cells were isolated from mice with DSS-induced colitis and treated with anti-CTLA4 alone or in combination with ECP. + Pre-gating was performed on receptor cells. Significance was calculated using the Mann-Whitney test. Mean values ​​(± sem) are shown.

[0309] Figures S14g and S14h show the histopathological grade of neutrophil (Figure S14g) and lymphocyte (Figure S14h) infiltration into the colon of mice with DSS-induced colitis and treated with anti-PD1 or anti-PD1 plus ECP. Mice were used as T cell donors for adoptive transfer of T cells into secondary recipients with colitis. Experiments were performed twice (mean ± sem), and p values ​​were calculated by Mann-Whitney test.

[0310] Figures S14i to S14qFigure 4 shows CD4 expression in pan-T cells isolated from mice with DSS-induced colitis. + and CD8 + T cells ( Figure S14i and Figure S14n )、KLRG1 + ( Figure S14j and Figure S14o ), CD47 + ( Figure S14k and Figure S14p ), BTLA + (Figure S14l and Figure S14q )T cells and FoxP3 + T reg (Figure S14m) Frequency (mean ± sem). Recipients were treated with anti-PD1 or anti-PD1 plus ECP. T cells were isolated on day 8 and adoptively transferred into colitis secondary recipients. Significance was calculated by Mann-Whitney or unpaired Student's t-test.

[0311] Figures S15a to S15g show that long-term steroid treatment reduced splenic immune cells.

[0312] Specifically, Figure S15a shows spleen weights of mice treated chronically with vehicle or prednisolone for 50 days or transplanted with ECP eight times a week. Spleens were isolated on day 50. Data from both experiments were combined (mean ± sem), and p values ​​were calculated using one-way ANOVA.

[0313] Figures S15b to S15g show the results of ECP transplantation (CD45.1 + Splenic CD45.2 isolated from untreated mice + Recipient monocytes (Figure S15b), neutrophils (Figure S15c), CD4 + FoxP3 + T reg Absolute counts of pDCs (Figure S15d), pDCs (Figure S15e), cDC1s (Figure S15f), and cDC2s (Figure S15g). Cells were isolated on day 50. The experiment was performed twice (mean ± sem), and p values ​​were calculated by one-way ANOVA.

[0314] Figures S16a to S16m These results suggest that the protective ECP effect did not reduce the anti-tumor immune response.

[0315] Specifically, Figure S16aThe experimental setup of the intravenous B16.F10 melanoma model treated with anti-PD1, steroids, and ECP is shown. Where applicable, mice received transplants of ECP-treated splenocytes from donor animals (arrows on days 13, 18, and 22). Arrows on days 1, 4, 8, 13, 16, and 22 indicate anti-PD1 treatment, and orange arrows on days 13 to 22 indicate steroid treatment.

[0316] Figure S16b Shown is a survival analysis of C57BL / 6 mice (C57BL / 6 background) that were intravenously transplanted with B16.F10 melanoma cells. Mice were treated with isotype control or anti-PD1, and one group received anti-PD1 and untreated donor splenocytes. Data are from multiple experiments. Significance was calculated by Mantel-Cox test.

[0317] Figures S16c and S16d show histopathological scoring of neutrophil (Figure S16c) and lymphocyte (Figure S16d) infiltration into the colon of melanoma-bearing mice with DSS-induced colitis. Analysis was performed on day 23 after melanoma implantation. The experiment was performed as described in (a) and shown in the Methods section, with the addition of 3% DSS from day 9 to day 12. The experiment was performed in duplicate (mean ± sem), and p values ​​were calculated by Kruskal-Wallis test.

[0318] Figure S16e and Figure S16f show the expression of mPmel (Figure S16e) and mDct (Figure S16f) in the lungs of healthy C57BL / 6 mice and in B16.F10 melanoma cells, expressed as a percentage of mPmel expression (mean ± sem). P values ​​were calculated using an unpaired Student's t-test.

[0319] Figures S16g to S16j Shown are CD4 T cells isolated from mice implanted intravenously with B16.F10 melanoma. + Splenic T cells and CD8 + The protein level of IFNγ in splenic T cells ( Figure S16g and Figure S16i ) and CD4 + Splenic T cells and CD8 + CD107a protein levels on splenic T cells ( Figure S16h and Figure S16j Mice were treated with anti-PD-1 alone or with anti-PD-1 and ECP. Analysis was performed on day 23 after tumor implantation. Splenocytes were restimulated before analysis. Significance was calculated using the Mann-Whitney or unpaired Student's t-test.

[0320] Figure S16kShown are subcutaneously treated MC38-OVA dim Spleen weights of mice treated with steroids for 10 days were calculated as shown. Spleens were isolated on day 16 after tumor injection. Data from two experiments were combined (mean ± sem) and p values ​​were calculated using one-way ANOVA.

[0321] Figure S16l Figure 2 shows the survival analysis of C57BL / 6 mice (C57BL / 6 background) that were intravenously transplanted with B16.F10 melanoma cells. Recipients received sublethally irradiated prior to tumor cell transplantation. If eligible, mice received adoptive transfer of T cells from B16.F10 melanoma-bearing mice treated with anti-PD1. Data from two separate experiments were combined. Significance was calculated by the Mantel-Cox test.

[0322] Figure S16m Intravenous transplantation of BRAF V600E - Survival analysis of C57BL / 6 mice (C57BL / 6 background) harboring mutant 4434 melanoma cells. Recipients were sublethally irradiated prior to tumor cell transplantation. If eligible, mice received adoptive transfer of T cells from anti-PD1-treated B16.F10 melanoma-bearing mice. The transferred T cells were specific for B16.F10 but not for 4434-BRAF. V600E No specificity. Data from two separate experiments were combined. Significance was calculated by Mantel-Cox test.

[0323] Figure S17a Figure S17h shows that ECP does not induce tolerogenic tumor-specific T cells.

[0324] Specifically, Figures S17a to S17e BTLA of each tumor mass is shown + ( Figure S17a ), CD47 + ( Figure S17b )、KLRG1 + ( Figure S17c ), PD-1 + ( Figure S17d ) or TIM-3 + ( Figure S17e ) Total number of OT-1 T cells. Cells were isolated from mice bearing subcutaneous B16.F10-OVA tumors (CD45.1) that had received adoptive transfer of OT-1 T cells (CD45.2) and were treated with anti-PD-1 or anti-PD-1 / ECP. Tumor-infiltrating T cells were isolated on day 16 after tumor implantation. Data from two experiments were combined (mean ± SEM). P values ​​were calculated using the Mann-Whitney test or unpaired Student's t-test.

[0325] Figures S17f to S17h show the BTLA for each tumor mass. + (Figure S17f), CD47 + (Figure S17g) and KLRG1 + (Figure S17h) OVA-Dextramer + CD8 + Total number of (tumor-specific) T cells. dim Mice with tumors were isolated and treated with anti-PD-1 or anti-PD-1 / ECP (mean ± sem). Tumor-infiltrating T cells were isolated on day 16 after tumor implantation. P values ​​were calculated using the Mann-Whitney test or unpaired Student's t-test.

[0326] Figures S18a to S18b Figure S18m Patient data from a prospective Phase Ib / II clinical trial are shown.

[0327] Specifically, Figure S18a shows a scatterplot bar graph showing the mean number of weeks (± sem) of corticosteroid pretreatment before initiation of ECP therapy for n = 14 patients enrolled in a prospective clinical phase Ib / II trial (range 2-16 weeks). Each data point represents one patient.

[0328] Figure S18b shows a scatter plot showing the number of ECP treatment courses (mean ± sem) per patient in n = 14 patients in a prospective clinical phase Ib / II trial (range 9-30). Each data point represents one patient. One patient received only nine ECP treatments before discontinuing treatment due to disease progression, while one patient continued ECP therapy after 16 treatments due to recurrence of colitis and responded to ECP again.

[0329] Figure S18c shows the tumor remission assessment during the ECP therapy in prospective clinical Ib / II phase trial.Bar graph shows the absolute number of patients with tumor remission before (week 0) and after 12 weeks (week 12) of ECP treatment.Bar shows the absolute number of patients in week 0 and week 12 respectively.One patient experienced tumor progression and died soon after week 6.The patient could not perform tumor remission assessment at week 12 and was therefore shown as non-evaluable (ne).According to immune-related RECIST criteria and based on radiological findings, tumor remission was divided into complete remission (CR), partial remission (PR), stable disease (SD) and progressive disease (PD).

[0330] Figure S18dFigure 5 Kaplan-Meier estimates of overall survival from the start of ECP therapy until death from any cause. Median survival was not reached, with a median follow-up of 8 months, as determined by the inverse Kaplan-Meier method.

[0331] Figure S18e The IrAE response assessments for colitis, hepatitis, and dermatitis after 6 weeks of ECP therapy are shown. The absolute number of patients with complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) is shown. The ORR percentage for each irAE is shown above each bar.

[0332] Figures S18f to S18i show lymphocyte subsets quantified by flow cytometry in peripheral blood of patients before the first ECP treatment and after the second ECP treatment. Lymphocyte counts are shown as percentages of peripheral blood leukocytes in patients enrolled in a prospective clinical phase Ib / II trial. Each dot represents one patient. CD4 + T cells (Figure S18f) and CD8 + T cells (Figure S18g), NK cells (Figure S18h), and B cells (Figure S18i). Significance was calculated using paired Student's t-test.

[0333] Figures S18j to S18m Figure 1 shows the quantification of lymphocyte subsets by flow cytometry at two different time points with at least 1 week between the two time points of sample collection in a prospective clinical phase Ib / II trial when ECP therapy was administered to patients with irAEs. The relative lymphocyte counts of peripheral blood leukocytes are shown. Each point represents one patient. CD4 + T cells ( Figure S18j ) and CD8 + T cells ( Figure S18k ), NK cells ( Figure S18l ) and B cells ( Figure S18m ). Significance was calculated using paired Student's t-test.

[0334] Figures S19a to S19d Patient data from a real-world patient cohort are shown.

[0335] Specifically, Figure S19a Shown is a scatter plot showing the mean number of weeks (± sd) of corticosteroid pretreatment before the start of ECP therapy for n = 7 patients enrolled in a real-world patient cohort and receiving ECP for irAEs at one center. Each dot represents one patient.

[0336] Figure S19bShown is a scatterplot bar graph showing the mean number of ECP courses (± sem) for n = 7 irAE patients enrolled in a real-world patient cohort and treated at one center. Each dot represents one patient.

[0337] Figure S19c The time course of AST (grey dots) and ALT levels (orange squares) in a representative patient with ICI-induced hepatitis before (negative values) and after (positive values) the start of ECP treatment is shown, expressed in U / l. The time intervals between the administration of systemic immunosuppression with corticosteroids and mycophenolate mofetil (MMF) and the administration of infliximab are shown above the graph. Overall, complete remission of immune-related hepatitis required seven ECPs.

[0338] Figure S19d Figure 3 shows tumor response assessments for n=7 patients in a real-world patient cohort before and after ECP treatment termination. Tumor responses were divided into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to immune-related RECIST criteria and based on radiological findings. At the time of analysis (ongoing), one patient was on ECP therapy. At the time of data cutoff, the patient's tumor was in complete response.

[0339] Figure S20a Figure S20o shows that a protective ECP effect was found in a humanized irAE model.

[0340] Specifically, Figure S20a Shows the use of Rag2 - / - γc - / - Experimental setup of humanized irAE model in recipient mice. 7 PBMCs from healthy individual donors were treated with human anti-PD-1. If eligible, mice received ECP-treated human PBMCs or prednisolone as therapy. Organs were harvested 15 days after the initial PBMC injection.

[0341] Figures S20b to S20g show scatter plots showing that immunodeficient Rag2 cells injected with human PBMCs and treated with human anti-PD1 - / - yc - / -Histopathological grading of neutrophil (Figure S20b, Figure S20d, and Figure S20f) and lymphocyte (Figure S20c, Figure S20e, and Figure S20g) infiltration into the liver (Figure S20b and Figure S20c), lung (Figure S20d and Figure S20e), and skin (Figure S20f and Figure S20g) of mice. Mice received ECP-treated human PBMCs or steroids if eligible. Analyses were performed on day 15 after the first PBMC transfer. Results from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Kruskal-Wallis test.

[0342] Figures S20h to S20o show scatter plots showing immunodeficient Rag2 cells that were left untreated or injected with human PBMCs and treated with vehicle or human anti-PD1. - / - yc - / - Histopathological grading of neutrophil (Figure S20h, Figure S20j, Figure S20l, and Figure S20n) and lymphocyte (Figure S20i, Figure S20k, Figure S20m, and Figure S20o) infiltration into the colon (Figure S20h and Figure S20i), liver (Figure S20j and Figure S20k), lung (Figure S20l and Figure S20m), and skin (Figure S20n and Figure S20o) of mice. Analyses were performed on day 15 after the first PBMC transfer. Results from two separate experiments were combined (mean ± sem). P values ​​were calculated using the Kruskal-Wallis test.

[0343] Figure S21 A graphical summary of the mechanism of action of ECP on ICI-induced colitis is shown.

[0344] Specifically, Figure S21 They showed that ECP induces apoptosis of leukocytes, which are then phagocytosed by intestinal macrophages. Uptake induces a transcriptional program that includes STAT6 and PPARγ activation, thereby enhancing adiponectin transcription. Intestinal macrophages polarize toward an immunosuppressive phenotype, resulting in the production of Arg1 and adiponectin. Subsequently, tolerogenic T cells increase in the inflamed intestine but not in melanoma tissue, preserving antitumor immunity. DETAILED DESCRIPTION

[0345] All citations in this patent and non-patent literature are hereby incorporated by reference in their entirety.

[0346] The present invention relates to adiponectin and / or an adiponectin receptor agonist (ARA) or a combination drug comprising adiponectin and / or an adiponectin receptor agonist (ARA), a photosensitizer and lymphocytes, which are obtained from a method comprising the following steps: (i) providing a sample obtained from an isolated blood sample of the subject, (ii) adding the photosensitizer to the sample, and (iii) subjecting the sample to UVA irradiation, preferably ECP treatment, for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in the subject.

[0347] In a preferred embodiment, the photosensitizer is administered ex vivo to a blood sample of the subject prior to in vitro UVA irradiation of the blood sample of the subject.In a preferred embodiment, the subject is receiving checkpoint blockade therapy, for example as a cancer therapy.

[0348] As used herein, the term "subject" refers to a human or non-human animal selected for treatment or therapy. A subject or patient, such as a subject in need of treatment or prevention, can be an animal, a vertebrate, a mammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), a murine (e.g., a mouse), a canine (e.g., a dog), a feline (e.g., a cat), an equine (e.g., a horse), a primate, a simian (e.g., a monkey or ape), a monkey (e.g., a marmoset, a baboon), an ape (e.g., a gorilla, a chimpanzee, an orangutan, a gibbon), or a human. The meanings of the terms "animal," "mammal," and the like are well known in the art and can be derived, for example, from Wehner und Gehring (1995; Thieme Verlag). The term subject may also refer to a "patient" and can be used interchangeably therewith herein. In the context of the present invention, it is particularly contemplated that animals of economic, agronomic, or scientific importance are treated. Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human.

[0349] In embodiments of the invention, the subject who has received checkpoint inhibitor therapy has cancer, such as malignant melanoma or another cancer treatable by checkpoint inhibitor therapy.

[0350] "Adiponectin" (also known as Acrp30, GBP28, apM1, or AdipoQ) is a protein hormone and adipokine (a cytokine secreted by adipose tissue) that is involved in regulating glucose levels and fatty acid breakdown. Adiponectin is known to bind to at least two receptors, two of which are homologous to G protein-coupled receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2), and one receptor is similar to the cadherin family, T-cadherin CDH13. The adiponectin receptor affects the downstream target AMP kinase, which is an important control point for cellular metabolic rate. The expression of the receptor is correlated with insulin levels. Adiponectin generally regulates multiple metabolic processes, such as glucose regulation and fatty acid oxidation, including glucose flux, decreased gluconeogenesis, increased glucose uptake, lipid catabolism, beta-oxidation, triglyceride clearance, protection from endothelial dysfunction (an important aspect of atherogenesis), insulin sensitivity, weight loss, control of energy metabolism, upregulation of uncoupling proteins, reduction of TNF-α, and promotion of reverse cholesterol transport. Adiponectin is known to self-associate into larger structures, starting with three adiponectin molecules forming a homotrimer, which then further self-associates into hexamers or dodecamers. Monomeric (30 kDa) adiponectin is not normally observed in the circulation and appears to be confined to adipocytes.

[0351] Irradiation and extracorporeal photopheresis (ECP)

[0352] Photopheresis, or extracorporeal photopheresis or ECP, is a form of apheresis and photodynamic therapy in which blood is treated with a photosensitizer and subsequently irradiated with light of a specific wavelength to achieve its effect. For example, the buffy coat (WBC + platelets) can be separated from whole blood, chemically treated with 8-methoxypsoralen (either dripped into a collection bag or taken orally beforehand), exposed to ultraviolet light (UVA), and transfused back into the patient. Activated 8-methoxypsoralen crosslinks DNA in exposed cells, ultimately leading to apoptosis of nucleated cells. The photochemically damaged T cells transfused back into the patient appear to induce a cytotoxic effect on T cell formation.

[0353] Photopheresis involving 8-methoxypsoralen was first described in a 1987 publication in the New England Journal of Medicine (Edelson, R et al. (1987). "Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy. Preliminary results", New England Journal of Medicine. 316(6):297-303). Photopheresis is currently a standard therapy approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma. There is evidence that this treatment may be effective in treating graft-versus-host disease. Photopheresis has also been successfully used to treat epidermolysis bullosa acquisitive when all other treatments have failed.

[0354] As used herein, ECP includes blood irradiation therapy. In embodiments, the ECP and ECP systems of the present invention relate to blood irradiation therapy and systems for blood irradiation therapy. In embodiments of the present invention, ECP relates to ECP other than blood irradiation therapy.

[0355] Blood irradiation therapy is a procedure in which the blood is exposed to low-intensity red light (usually a laser) for therapeutic reasons. Blood irradiation therapy can be administered in three ways. In vitro, blood is drawn and irradiated in a special cuvette. This method is used to perform ultraviolet (UV) blood irradiation (UVBI) using a UV lamp. The laser is monochromatic, that is, it has a wavelength that allows the light to be brought into an optical fiber and irradiated intravenously through a catheter in the vein. This method is simpler and more effective. Blood irradiation therapy is also administered topically by targeting the surface projection area of ​​large blood vessels.

[0356] Intravenous or intravascular laser blood irradiation (ILBI) involves internal irradiation of the blood by delivering low-intensity laser light at a wavelength of 632.8 nm, generated by a 1-3 mW helium-neon laser, into vascular channels (typically veins in the forearm), under the assumption that any therapeutic effects will circulate through the circulatory system. Wavelengths of 365 nm, 405 nm, 525 nm, and 635 nm and a power of 2.3 mW are most commonly used. This technique is currently widely used in Russia, to a lesser extent in Asia, and not widely used in other parts of the world. Results have shown that ILBI improves blood flow and transport activity, thereby improving tissue nutritional status and having positive effects on the immune system and cellular metabolism. This issue has been questioned, and there have been calls for increased research on this topic. Transcutaneous therapy involves applying laser light to intact skin in areas with dense blood vessels, such as the forearm. Because the skin acts as a barrier to the blood, absorbing low-level laser energy, the laser power is often increased to compensate. This problem can be overcome by using a pulsed matrix laser source. External irradiation is used exclusively for ultraviolet blood irradiation, where blood is drawn through a vein and irradiated outside the body.

[0357] Despite being promoted as a cancer treatment, a 1952 review in the Journal of the American Medical Association and another 1970 review by the American Cancer Society concluded that the treatment was ineffective.

[0358] "Extracorporeal photopheresis (ECP)", also known as "extracorporeal photoimmunotherapy" or "photochemotherapy", is a leukapheresis-based therapy that was initially used for patients with cutaneous T-cell lymphoma (CTCL). Specifically, ECP was approved by the FDA (U.S. Food and Drug Administration) in 1988 for the treatment of refractory CTCL patients with the leukemia variant Sézary Syndrome. During ECP, the patient's whole blood is collected via an antecubital vein or a permanently implanted catheter to separate the white blood cells from the plasma and anucleated cells. Using a device specially constructed for the procedure, the collected white blood cells (the so-called buffy coat) are then exposed to ultraviolet A (UVA) irradiation in the presence of a photosensitizer (8-methoxypsoralen) and then re-infused into the patient.

[0359] Two fundamentally different methods for performing the ECP procedure have been described and encompassed herein. These differ in the apparatus used for leukocyte collection and UVA irradiation: a "closed system" and a so-called "open system." The closed system, based on the original design by Edelson and colleagues, is the only FDA-approved system.

[0360] In a closed ECP system, a white blood cell layer containing lymphocytes and platelets is separated from the blood by centrifugation. When red blood cells and plasma are reintroduced into the vein of the experimenter (via an intravenous catheter), the separated white blood cell layer is mixed with a photosensitive substance (preferably 8-methoxypsoralen) and then subjected to ultraviolet A (UVA) irradiation (320nm-400nm). The UVA irradiation induces the photosensitive substance to be incorporated into the DNA of the lymphocytes. Subsequently, the treated white blood cell layer is reinfused into the patient through an intravenous catheter.

[0361] Open systems are systems that combine different separation instruments and are primarily used outside the U.S. Although ECP has been an effective treatment for 30 years and more than 2 million treatments have been performed, no adverse cytogenetic effects have been reported to date.

[0362] Since its introduction, the range of indications for ECP has continued to expand. ECP treatment is generally well tolerated by patients and has few significant adverse side effects. Overall, ECP offers an excellent combination of safety and efficacy.

[0363] Extracorporeal photopheresis (sometimes also referred to as extracorporeal photochemotherapy) is a process that includes the following steps: (1) collecting mononuclear cells (MNCs) from a patient, (2) subjecting the collected MNCs to a photoactivation treatment; and (3) re-infusing the treated cells (MNCs) back into the patient. More specifically, ECP involves the in vitro exposure of peripheral blood mononuclear cells in combination with a photoactive compound (such as 8-methoxypsoralen or "8-MOP"), which is then photoactivated by ultraviolet light, followed by re-infusion of the treated monocytes. It is believed that the combination of 8-MOP and UV irradiation causes apoptosis, or programmed cell death, of the ECP-treated T cells.

[0364] Although the exact mechanism of action of ECP treatment (in different disease states) is not fully understood, according to early theories, it is believed that light activation causes 8-MOP to irreversibly covalently bind to DNA chains contained in the nucleus of T cells. When the photochemically damaged T cells are re-infused, a cytotoxic effect is induced. For example, cytotoxic T cells or "CD8+ cells" release cytotoxins when exposed to infected or damaged cells, or otherwise attack cells that carry certain foreign or abnormal molecules on their surface. The cytotoxins target the membranes of damaged cells and enter the target cells, ultimately leading to apoptosis, or programmed cell death, of the target cells. In other words, after the treated monocytes are returned to the body, the immune system recognizes the dying abnormal cells and begins to produce healthy lymphocytes (T cells) to fight these cells.

[0365] In addition to the above, it is theorized that extracorporeal photopheresis also induces monocytes (a type of mononuclear cell) to differentiate into dendritic cells that can phagocytose and process apoptotic T cell antigens. When these activated dendritic cells are reinfused into the systemic circulation, they can elicit a systemic cytotoxic CD8+ T lymphocyte-mediated immune response against the apoptotic T cell antigens processed as described above. It should be understood that other possible mechanisms of action may be involved in achieving the benefits that have been observed from ECP treatment of monocytes and the subsequent benefits to patients receiving ECP-based therapies.

[0366] Recently, some have hypothesized that ECP may induce an immune tolerance response in patients. For example, in the case of graft-versus-host disease, the infusion of apoptotic cells can stimulate the production of regulatory T cells, inhibit the production of inflammatory cytokines, cause the loss of effective T cells, and lead to other responses. See Peritt, "Potential Mechanisms of Photopheresis in Hematopoietic Stem Cell Transplantation," Biology of Blood and Marrow Transplantation 12:7-12 (2006). Although the immune tolerance response theory seems to be one of the main explanations, there are other theories about the mechanism of action of ECP in graft-versus-host disease and other disease states.

[0367] Systems that perform ECP include, for example, the UVAR XTS Photopheresis System and the CellEx Photopheresis System, available from Therakos, Inc. of Exton, Pa. Further details of performing ECP on the Therakos system can be found, for example, in US Patent No. 5,984,887.

[0368] There are two common methods for performing photopheresis—online and off-line systems and methods.

[0369] In the online approach, the entire treatment, including the reinfusion of processed MNCs, is performed using a dedicated photopheresis device, such as the Therakos device described above. Such devices are "dedicated" photopheresis devices, designed only to perform photopheresis and are not capable of performing other collection protocols required in a hospital or blood processing environment, including, for example, multifunctional apheresis protocols for collecting platelets, plasma, RBCs, granulocytes, and / or performing plasma / RBC exchange protocols.

[0370] In the offline photopheresis method, a multifunctional apheresis device is used to collect mononuclear cells. The collected MNCs, typically contained in one or more collection containers, are cut or otherwise separated from the tubing used during the collection process, subsequently processed in a separate irradiation or UVA light device, and the treated cells are then manually re-infused into the patient. However, in this offline method, when the cells are transferred from the apheresis device to the irradiation device (which can be located in another room or laboratory), the connection with the donor must be cut off, so the cells are detached from the donor. Therefore, additional traceability procedures are required to ensure that the processed MNC product is ultimately re-infused into the correct donor.

[0371] In embodiments, ECP employs (a) apheresis, wherein peripheral blood mononuclear cells are collected ex vivo, (b) photoactivation, wherein leukocyte-rich plasma is exposed to the photosensitizer 8-methoxypsoralen and ultraviolet A light, and (c) re-infusion of such modified ECP-treated cells into the patient.

[0372] According to the present invention, the expression "extracorporeal blood purification" preferably refers to a process of removing substances from body fluids by removing these substances from the flowing blood in a diverting circuit outside the patient's body (extracorporeally). The substances may include endogenous toxins (i.e., uremic toxins), exogenous poisons (i.e., ethylene glycol or mycotoxins), administered drugs, viruses, bacteria, antibodies, metabolites and proteins (i.e., IMHA, myasthenia gravis), abnormal cells (i.e., leukemia) and excess water. Therapeutic procedures include hemodialysis, including intermittent hemodialysis (HD, HDF, HF) and continuous renal replacement therapy (CRRT); hemoperfusion; plasma exchange and therapeutic blood separation. Such methods are known to the skilled person and can be combined with the device of the present invention accordingly.

[0373] As used herein, the expression "blood" refers to whole blood containing all the components of the blood of an organism, including red blood cells, white blood cells and platelets suspended in plasma. The expression "plasma" refers to a fluid consisting of about 92% water, 7% proteins (such as albumin, gamma globulin, fibrinogen, complement factors, coagulation factors) and 1% mineral salts, sugars, fats, electrolytes, hormones and vitamins, which forms a part of whole blood but no longer contains red blood cells, white blood cells and platelets. In the context of the present invention, the expression "plasma" refers to a specific fraction of plasma defined above in its standard meaning, such as serum.

[0374] In therapeutic apheresis, whole blood may be processed or separated into its component fractions, for example by centrifugation or with the aid of plasma membranes or filters, and the fractions containing the solutes to be removed are specifically treated before being returned to the patient.

[0375] The present invention provides an apheresis treatment in which whole blood or plasma (containing the target protein) is removed from a patient's circulating blood and returned to the patient after contact with an apheresis device or matrix.

[0376] ICB treatment

[0377] "Immune checkpoints" are an important part of the immune system because they prevent excessive immune responses that can lead to autoimmune reactions or generally damage healthy cells of the body. Immune checkpoints are effective when proteins on the surface of T cells (such as PD-1) recognize and bind to checkpoint proteins (such as PD-L1) on other cells (such as tumor cells). The binding of checkpoint proteins to partner proteins on T cells can lead to T cell inhibition and prevent the T cells from killing other cells. This mechanism not only prevents the autoimmune effects of T cells, but also prevents the killing of tumor cells.

[0378] "Immune checkpoint blockade" (ICB) therapy, "checkpoint inhibitor therapy" or "immune checkpoint inhibitors" block (immune) checkpoint proteins from binding to their partner proteins on T cells, thereby preventing the inhibition of T cell-mediated killing of, for example, cancer cells. Immune checkpoint blockade therapy may include treatment with anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (ipilimumab and tremelimumab), anti-programmed cell death receptor (PD-1) (nivolumab and pembrolizumab) or anti-PD ligand (PD-L1) (durvalumab, atezolizumab and avelumab) monoclonal antibodies. ICBs use the infiltration of immune cells into the tumor or tumor environment to initiate and / or restore an effective anti-tumor immune response. The PD-1 / PD-L1 signaling pathway is considered to be critical for the interaction between immune cells, stromal cells and tumor cells. In this article, the terms "immune checkpoint blockade" (ICB) therapy, "checkpoint inhibitor therapy" or "immune checkpoint inhibitor" therapy are used interchangeably.

[0379] "Immune checkpoint inhibitor" or "ICB" therapy can cause a variety of side effects that can depend on the patient's health at the start of treatment, the type of cancer being treated, the cancer stage, the drug administered, and its dose. A group of side effects can be summarized as immune-related adverse events (irAEs).

[0380] Immune-related adverse events (irAEs)

[0381] As used herein, the term "immune-related adverse event" (irAE) refers to any specific side effect that occurs specifically in the context of treatment with immune checkpoint inhibitors, for example, in the context of cancer therapy. IrAEs are unique and different from adverse events that occur in the context of traditional cancer therapies, typically with a delayed onset and duration. IrAEs can involve any organ or system. These effects are usually mild, treatable, and reversible; however, some side effects can be severe and lead to permanent disease. Treatment is mainly based on corticosteroids and other immunomodulators, which should be used with caution to reduce the possibility of short-term and long-term complications.

[0382] Common immune-related adverse events (irAEs) include gastrointestinal, endocrine, and skin toxicities. Rare but more severe and potentially fatal irAEs may include neurotoxicity, cardiotoxicity, and pulmonary toxicity. Observed cytotoxicity is typically graded according to four severity levels (currently based on the Common Terminology Criteria for Adverse Events, the European Society for Medical Oncology guidelines, and the American Society of Clinical Oncology guidelines; see, for example, Brahmer et al., 2018; Haanen et al., 2018). Higher-grade irAE cytotoxicity is typically treated by discontinuing ICB therapy and / or initiating glucocorticoid therapy (e.g., including administration of prednisone).

[0383] The term irAE specifically includes symptoms of autoimmune diseases, as well as autoimmune diseases such as (autoimmune) colitis, (autoimmune) hepatitis, (autoimmune) thyroiditis and (autoimmune) dermatitis. There is no particular limitation on irAEs caused by the administration of checkpoint inhibitor therapy in the present invention. irAEs are understood to be adverse events that are presumed to be immune-related: irAEs (e.g., see the revised April 2016 Pre-medication evaluation interview form for intravenous infusion 20mg-100mg (9th edition); anti-CTLA-4 antibody ipilimumab The nature and management of adverse events of sedatives, published by the Japan Dermatological Association Malignant Melanoma New Drug Safety Committee on August 24, 2015).

[0384] The specific embodiment of the irAE of the present invention includes interstitial lung disease, myasthenia gravis, myositis, colitis, type 1 diabetes, liver dysfunction (liver disease), lung disease such as hepatitis (for example, autoimmune pneumonia), pituitary dysfunction such as hypopituitarism or hypophysitis, thyroid dysfunction such as hypothyroidism, neuropathy, nephropathy, encephalitis, adrenal disease such as adrenal insufficiency, severe skin disease, venous thromboembolism, infusion reaction, psoriasis, psoriatic rash, diarrhea (for example, severe diarrhea), rheumatoid arthritis, uveitis, episcleritis, bursitis, radiation dermatitis increases, chronic inflammatory demyelinating polyneuropathy (hereinafter also referred to as demyelinating polyneuropathy), biliary tract disease or nephritis, preferably pituitary dysfunction. It is generally known that immune-related adverse events (IrAEs) occur after 8 to 12 weeks after administering ICB. Immune-related adverse events can be evaluated with "grade" or "IrAE evaluation". Here, "irAE score" is an indicator of disease severity, expressed on a scale of 1 to 3. In the irAE score, 1 represents "no additional therapeutic intervention is required due to the irAE," 2 represents "medication intervention is required due to the irAE, but hospitalization or treatment interruption is not required," and 3 represents "medication intervention is required, accompanied by hospitalization due to the irAE, and treatment interruption is required." The correspondence between "irAE score" and "grade" varies for each disease.

[0385] Immune checkpoint molecules and checkpoint modulators

[0386] In the context of the present invention, immune checkpoint inhibitors are drugs that activate immune cells by regulating immune checkpoint molecules.

[0387] Immune checkpoint molecules are molecules in the immune system that either enhance signals (co-stimulatory molecules) or reduce the signals provided to immune effector cells. Therefore, immune checkpoint molecules can be subdivided into co-stimulatory checkpoint molecules or co-inhibitory checkpoint molecules. Co-stimulatory checkpoint molecules include co-stimulatory lymphocyte receptors, which are lymphocyte surface receptors that can lead to the activation or stimulation of lymphocyte effector functions. Co-inhibitory checkpoint molecules include co-inhibitory lymphocyte receptors, which are lymphocyte surface receptors that can lead to the inhibition of lymphocyte effector functions.

[0388] Co-stimulatory checkpoint molecules include, but are not limited to, HVEM, CD27, CD40, OX40, GITR, CD137, CD28, and ICOS.

[0389] In a preferred embodiment of the present invention, the term co-stimulatory lymphocyte receptor does not refer to CD122.

[0390] HVEM (herpesvirus entry mediator, CD270), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a receptor of the TNF-receptor superfamily that binds to BTLA.

[0391] CD27 supports antigen-specific expansion of naive T cells and is crucial for the generation of T cell memory. It is also a memory marker for B cells. CD27 activity is controlled by the transient availability of its ligand, CD70, on lymphocytes and dendritic cells. CD27 co-stimulation is known to inhibit Th17 effector cell function. The agonistic monoclonal antibody CDX-1127 / varirizumab targeting CD27 has been shown to be effective in the context of T cell receptor stimulation in animal models. CD28 is constitutively expressed on nearly all human CD4+ T cells and approximately half of all CD8 T cells. Binding of one of its two ligands, CD80 and CD86, expressed on dendritic cells, for example, promotes T cell expansion.

[0392] CD40 is expressed on a variety of immune system cells, including antigen-presenting cells. Its ligand is called CD40L, also known as CD154, and is transiently expressed on the surface of activated CD4+ T cells. CD40 signaling is known to "license" dendritic cells to mature, thereby triggering T cell activation and differentiation.

[0393] 4-1BB (CD137) binds to the CD137 ligand, leading to T cell proliferation. It is also known that CD137-mediated signaling protects T cells, particularly CD8+ T cells, from activation-induced cell death. The fully human IgG2 agonist monoclonal antibody Utolumab (PF-05082566) targets 4-1BB to stimulate the immune system to launch a stronger attack on cancer.

[0394] OX40 (CD134) has OX40L (CD252) as its ligand. OX40 promotes the expansion of effector and memory T cells and is also known for its ability to inhibit the differentiation and activity of T regulatory cells. OX40 is transiently expressed following T cell receptor engagement, which is why it is only upregulated on recently antigen-activated T cells within inflammatory lesions, and why OX40 is a valuable drug target. Agonistic anti-OX40 monoclonal antibodies have been shown to have clinical utility in advanced cancers. Pharmaceutical company AstraZeneca has three drugs in development targeting OX40: MEDI0562, a humanized OX40 agonist; MEDI6469, a murine OX40 agonist; and MEDI6383, an OX40 agonist.

[0395] GITR (glucocorticoid-induced TNFR family-related gene) promotes T cell expansion. GITR's ligand (GITRL) is primarily expressed on antigen-presenting cells. Antibodies to GITR have been shown to promote anti-tumor responses by destabilizing the Treg lineage.

[0396] ICOS (inducible T cell co-stimulator, also known as CD278) is expressed on activated T cells. Its ligand is ICOSL, which is mainly expressed on B cells and dendritic cells. This molecule appears to be important in T cell effector function. Co-inhibitory checkpoint molecules include but are not limited to A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, TIGIT and VISTA.

[0397] A2AR (adenosine A2A receptor) is considered an important checkpoint in cancer therapy because adenosine in the immune microenvironment leading to A2A receptor activation is a negative immune feedback loop, and the tumor microenvironment has a relatively high concentration of adenosine.

[0398] B7-H3, also known as CD276, was originally considered a co-stimulatory molecule but is now considered a co-inhibitory molecule. MacroGenics is investigating MGA271 (enotuzumab), an Fc-optimized monoclonal antibody targeting B7-H3.

[0399] B7-H4 (or VTCN1) is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor evasion. BTLA (B and T lymphocyte attenuator, also known as CD272) is a co-inhibitory receptor that has HVEM (herpes virus entry mediator) as its ligand. During the differentiation of human CD8+ T cells from a naive cell phenotype to an effector cell phenotype, the surface expression of BTLA is gradually downregulated, whereas tumor-specific human CD8+ T cells express high levels of BTLA.

[0400] IDO (indoleamine 2,3-dioxygenase) is a tryptophan-catabolizing enzyme with immunosuppressive properties. Another important molecule is TDO, or tryptophan 2,3-dioxygenase. IDO is known to inhibit T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis.

[0401] KIR (killer cell immunoglobulin-like receptor) is a receptor for MHC class I molecules on natural killer cells. Lirelumab is a monoclonal antibody against KIR.

[0402] LAG-3 (lymphocyte activation gene-3) suppresses immune responses through both effects on Tregs and direct effects on CD8+ T cells.

[0403] "Cytotoxic T lymphocyte-associated protein 4" (CTLA-4 or CTLA4 or CD152-cluster of differentiation 152) is a protein receptor constitutively expressed on regulatory T cells that acts as an immune checkpoint, downregulating T cell-mediated immune responses. When CTLA-4 binds to CD80 or CD86 on the surface of antigen-presenting cells, it can inactivate T cell effector function. CTLA-4 is a regulatory molecule that inhibits T cell effector function after initial activation by co-stimulatory signals. Human monoclonal antibodies targeting CTLA-4 are used in ICB therapy and can increase T cell function and anti-reversal or immune response. Immune checkpoint blockade (ICB) therapy may include treatment with anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (ipilimumab and tremelimumab) monoclonal antibodies.

[0404] "Programmed cell death protein 1" (PD-1, or CD279—cluster of differentiation 279) is a cell surface protein / receptor on B cells and T cells that plays a role in the regulation of autoimmune responses by inhibiting T cell inflammatory activity and promoting self-tolerance. PD-1 has two ligands, PD-L1 and PD-L2. PD-1 is an immune checkpoint that protects against autoimmunity through two mechanisms: promoting apoptosis of antigen-specific T cells in the lymph nodes, and reducing apoptosis of regulatory T cells. The advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. Immune checkpoint blockade (ICB) therapy can include treatment with anti-programmed cell death receptor (PD-1) monoclonal antibodies, such as nivolumab (Opdivo—BristolMyers Squibb), pembrolizumab (Keytruda, MK-3475, Merck), pidilizumab (CT-011, Cure Tech), and BMS-936559 (BristolMyers Squibb). Atezolizumab (MPDL3280A, Roche) and avelumab (Merck, Darmstadt, Germany, and Pfizer) are monoclonal antibodies that target the PD-1 ligand, PD-L1. The transmembrane protein "programmed death ligand 1" (PD-L1 or CD274—cluster of differentiation 274 or B7-H1—B7 homolog 1) is encoded by the CD274 gene in humans. IFN-γ stimulation induces PD-L1 expression on T cells, NK cells, macrophages, myeloid dendritic cells, B cells, epithelial cells, and vascular endothelial cells. Binding of PD-L1 to its receptor, PD-1, present on activated T cells, B cells, and myeloid cells regulates immune cell activation or suppression. Binding of PD-L1 to its receptor, PD-1, on T cells initiates activation signals that inhibit TCR-mediated T cell proliferation and IL-2 production. The interaction between PD-1 and its ligand, PD-L1, prevents autoimmunity. PD-L1 is highly expressed in many cancers, particularly lung cancer. Immune checkpoint blockade (ICB) therapy can include treatment with monoclonal antibodies against PD-L1 (durvalumab, atezolizumab, and avelumab).

[0405] TIM-3 (T cell immunoglobulin domain and mucin domain 3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1 / Th17 function by triggering cell death after interacting with its ligand galectin-9.

[0406] VISTA (V-domain Ig inhibitor of T-cell activation) is a protein primarily expressed on hematopoietic cells, so the sustained expression of VISTA on intratumoral leukocytes may make VISTA blockade effective against a wide range of solid tumors.

[0407] TIGIT (T-cell immunoreceptor with Ig and ITIM domains, also known as WUCAM and Vstm3) is an immunoreceptor present on some T cells and natural killer cells and regulates T cell-mediated immunity. TIGIT can bind with high affinity to CD155 on DCs and macrophages and with lower affinity to CD112.

[0408] The co-inhibitory lymphocyte receptors of the present invention include PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, BTLA or VISTA. The co-stimulatory lymphocyte receptors of the present invention include OX40, 4-1BB, GITR, CD27, HVEM, CD28 or CD40.

[0409] Receptor inhibitors prevent the corresponding receptor from generating a signal. Thus, inhibitors of co-inhibitory lymphocyte receptors are molecules that prevent the activation of the corresponding receptor and thus prevent the generation of inhibitory signals. Conversely, activators of receptors induce the corresponding receptor to generate a signal, while activators of co-stimulatory lymphocyte receptors result in the generation of stimulatory signals.

[0410] Checkpoint modulators are molecules that interfere with the activity of immune checkpoint molecules by either stimulating or inhibiting their activity.

[0411] Soluble checkpoint modulators are molecules that are able to diffuse freely and, for example, are not bound to cell membranes or retained within the cell.

[0412] In the sense of the present invention, check point inhibitors include lymphocyte stimulation check point regulators, which are molecules that cause lymphocyte (preferably effector T cells) activation by the activation of costimulatory check point molecules or by the inhibition of co-inhibitory check point molecules. In addition, soluble lymphocyte stimulation check point regulators include molecules that interfere with the activation of membrane-bound immune check point molecules, such as soluble forms of corresponding immune check point molecules.

[0413] Checkpoint modulators can be naturally occurring or engineered molecules that interfere with or modulate the activity of immune checkpoint molecules. Checkpoint modulators include, for example, antibodies or antibody fragments that have agonist or antagonist activity against immune checkpoint molecules, as well as ligands or modified ligands of immune checkpoint molecules.

[0414] The present invention encompasses both therapeutic and prophylactic treatment of subjects. "Prophylactic" treatment is treatment administered to subjects who do not show signs of disease or who show only early signs of disease, with the goal of reducing the risk of developing the condition. Prophylactic treatment is administered to prevent the progression, acceleration, escalation, and / or onset of disease.

[0415] "Glucocorticoids", which are further referred to as "glucocorticoids", "corticosteroids" or simply "steroids", are the most effective anti-inflammatory substances currently used to treat chronic inflammatory, autoimmune and immune diseases. Glucocorticoids induce immunosuppression, mainly including a decrease in lymphocyte function and quantity of both B cells and T cells. Glucocorticoids are lipophilic hormones that can be subdivided into glucocorticoids produced in the zona fasciculata of the adrenal cortex, mineralocorticoids produced in the zona glomerulosa, and sex hormones produced in the zona reticularis and to a greater extent in the gonads. A variety of synthetic glucocorticoids can be used for therapeutic purposes. Examples of glucocorticoids are Cortisol (hydrocortisone), cortisone, prednisone, prednisolone, methylprednisolone and dexamethasone.

[0416] immune cells

[0417] Immune cells as described herein relate to biological cells participating in the immune response in a subject.Immune cells are preferably selected from T cells, B cells, dendritic cells, granulocytes, innate lymphocytes (ILC), megakaryocytes, monocytes / macrophages, natural killer (NK) cells, platelets, red blood cells (RBC) and / or thymocytes.Term " immune cell " includes the MNC comprised in blood, also can be referred to as peripheral blood mononuclear cells (PBMC).PBMC includes any peripheral blood cell with round nucleus, and is mainly composed of lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets do not have nuclei, and granulocytes (neutrophils, basophils and eosinophils) have multilobed nuclei.In people, lymphocytes (lymphoid cells) account for the major part of PBMC colony, followed by monocytes, and only a small part is dendritic cells. These cells can be extracted from whole blood using ficoll (a hydrophilic polysaccharide that separates blood layers) and "density gradient centrifugation," which separates the blood into a top layer of plasma, followed by a PBMC layer and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and red blood cells. The polymorphonuclear cells can be further isolated by lysing the red blood cells. Basophils are sometimes present in both the denser fraction and the PBMC fraction.

[0418] In a preferred embodiment of the present invention, the leukocytes may particularly refer to lymphocytes, wherein the leukocytes or lymphocytes may preferably include or refer to T cells.

[0419] The "buffy coat" can be separated from plasma and red blood cells by centrifugation. The buffy coat can also be referred to as the fraction of the anticoagulated blood sample that contains most white blood cells (leukocytes) and platelets after centrifugation. It is rich in many immune cells, including platelets and white blood cells (leukocytes), such as lymphocytes, granulocytes, monocytes and macrophages. The buffy coat can be used to enrich a large number of leukocyte subsets from peripheral blood mononuclear cells (PBMC). PBMC is a separate lymphocyte and monocyte that can be separated from the residual whole blood sample by a process called density gradient centrifugation (see above).

[0420] " T cell " or T lymphocyte is a class of lymphocytes (subtype of leukocyte) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes such as B cells and natural killer cells by the presence of T cell receptors on the cell surface. Several subgroups of T cells each have different functions. T cell subtypes include but are not limited to type 1 T helper (Th1) cells, type 2 T helper (Th2) cells, type 9 T helper (Th9) cells, type 17 T helper (Th17) cells, type 22 T helper (Th22) cells, follicular helper T (Tfh) cells, regulatory T (Treg) cells, natural killer T (NKT) cells, γ δ T cells, CD8+ cytotoxic T lymphocytes (CTL). Other non-limiting embodiments of T cells include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells) or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3 and CD56 positive, non-major histocompatibility complex (MHC) restricted, natural killer (NK)-like T lymphocytes. T cells can be CD4+T cells, cytotoxic T cells (CTL; ​​CD8+T cells), CD4+CD8+T cells, CD4CD8 T cells or any other T cell subsets. "Natural killer (NK) cells" are cytotoxic innate effector cells similar to the cytotoxic T cells of the adaptive immune system. NK cells are a group of innate immune cells that show spontaneous cytolytic activity against stress cells, for example, cancer cells or virus-infected cells. Upon activation, NK cells can secrete cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF) and chemokines (CCL1, CCL2, CCL3, CCL4, CCL5 and CXCL8), which can regulate the functions of other innate and adaptive immune cells. NK cells in humans are usually labeled CD3 - CD56 +Cells. They are distributed throughout the blood, organs and lymphoid tissues and constitute approximately 15% of peripheral blood lymphocytes. NK cells play a role in the rapid elimination of tumor surveillance and virus-infected cells. They do not require the missing "self" signal of MHC class I and can recognize stress cells in the absence of antibodies, allowing them to react faster than the adaptive immune system. Natural killer (NK) cells play a key role in host immunity against cancer. In response, cancer develops mechanisms to evade NK cell attack or induce defective NK cells (Cheng M et al., Cell Mol Immunol, May 2013; Vol. 10 No. 3: pp. 230-252. doi:10.1038 / cmi.2013.10. Epub, April 22, 2013.).

[0421] The present invention particularly relates to immunoregulatory T cells (which may also be referred to as immunoregulatory T cells) for use in treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy (preferably as cell therapy).

[0422] In embodiments, the generation or induction of immunoregulatory T cells in a sample can be measured by comparing the phenotype and population distribution of monocytes and T cells in the sample before and after performing the methods of the invention, for example, by flow cytometry, mass spectrometry analysis of gene expression and / or protein abundance. 低 Arginase-1 高 The shift in monocytes indicates the induction of an immunoregulatory phenotype. In addition, a decrease in the expression of GM-CSF, IFN-γ, TNF and / or IL-2 in a T cell population also indicates the induction of immunoregulatory T cells. The skilled person is aware of suitable methods and protocols for identifying different monocyte and T cell populations within a sample. For example, in a blood-derived sample, total monocyte and T cell populations can be preferably identified by flow cytometry and defined as tolerogenic KLRG1 + BTLA + CD47 + TOX 低 T cell populations.

[0423] Additional characteristics and definitions of immunoregulatory T cells are well known in the art and are the subject of numerous studies and review articles known to or identifiable by the skilled artisan.

[0424] The term "immunomodulatory function" relates to a function or property of a molecule or cell that induces a change or modulation of the function, action or state of any component of the immune system.

[0425] Cell therapy typically involves administering immune cells isolated from a patient's blood. Cell types that can be used in this manner include, but are not limited to, natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, monocytes, macrophages, granulocytes, and dendritic cells. Dendritic cell therapy stimulates an anti-tumor response by having dendritic cells present tumor antigens. Dendritic cells present the antigens to lymphocytes, which activates them and triggers them to kill other cells that presented the antigen.

[0426] cancer

[0427] In the context of the present invention, the term "cancer" relates to the treatment of all kinds of cancer, regardless of whether the cancer is associated with the formation of solid tumors or whether the cancer cells do not form solid tumors, as is the case with certain leukemias.

[0428] Cancer includes a group of diseases that can affect any part of the body and is caused by abnormal cell growth and proliferation. These proliferating cells have the potential to invade surrounding tissues and / or spread to other parts of the body where they form metastases. In 2012, there were 14 million new cancer cases and 8.2 million cancer-related deaths worldwide (World Cancer Report 2014). Most cancers are caused by environmental signals involving tobacco use, obesity, and infection, while about 5%-10% are hereditary cases. Cancer can be divided into subclasses based on the cell of origin. The most common subclasses are epithelial cell carcinoma, connective tissue sarcoma, and lymphomas and leukemias from hematopoietic cells. Cancer is associated with a variety of local and systemic symptoms and cannot be cured in many cases. In view of the high number of cases of new cancer patients and cancer-related deaths, new treatment strategies are needed.

[0429] Cancer according to the present invention refers to all types of cancers or neoplasms or malignancies found in mammals, including leukemias, sarcomas, melanomas and carcinomas. Any solid tumor and / or liquid tumor (such as leukemia or lymphoma) can be treated.

[0430] Melanomas include, but are not limited to, acral lesional melanoma, amelanotic melanoma, benign juvenile melanoma, Claudemann melanoma, S91 melanoma, Hardin-Pass melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0431] Leukemias include, but are not limited to, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic granulocytic leukemia, cutaneous leukemia, stem cell leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hematopoietic leukemia, hematoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenia Leukemia, lymphoid leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myelogranulocytic leukemia, chronic myelomonocytic leukemia, Negley's leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, Liddle's cell leukemia, Schilling's leukemia, stem cell leukemia, subcellular leukemia and undifferentiated cell leukemia.

[0432] Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Albernethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chlorocarcinosarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukocytic sarcoma, malignant mesenchymal sarcoma, extraperiosteal sarcoma, reticulum cell sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0433] Cancer includes, but is not limited to, acinar carcinoma, acinar carcinoma, adenoid cystic carcinoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basaloid cell tumor, basaloid carcinoma, basosquamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchial carcinoma, encephaloid carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, armored carcinoma, skin carcinoma, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, medullary carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, explant carcinoma, ulcerative carcinoma, fibrocarcinoma, colloid carcinoma, colloid carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, maternal carcinoma, hematopoietic carcinoma, hepatocellular carcinoma, Hutt's cell, colloid carcinoma, adrenal carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, cleavage Rompchel's carcinoma, Kulczycki cell carcinoma, large cell carcinoma, lenticular carcinoma, lens carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma, soft tissue carcinoma, mucinous carcinoma, mucinous carcinoma, mucocellular carcinoma, mucoepidermoid carcinoma, mucinous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, acanthocyte carcinoma, medullary carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneider's carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, potato-shaped carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, linear carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, nodular cutaneous carcinoma, verrucous carcinoma, and villous carcinoma.

[0434] Additional cancers include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung cancer, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid tumor, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignancy, cervical cancer, endometrial cancer, adrenocortical carcinoma, and prostate cancer.

[0435] In some embodiments, "tumor" shall include, but is not limited to, prostate tumors, pancreatic tumors, squamous cell carcinomas, breast tumors, melanomas, basal cell carcinomas, hepatocellular carcinomas, cholangiocarcinomas, testicular cancer, neuroblastomas, gliomas, or malignant astrocytic tumors such as glioblastoma multiforme, colorectal tumors, endometrial cancer, lung cancer, ovarian tumors, cervical tumors, osteosarcomas, rhabdomyosarcomas / leiomyosarcomas, synovial sarcomas, angiosarcomas, Ewing's sarcoma / PNET, and malignant lymphomas. These tumors include primary tumors as well as metastatic tumors (both vascularized and non-vascularized).

[0436] Therapeutic applications of the invention

[0437] Drug combinations or combination drugs

[0438] According to the present invention, a "drug combination" or "combination drug" is a combination of adiponectin and / or an adiponectin receptor agonist (ARA) and a photosensitizer and / or lymphocytes obtained by ECP treatment according to the present invention, in close proximity to each other. In one embodiment, the combination is suitable for combined administration.

[0439] In one embodiment, the pharmaceutical combination or combination medicament as described herein is characterized in that adiponectin and / or adiponectin receptor agonist (ARA) is in a pharmaceutical composition mixed with a pharmaceutically acceptable carrier, and the photosensitizer is in a separate pharmaceutical composition mixed with a pharmaceutically acceptable carrier, and the blood sample or lymphocytes are in a separate pharmaceutical composition mixed with a pharmaceutically acceptable carrier. Thus, in some embodiments, the combination medicament or pharmaceutical combination of the present invention may involve the presence of two or three or even more separate compositions or dosage forms in close proximity to each other. The combined agents need not be present in a single composition.

[0440] Combined administration

[0441] According to the present invention, the term "combination administration", also referred to as co-administration or combination therapy, encompasses in some embodiments the administration of separate formulations of the compounds described herein, wherein the treatments can occur within a few minutes of each other, in the same hour, on the same day, in the same week, or in the same month. The alternating administration of two agents is considered to be an embodiment of combination administration. The term combination administration encompasses staggered administration, wherein one agent can be administered first, followed by a subsequent administration of a second agent, optionally followed by administration of the first agent, and so on. The simultaneous administration of multiple agents is considered to be an embodiment of combination administration. In some embodiments, simultaneous administration encompasses, for example, the simultaneous administration of multiple compositions comprising multiple agents, for example, by taking separate tablets orally at the same time. Combination drugs, such as single formulations comprising multiple agents disclosed herein and optional additional anticancer and / or immunosuppressive (e.g., steroids) or immunomodulatory drugs, can also be used to administer the various components together in a single administration or dose.

[0442] In the context of the present invention, in preferred embodiments, the combined administration or combined use may comprise administering lymphocytes to a subject and / or administering adiponectin and / or an adiponectin receptor agonist (ARA) to a subject and administering a photosensitizer ex vivo / in vitro to a sample / cell of the subject.

[0443] The combination therapy or combined administration of a kind of medicament can be carried out before or after treatment with another kind of medicament to be combined, and interval range is several minutes to several weeks.In the embodiment that the second medicament and the first medicament are used separately, people usually will ensure that between the time of each delivery, not more than significant time period, so that the first medicament and the second medicament can still play a favorable combination synergistic effect to treatment site.In such cases, it is considered that in about 12 hours -96 hours, preferably in about 6 hours -48 hours, contact subject with two kinds of modalities in embodiments.However, in some cases, it may be necessary to significantly extend the time period for treatment, wherein between corresponding administration, intervals of several days (2 days, 3 days, 4 days, 5 days, 6 days or 7 days) to several weeks (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks).

[0444] In embodiments, the therapeutic agent or combination drug is administered from day 1, day 2, day 3, day 4, day 5, day 6, or day 7, or for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, or 50 weeks, or from day 1 to at least 3 weeks after the onset of irAE symptoms. In embodiments, the therapeutic agent or combination drug is administered between 1 day and 5 days prior to administration of ICB therapy, or for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, or 50 weeks prior to administration of ICB therapy. In preferred embodiments, the therapeutic agent or combination drug is administered between 1 day and 14 days prior to administration of ICB therapy, more preferably for 1 day to 5 days.

[0445] In the meaning of the present invention, combined administration encompasses any form of administration of the various agents described herein such that a beneficial additional therapeutic effect, preferably a synergistic effect, is achieved by the combined administration of two drugs, preferably two agents, and an ECP.

[0446] It is understood that substituents and substitution patterns of the compounds described herein can be selected by one of ordinary skill in the art to provide chemically stable compounds that can be readily synthesized by techniques known in the art and further by the methods described herein.

[0447] Another aspect of the present invention includes preparing "pharmaceutical compositions" for administration to subjects, which contain one or more compounds of the compounds disclosed herein in a "therapeutically effective amount". In certain embodiments, the pharmaceutical composition can be used to treat immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in subjects. The therapeutically effective amount of the disclosed compound (e.g., a medicament, a substance, a blood sample, or a cell) will depend on the route of administration, the species of the subject, and the physical characteristics of the subject being treated. Specific factors that may be considered include disease severity and staging, body weight, diet, and simultaneous administration. Those skilled in the art understand the relationship between these factors and determining the therapeutically effective amount of the disclosed compound.

[0448] In addition to the selected molecule, the pharmaceutical composition for use in a subject may also include at least one other pharmaceutically acceptable additive, such as a carrier, a thickening agent, a diluent, a buffer, a preservative, a surfactant, etc. The pharmaceutical composition may also include one or more additional active ingredients, such as an antimicrobial agent, an anti-inflammatory agent, an anesthetic, etc. The pharmaceutically acceptable carriers that can be used for these preparations are conventional. "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, PA), 19th edition (1995), compiled by E.W. Martin, describes compositions and preparations suitable for drug delivery of the compounds disclosed herein.

[0449] In general, the property of the carrier will depend on the specific mode of administration adopted. For example, parenteral formulations usually contain injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, normal saline, balanced salt solution, aqueous glucose solution, glycerol, etc. as solvents. For solid compositions (for example, powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may include trace amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, for example, sodium acetate or sorbitan monolaurate.

[0450] The pharmaceutical composition can be used by intramuscular, intraocular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intrathecal, intraventricular or parenteral route. Optionally, the composition can be administered by a variety of mucosal modes of administration, including oral, rectal, intraocular, intranasal, intrapulmonary or transdermal delivery, or applied to a subject by topical delivery to other surfaces. Compositions according to the present invention can alternatively comprise pharmaceutically acceptable carrier materials necessary to approach physiological conditions, such as pH adjustment and buffer, tension regulator, wetting agent, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used, and these carriers include for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, talcum, cellulose, glucose, sucrose, magnesium carbonate etc.

[0451] In accordance with the methods of treatment of the present invention, the compounds can be delivered to the subject in a manner consistent with conventional methods associated with the management of the condition for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the compound and / or other biologically active agent is administered to a subject in need of such treatment for a period of time and under conditions sufficient to prevent, inhibit and / or ameliorate a selected disease or condition or one or more symptoms thereof.

[0452] "Administering" a compound and "administering a" compound should be understood to mean providing a compound, a prodrug of a compound, a combination of at least two compounds, or a pharmaceutical composition as described herein. The compound or composition can be administered to a subject by another person (e.g., intravenously) or can be self-administered by the subject (e.g., a tablet).

[0453] The dosage can be varied by the attending physician to maintain the desired concentration at the target site (e.g., lung or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, such as intravenous or subcutaneous delivery, transdermal, rectal, oral, pulmonary or intranasal delivery. The dosage can also be adjusted based on the release rate of the formulation being administered, such as intrapulmonary sprays and powders, sustained-release oral formulations and injectable granular formulations, or transdermal delivery formulations.

[0454] The present invention also relates to a method of treating a subject suffering from a medical condition disclosed herein. The method of treatment preferably comprises administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein.

[0455] "Therapeutically effective amount" refers to the amount of the compound sufficient to achieve the desired effect in a subject treated with a particular compound. For example, this can be the amount of the compound disclosed herein that can be used to treat the condition of immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject. The therapeutically effective amount or diagnostically effective amount of the compound will depend on the subject being treated, the severity of the pain, and the mode of administration of the therapeutic composition. The dosage regimen can be adjusted to provide optimal prevention or treatment response. A therapeutically effective amount is also the amount in which any toxic or harmful side effects of the compound and / or other bioactive agents are clinically offset by the beneficial effects of the treatment.

[0456] A non-limiting range of a therapeutically effective amount of the compound adiponectin or ARA and / or other biologically active agent within the methods and formulations of the present invention is about 0.001 mg / kg body weight to 50 mg / kg body weight, 0.01 mg / kg body weight to about 20 mg / kg body weight, such as about 0.05 mg / kg body weight to about 5 mg / kg body weight, or about 0.2 mg / kg body weight to about 2 mg / kg body weight, or about 1*10^4 cells / kg body weight.

[0457] A non-limiting range of a therapeutically effective amount of tolerogenic T cells is about 1*10^3 cells / kg body weight, about 5*10^3 cells / kg body weight, about 1*10^4 cells / kg body weight, about 5*10^4 cells / kg body weight, about 1*10^5 cells / kg body weight, about 5*10^5 cells / kg body weight, about 1*10^6 cells / kg body weight, about 5*10^6 cells / kg body weight, about 1*10^7 cells / kg body weight, about 5*10^7 cells / kg body weight, or about 1*10^8 cells / kg body weight or more. A non-limiting range of a therapeutically effective amount of a photosensitizer, preferably 8-methoxypsoralen, is between 1 mg / mL and 10 mg / mL of sample, preferably about 2 mg / mL of sample.

[0458] As used herein, the term "about" or "approximately" is used to describe and explain small variations. For example, the term may refer to less than or equal to 10, such as less than or equal to as low as 1, when appropriate, and the term may also refer to greater than or equal to 10, such as greater than or equal to as high as 100 or more, when appropriate. It should be understood that the range format is used for simplicity and brevity and should be interpreted flexibly to include the values ​​explicitly stated as the limits of the range, encompassing each value and sub-range.

[0459] The present invention also includes kits, packages, and multi-container units containing the pharmaceutical compositions, active ingredients described herein, and / or means for administering the kits, packages, and multi-container units for preventing and treating diseases and other conditions in mammalian subjects.

[0460] Against the above background, the following consecutively numbered embodiments provide further specific aspects of the present invention:

[0461] 1. Adiponectin and / or adiponectin receptor agonists (ARAs) for the treatment or prevention of immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in subjects.

[0462] 2. Adiponectin and / or ARA for use according to embodiment 1, wherein the subject is receiving checkpoint blockade therapy.

[0463] 3. Adiponectin and / or ARA for use according to any one of the preceding embodiments, wherein the adiponectin and / or ARA is administered at least 1 hour before, during and / or at least one hour after administration of the checkpoint blockade therapy.

[0464] 4. Adiponectin and / or ARA for use according to any one of the preceding embodiments, wherein the checkpoint blockade therapy comprises the administration of at least one antibody, preferably at least two antibodies.

[0465] 5. Adiponectin and / or ARA for use according to any of the preceding embodiments, wherein the at least one antibody is selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3).

[0466] 6. Adiponectin and / or ARA for use according to any of the preceding embodiments, wherein the irAE may involve any organ, preferably the gastrointestinal tract, lungs, endocrine glands, skin and liver.

[0467] 7. Adiponectin and / or ARA for use according to any one of the preceding embodiments, wherein the irAE comprises ICB-induced colitis.

[0468] 8. Adiponectin and / or ARA for use according to any one of the preceding embodiments, wherein said administration of adiponectin and / or ARA reduces ICB-induced colitis.

[0469] 9. Adiponectin and / or ARA for use according to any of the preceding embodiments, wherein the administration of adiponectin and / or ARA does not interfere with the anti-tumor response induced by the checkpoint blockade therapy, preferably anti-PD1 treatment, due to tissue-specific adiponectin induction.

[0470] 10. A combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, the combination drug comprising at least two of the following components:

[0471] a. Photosensitizers and

[0472] b. adiponectin and / or adiponectin receptor agonist (ARA),

[0473] and wherein the blood sample of the subject is subjected to ultraviolet A (UVA) irradiation in vitro, preferably by external photopheresis (ECP) therapy

[0474] 11. The combination for use according to embodiment 10, wherein the photosensitizer is a psoralen agent, preferably 8-methoxypsoralen.

[0475] 12. The combination for use according to embodiments 10 to 11, wherein the wavelength of the UVA irradiation is a wavelength of 3200 angstroms to 4000 angstroms.

[0476] 13. The combination for use according to embodiments 11 to 12, wherein the subject suffers from cancer.

[0477] 14. The combination for use according to embodiments 10 to 13, wherein the checkpoint blockade therapy comprises the administration of at least one antibody, preferably at least two antibodies.

[0478] 15. The combination for use according to embodiments 10 to 14, wherein the at least one antibody is selected from the group consisting of cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3).

[0479] 16. The combination drug for use according to embodiments 10 to 15, wherein the irAE may involve any organ, preferably the gastrointestinal tract, lungs, endocrine glands, skin and liver.

[0480] 17. The combination drug for use according to embodiments 10 to 16, wherein the irAE comprises ICB-induced colitis.

[0481] 18. The combination for use according to embodiments 10 to 17, wherein the combination induces the expression of adiponectin, arginase-1 (Arg1) and tolerogenic KLRG1 in the inflamed intestine of the subject. + BTLA + CD47 + TOX 低Increase of T cells but not induction of adiponectin, arginase-1 (Arg1), and tolerogenic KLRG1 in cancer tissues + BTLA + CD47 + TOX 低 Increase in T cells.

[0482] 19. Adiponectin and / or adiponectin receptor agonist (ARA) for use in treating a tumor patient (or: a subject with cancer), wherein the patient has experienced immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB), wherein the treatment comprises the combined administration of adiponectin and / or ARA and anti-tumor immunotherapy, wherein the immunotherapy comprises the administration of immunoregulatory T cells (or more generally: a blood sample).

[0483] 20. Adiponectin and / or ARA for treating tumor patients according to the above embodiment, wherein the immunoregulatory T cells (or: blood sample)

[0484] Obtained from a method comprising the steps of:

[0485] - providing a sample derived from an isolated blood sample of said patient,

[0486] - adding a photosensitizer to the sample, and subjecting the sample to irradiation.

[0487] 21. Adiponectin and / or ARA for use in treating a tumor patient according to embodiments 19 and 20, wherein the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation.

[0488] 22. A method for treating or preventing immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB), the method comprising administering adiponectin and / or an adiponectin receptor agonist (ARA) to a subject, wherein the subject is receiving checkpoint blockade therapy.

[0489] 23. The method of embodiment 22, wherein adiponectin and / or ARA is administered at least one hour before, simultaneously with, and / or at least one hour after administration of the checkpoint blockade therapy.

[0490] 24. The method of embodiment 22 or 23, wherein the checkpoint blockade therapy comprises administering at least one antibody.

[0491] 25. The method of embodiment 24, wherein the at least one antibody is selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3).

[0492] 26. The method of any one of embodiments 22 or 23, wherein the checkpoint blockade therapy comprises administering two or more antibodies.

[0493] 27. A method according to embodiment 26, wherein at least one of the two or more antibodies is selected from the group consisting of: cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3).

[0494] 28. A method according to embodiment 26 or 27, wherein the two or more antibodies are selected from the group consisting of: cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3).

[0495] 29. The method of any one of embodiments 22 to 28, wherein the irAE involves any organ.

[0496] 30. The method of embodiment 29, wherein the organ is selected from the group consisting of the gastrointestinal tract, lungs, endocrine glands, skin, and liver.

[0497] 31. The method of any one of embodiments 22 to 30, wherein the irAE comprises ICB-induced colitis.

[0498] 32. The method of any one of embodiments 22 to 31, wherein said administration of adiponectin and / or ARA reduces ICB-induced colitis.

[0499] 33. The method of any one of embodiments 22 to 32, wherein the administration of adiponectin and / or ARA does not interfere with the anti-tumor response induced by the checkpoint blockade therapy due to tissue-specific adiponectin induction.

[0500] 34. The method of any one of embodiments 22 to 33, wherein the checkpoint blockade therapy is anti-PD1 treatment.

[0501] 35. A method of treating an immune-related adverse event (irAE) induced by immune checkpoint blockade (ICB) in a subject, the method comprising administering to the subject components a and b:

[0502] (a) Photosensitizer and

[0503] (b) adiponectin and / or adiponectin receptor agonist (ARA),

[0504] And wherein the subject's blood sample is subjected to ultraviolet A (UVA) irradiation in vitro.

[0505] 36. The method of embodiment 35, wherein the ultraviolet A (UVA) irradiation is extracorporeal photopheresis (ECP) therapy.

[0506] 37. The method of embodiment 35 or 36, wherein the photosensitizer is a psoralen agent.

[0507] 38. The method of embodiment 37, wherein the psoralen agent is 8-methoxypsoralen.

[0508] 39. The method of any one of embodiments 35 to 38, wherein the UVA irradiation has a wavelength of 3200 angstroms to 4000 angstroms.

[0509] 40. The method of any one of embodiments 35 to 39, wherein the subject has cancer.

[0510] 41. The method of embodiment 40, wherein the cancer is malignant melanoma or breast cancer.

[0511] 42. The method of any one of embodiments 35 to 41, wherein the subject is receiving checkpoint blockade therapy.

[0512] 43. The method of embodiment 42, wherein the checkpoint blockade therapy comprises administering at least one antibody.

[0513] 44. A method according to embodiment 43, wherein the at least one antibody is selected from the group consisting of: cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3).

[0514] 45. The method of embodiment 42, wherein the checkpoint blockade therapy comprises administering two or more antibodies.

[0515] 46. ​​A method according to embodiment 45, wherein at least one of the two or more antibodies is selected from the group consisting of: cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3).

[0516] 47. A method according to embodiment 45 or 46, wherein the two or more antibodies are selected from the group consisting of: cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3).

[0517] 48. The method of any one of embodiments 35 to 47, wherein the irAE involves any organ.

[0518] 49. The method of embodiment 48, wherein the organ is selected from the group consisting of the gastrointestinal tract, lungs, endocrine glands, skin, and liver.

[0519] 50. The method of any one of embodiments 35 to 49, wherein the irAE comprises ICB-induced colitis.

[0520] 51. The method of any one of embodiments 35 to 50, wherein said administration of adiponectin and / or ARA reduces ICB-induced colitis.

[0521] 52. The method of any one of embodiments 35 to 51, wherein the administration of adiponectin and / or ARA does not interfere with the anti-tumor response induced by the checkpoint blockade therapy due to tissue-specific adiponectin induction.

[0522] 53. The method of embodiment 51, wherein the checkpoint blockade therapy is anti-PD1 treatment.

[0523] 54. The method of any one of embodiments 35 to 53, wherein the treatment induces adiponectin, arginase-1 (Arg1), and tolerogenic KLRG1 in the inflamed intestine of the subject. + BTLA + CD47 + TOX 低 Increase of T cells but not induction of adiponectin, arginase-1 (Arg1), and tolerogenic KLRG1 in cancer tissues + BTLA + CD47 + TOX 低 Increase in T cells.

[0524] 55. A method for treating a cancer patient comprising administering adiponectin and / or an adiponectin receptor agonist (ARA) in combination with anti-tumor immunotherapy, wherein the immunotherapy comprises administering immunoregulatory T cells and the patient has immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB).

[0525] 56. The method according to embodiment 55, wherein the immunoregulatory T cells are prepared by the following steps:

[0526] (a) obtaining a sample derived from an isolated blood sample of said patient,

[0527] (b) adding a photosensitizer to the sample, and

[0528] (c) subjecting the sample to irradiation.

[0529] 57. The method of embodiment 56, wherein the photosensitizer is 8-methoxypsoralen.

[0530] 58. The method of embodiment 56 or 57, wherein the irradiation is ultraviolet A (UVA) irradiation.

[0531] 59. The method of any one of embodiments 55 to 58, wherein the patient has cancer and the cancer is malignant melanoma or breast cancer.

[0532] Example

[0533] The present invention is further described by the following examples. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention provided to better illustrate the disclosure described herein.

[0534] Example 1

[0535] method:

[0536] Patient irAE remission evaluation

[0537] The severity of irAEs was assessed by clinical evaluation of symptoms and was performed before the start of ECP and at each patient visit after the start of therapy. Definitions of irAE severity and resolution were established based on the ASCO guidelines.

[0538] irAEs were graded according to the Common Terminology Criteria for Adverse Events (CTC-AE v5). Complete response (CR) was defined as complete resolution of all irAE-related symptoms. Partial response (PR) was defined as a decrease of at least 1 grade in at least one irAE organ manifestation based on CTC-AE. Stable disease (SD) was defined as no change in the CTC-AE grade of all irAEs. Progressive disease (PD) was defined as an increase of at least 1 grade in at least one irAE organ manifestation based on CTC-AE. CR, PR, and PD were diagnosed only when the severity of symptoms remained stable for at least 5 consecutive days.

[0539] The full analysis dataset (FAS) and safety dataset (SAF) included all patients who received at least one ECP administration. Descriptive statistics were used to report the frequency and percentage of continuous variables and discrete variables. Based on the binomial distribution, the exact two-sided 95% confidence interval of ORR was calculated. Overall survival (OS) and progression-free survival (PFS) were defined as the time from the start of treatment with ECP until death (OS) / death or disease progression (PFS), and the events of interest during the treatment observation time did not occur as censored. The analysis of OS and PFS was performed using the Kaplan Meier method.

[0540] The cell line B16.F10 mouse melanoma cell line (C57BL / 6 background) was provided by Hanspeter Pircher (University of Freiburg, Germany). luc / GFP Cells were established as described in Mastroianni, J. et al., “miR-146a Controls Immune Response in the Melanoma Microenvironment”, Cancer Res, Vol. 79: 183-195 (2019). The BRAF mutant 4434 murine melanoma cell line is an established C57BL / 6LSL-Braf V600E ;Tyr::CreERT + / o mice (Dhomen, N. et al., “Oncogenic Braf induces melanocyte senescence and melanoma in mice”, Cancer Cell, Vol. 15: pp. 294-303 (2009)) and kindly provided by Richard Marais (Manchester Cancer Research Institute, Manchester, UK). MC38 and MC38 OVACell lines were kindly provided by Karen Dixon and Vijay Kuchroo (Harvard Medical School, USA). All cell lines were cultured in DMEM high glucose medium (Gibco) supplemented with 10% FCS (Sigma) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO2 in a humidified atmosphere. The culture medium of the MC38 line was additionally supplemented with 1% sodium pyruvate (Gibco). After thawing, B16.F10luc / GFP cells were subcultured twice with 2 mg / mL of the neomycin analogue Geneticin (G-418 sulfate; Genaxon). The immortalized small intestinal epithelial cell line MODE-K26 was cultured in RPMI 1640 (Gibco) supplemented with 10% FCS (Sigma), 1% penicillin / streptomycin (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 0.1 mM MEM non-essential amino acids (Gibco), and 50 μM 2-mercaptoethanol (Gibco) at 37° C. and 5% CO in a humidified atmosphere. The cells were repeatedly tested for mycoplasma contamination and found to be negative.

[0541] ECP treatment, mouse model

[0542] To model in vivo ECP transplantation, spleen cells from donor mice were used. Donor animals were treated in the same manner as the corresponding recipient groups. Spleens were harvested, sieved, and lysed. Splenocytes were resuspended in RPMI supplemented with 10% FCS and 1% P / S to a density of 3 × 106 cells / mL to 5 × 106 cells / mL and incubated with 200 ng / mL 8-methoxypsoralen (8-MOP; After incubation, cells were placed in a dose-controlled mode (Opsytec Dr ) were exposed to UVA light (2 J / cm2) in a calibrated BS-02 irradiation chamber operated by . The ECP-treated splenocytes were resuspended in PBS, and 107 cells were injected intravenously into recipient mice. If necessary, PBMCs were treated with ECP in a manner comparable to splenocytes, and 107 PBMCs were injected intravenously into recipient mice. PBMCs from mouse EDTA blood were separated using Ficoll-Paque PREMIUM 1.084 (Cytiva). For ECP treatment of epithelial cells, 6×106 MODE-K cells were seeded onto 10 cm culture dishes the day before ECP treatment. The next day, the cells were treated with 8-methoxypsoralen and UVA light in a calibrated BS-02 irradiation chamber. The cells were washed with PBS, and 2×106 ECP-treated MODE-K cells were injected intravenously into recipient mice.

[0543] DSS-induced colitis model

[0544] To induce acute colitis, mice received drinking water containing 3% dextran sulfate sodium (DSS colitis grade, 36kDa to 50kDa; MP Biomedicals) from day 0 to day 3, and then received normal drinking water. On days 0, 3, and 6 of the experiment, mice were injected intraperitoneally with 0.2 mg of immune checkpoint inhibitors (ICIs) or corresponding isotype controls. On days 3 and 6, 107 ECP-treated donor splenocytes were transplanted intravenously into the recipient. In some experiments, steroids, adiponectin, or AdipoRon were applied daily from day 3 to day 7. Arginase-1 inhibitors were applied twice a day from day 4 to day 7. STAT6 or PPARγ inhibitors were applied from day 4 to day 7. Clodrosomes were injected into mice on days 1, 3, 5, and 7. If not indicated differently, mice were sacrificed on day 8 by retro-orbital bleeding to harvest serum and organs for histopathological scoring and gene expression analysis.

[0545] ECP treatment using enriched immune cells

[0546] Colitis was induced using 3% DSS and ICI treatment as described above. If indicated, ECP-treated splenocytes were FACS-sorted for T cells (CD45+CD3+; 3×106) or myeloid cells (CD45+CD3-CD19-CD11b+; 1×106) and then injected intravenously into recipient mice. The antibodies used for sorting are summarized in Table S8. Table S8 is shown below.

[0547] Table S8: Antibodies used for FACS sorting

[0548] antigen clone Isotype dilution supplier Fluorophore CD45 30-F11 Rat IgG2b, kappa 1:200 Biolegend Pacific Blue CD3 14A2 Rat IgG2b, kappa 1:200 Biolegend Alexa Fluor 488 CD11b M1 / 70 Rat IgG2b, kappa 1:100 BioLegend PE

[0549] Generation of hematopoietic chimeras

[0550] To generate mice lacking arginase-1 in the hematopoietic compartment, 5 × 106 Arg1- / - or C57BL / 6WT bone marrow (BM) cells were transplanted intravenously into WTC57BL / 6 recipients in the tail vein after 12 Gy (2 × 6 Gy) total body irradiation (TBI) on day -30. The mice were used as ECP recipients or donors in DSS-induced colitis experiments.

[0551] B16.F10 intravenous / metastatic tumor model

[0552] C57BL / 6 mice were injected intravenously with 10,000 B16.F10luc / GFP melanoma cells in the tail vein and treated with 0.2 mg of ICI or corresponding isotype control on days 1, 4, 8, 16, and 22. ECP-treated splenocytes (107; intravenous) were transplanted on days 13, 18, and 22, and steroids were administered daily (intraperitoneally) from day 13 to day 22. Mice were monitored for survival until day 60.

[0553] 4434 Intravenous Tumor Model

[0554] 2 x 106 4434 melanoma cells were transplanted intravenously into C57BL / 6 recipients in the tail vein, and the treatment schedule was comparable to that described for the B16.F10 (iv) model.

[0555] MC38-OVAdim subcutaneous tumor model

[0556] 1 × 10 MC38-OVAdim or 0.5 × 10 MC38 cells were subcutaneously transplanted into the shaved right flank of C57BL / 6 recipients. Recipients were treated intraperitoneally with 0.2 mg ICI on days 1, 4, 8, 11, and 15. Splenocytes (10 7 ; intravenously) treated with ECP were transplanted on days 6, 11, and 15, and steroids were given daily (intraperitoneally) from day 6 to day 15. On day 16, serum samples were harvested by retro-orbital bleeding, mice were euthanized, and tumors were excised for further study.

[0557] B16.F10-OVA subcutaneous model with adoptive T cell transfer

[0558] 1×10 B16.F10-OVA cells were transplanted subcutaneously into the shaved right flank of CD45.1 recipients and treated intraperitoneally with 0.2 mg ICI on days 1, 4, 8, 11, and 15. ECP-treated splenocytes (10; intravenous) were transplanted on days 6, 11, and 15, and steroids were given daily (intraperitoneally) from day 6 to day 15. OT-I splenocytes (CD45.2) were activated at a density of 2×10 cells / mL in RPMI supplemented with 10% FCS, 1% P / S, 55 μM β-mercaptoethanol (Gibco), and 4 mM glutamine (Gibco) on day 2 in the presence of 100 U / mL rmIL-2 (Peprotech) and 0.1 μg / mL OVA257-264 peptide (Sigma). On day 4, splenocytes were washed and replated with 100 U / mL rmIL-2 at 2 × 10 cells / mL. On day 5, OT-I splenocytes were washed and 5 × 10 cells were injected intravenously into the tail vein of tumor-bearing mice. On day 16, serum samples were harvested by retro-orbital bleeding, mice were euthanized, and tumors were excised for further study.

[0559] Combined colitis and B16.F10 tumor model

[0560] C57BL / 6 mice were injected intravenously with 10,000 B16.F10luc / GFP melanoma cells in the tail vein and received drinking water containing 3% dextran sulfate sodium (DSS colitis grade, 36kDa-50kDa; MP Biomedicals) from day 9 to day 12, followed by normal drinking water. Mice were treated with 0.2 mg of ICI or corresponding isotype control on days 1, 4, 8, 16, and 22. ECP-treated splenocytes (107; intravenous) were transplanted on days 13, 18, and 22, and steroids were administered daily (intraperitoneally) from day 13 to day 22. Mice were imaged using BLI on day 23, and organs and serum were harvested on day 24.

[0561] Recall Immunity Experiment To evaluate recall immunity, T cell donor mice were challenged with 104 B16.F10 melanoma cells (intravenously) and treated with anti-PD1 (day 1, day 3, day 6, day 9, day 12), ECP (day 3, day 7, day 12) or steroids (day 3 to day 12). Secondary recipients were sublethally irradiated with 6Gy TBI and injected intravenously with 104 B16.F10 or 2×106 4434 melanoma cells. Splenic T cells were enriched from T cell donors using the Pan T Cell Isolation Kit II (Mouse, Miltenyi) on day 13, and 1×105 T cells were transplanted intravenously into secondary recipients on day 3 after tumor injection. Untreated melanoma mice were used as controls.

[0562] In vivo migration assay To analyze tissue-specific migration and phagocytosis of ECP-treated cells, mice were fed an alfalfa-free diet (D10012Gi AIN-93, Research Diets) throughout the experiment to reduce background fluorescence. 1×10 6 B16.F10 melanoma cells were subcutaneously transplanted into the shaved right flank of recipients. Mice were treated with 0.2 mg of anti-PD-1 on days 1, 5, 8, and 11 and received drinking water containing 3% DSS (colitis grade, 36 kDa-50 kDa; MP Biomedicals) from day 5 to day 8. Donor splenocytes were isolated on day 12, ECP-treated, and stained with VivoTrack 680 (PerkinElmer) according to the manufacturer's instructions. MODE-K intestinal epithelial cells were treated with ECP and VivoTrack 680 staining, if indicated. The cells were resuspended in PBS and 107 ECP-treated VivoTrack 680-stained spleen cells were transplanted intravenously. For epithelial cell transfer experiments, 2×106 MODE-K cells or spleen cells were transplanted intravenously. Recipients were euthanized at defined time points after transplantation, and tumor and colitis tissues were harvested for ex vivo fluorescence imaging (FLI). FLI was performed using an IVIS Lumina III (PerkinElmer). Thereafter, colitis and tumor tissues were processed into single cell suspensions as described above. Adoptive transfer of adiponectin-treated T cells Splenic T cells were isolated from C57BL / 6 donors using the Pan T Cell Isolation Kit II (Miltenyi) and activated using anti-CD3 / CD28 beads (ThermoScientific) in the presence of 30 U / mL rmIL2 (Peprotech). T cells were treated with 9 μg / mL recombinant murine adiponectin (GenScript) or vehicle for 72 hours. C57BL / 6 recipients received 1% DSS (MP Biomedicals) in their drinking water from day 0 to day 3, followed by normal drinking water to induce mild colitis. On days 0, 3, and 6 of the experiment, mice were injected intraperitoneally with 0.2 mg of anti-PD-1. On day 4, the activation beads were removed, the T cells were washed, and 5 × 106 T cells were injected intravenously into the colitis animals. Colitis tissue was harvested on day 8. T cells were stained to determine activation by CD25 surface expression.

[0563] Adoptive transfer of T cells from colitis donors C57BL / 6 donor mice received drinking water containing 3% DSS and anti-PD-1 treatment according to the schedule described above. Splenic T cells from the donor cohort were isolated on day 8 using the Pan T Cell Isolation Kit II (Miltenyi). The C57BL / 6 recipient cohort received drinking water containing 1% DSS (MP Biomedicals) from day 0 to day 3, followed by normal drinking water to induce mild colitis. On days 0, 3, and 6 of the experiment, mice were injected intraperitoneally with 0.2 mg of anti-PD-1. On day 4, mice were injected intravenously with splenic T cells from the donor animals. Colitis tissue from the recipient cohort was harvested on day 8.

[0564] Xenograft-induced irAE model

[0565] Xenograft-induced irAEs were induced as described in Perez-Ruiz, E. et al., “Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4and PD-1 immunotherapy”, Nature, Vol. 569, pp. 428-432 (2019). Briefly, Rag2- / -γc- / - were injected intraperitoneally with 107 human PBMCs in 200 μL PBS. Healthy donor PBMCs were isolated by density gradient separation (lymphocyte separation medium, Anprotec). On days 4, 7, 10, and 13, mice were treated intraperitoneally with 200 μg of pembrolizumab, and 107 ECP-treated human PBMCs were transplanted intravenously into the mice, depending on the treatment cohort. Steroids were administered intraperitoneally every day from day 4 to day 13. Mice were sacrificed on day 15 to harvest serum and organs.

[0566] Long-term steroid and ECP treatment

[0567] WT C57BL / 6 (CD45.2) mice were injected daily with steroids (1 mg / kg body weight) or vehicle for 50 days, or received eight weekly ECP transplants. Splenocytes for ECP transplantation were isolated from naive CD45.1 donors. Recipient mice were sacrificed on day 50, and spleens were processed for FACS analysis.

[0568] In vivo apoptosis analysis

[0569] To better understand whether ECP-treated splenocytes also undergo apoptosis in vivo, C57BL / 61uc mice were treated with B16.F10 melanoma (intravenously) and 0.2 mg of anti-PD1 (d1, d4, d8). Splenocytes were isolated and treated with or without ECP. 7 Splenocytes were injected intravenously into Rag2 - / - γc - / - The expansion of the cells was monitored by BLI. The recipients were sacrificed on day 30 and the colon, lung, liver and spleen were subjected to ex vivo BLI.

[0570] In vivo processing

[0571] Treatment time points are shown in the accompanying drawings and methods. ICI treatment consists of 0.2 mg anti-PD1 (mouse, clone RMP1-14; Ichorbio) or 0.2 mg anti-CTLA4 (mouse, clone 9D9; Ichorbio) injected intraperitoneally each time. Controls were rat IgG2a isotype control (clone 1-1, Ichorbio) and mouse IgG2b isotype control (clone MPC-11, Ichorbio), respectively. 0.2 mg pembrolizumab (Keytruda, anti-human PD1; MSD) was injected intraperitoneally. Steroids (prednisolone, Solu-Decortin H; Merck) were dissolved in PBS applied intraperitoneally at 1 mg / kg body weight. Adiponectin / Acrp30 (GenScript) was reconstituted in H2O, diluted in PBS, and applied intraperitoneally at 4 μg per injection. AdipoRon (MedChemExpress) was prepared in DMSO / corn oil (5% / 95%) and orally administered at 50 mg / kg body weight. Arginase-1 inhibitor CB-1158 (Numidargistat) dihydrochloride (MedChemExpress) was orally administered at 100 mg / kg body weight in H2O twice a day. STAT6 inhibitor AS1517499 (MedChemExpress) and PPARγ inhibitor GW9662 (MedChemExpress) were dissolved in DMSO / PEG300 / Tween80 / PBS (10% / 40% / 5% / 45%) and applied intraperitoneally at 10 mg / kg and 1 mg / kg, respectively. Using a standard macrophage depletion kit (Encapsula NanoSciences), phagocytes were depleted by intravenous injection of 0.2 mL of liposomes (Clodrosome) containing clodronate or control liposomes (Encapsome). Macrophage depletion was confirmed by FACS and qPCR.

[0572] Histopathological analysis of irAE target organs

[0573] Colon, liver, skin, and lung were harvested, fixed with formalin, and embedded in paraffin. Sections were stained with H&E and scored by experienced pathologists on a scale of 0 (absent) to 2 (abundant) based on histopathological features of human irAEs (including lymphocyte and neutrophil infiltration, crypt abscesses, and apoptotic cells). Scoring was performed without knowledge of the experimental groups.

[0574] Histopathological scoring of irAEs in colonic patient tissues

[0575] Intestinal biopsy specimens (small intestine, large intestine) were prepared at the Department of Surgical Pathology in Freiburg according to standardized procedures for formalin-fixed and paraffin-embedded tissue. Briefly, 2 μm sections were generated and H&E stained. The scoring of each specimen depended on the following parameters: eosinophilia, apoptosis, ulceration, cryptitis or crypt abscess formation, lymphoplasmacytic infiltration and signs of regeneration, focal or diffuse disease manifestations (each parameter was evaluated as present (0) or absent (1)). An experienced pathologist calculated the total score (0-9) for each individual sample in a blinded manner.

[0576] In vivo bioluminescence imaging (BLI)

[0577] Luciferin (D-luciferin, potassium salt (S)-4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid; Biosynth) was dissolved in H2O and injected intraperitoneally at a concentration of 150 μg / g body weight. Ten minutes later, mice were imaged using an IVISLumina III in vivo imaging system (PerkinElmer) with a 2-minute exposure time. Luciferase signal was quantified as photons per second per mouse. BLI acquisition, analysis, and visualization were performed using Living Image software (PerkinElmer).

[0578] Intestinal leukocyte separation

[0579] Intestinal leukocytes were isolated using a lamina propria dissociation kit (Miltenyi). Briefly, colon samples were harvested and Paneth's patches were removed. The samples were rinsed with PBS to remove feces, opened longitudinally and cut into 5mm-10mm fragments. Epithelial cells were removed using a pre-digestion solution. Colon tissue was transferred to a gentleMACS C tube (Miltenyi) containing an enzyme mixture and digested on a gentleMACS Octo dissociator (Miltenyi) with a heater using the appropriate procedure. The cells were washed with PBS and applied to a 100μm filter before use in further downstream applications.

[0580] Tumor digestion

[0581] Under 37 ℃ and 1,400rpm, with the 1mg / mL collagenase IA (Sigma) and 50 μ g / mL DNA enzyme I (Sigma) digestion in the DMEM culture medium for 1 hour, from B16.F10 tumor tissue, obtain single cell suspension.By carrying out density gradient centrifugation with 70% (3mL), 40% (4mL) and 30% (3mL) Percoll (Cytiva) and come enrichment of hematopoietic cells, wherein the latter contains the tumor of digestion.After centrifugation (680g, acc 3, without braking, room temperature), above the bottom phase, collect the cell of enrichment.Under 37 ℃ and 1,000rpm, with the 20 μ g / mL Liberase TM (Roche) and 50 μ g / mL DNA enzyme I (Sigma) in the DMEM culture medium, MC38 tumor digestion 30 minutes.By carrying out 20 minutes (without braking) density gradient centrifugation with 800g with 30% Percoll (Cytiva), come enrichment of immune cells.

[0582] Mass cytometry (CyTOF)

[0583] For multidimensional protein analysis, C57BL / 6 (CD45.2) mice were treated with DSS and anti-PD1 to induce colitis as described above, however one group received ECP splenocytes from CD45.1 donors. Organs were harvested on day 8 for analysis. The spleens were sieved and lysed for red blood cells; colon tissue was digested as described above and stained using 3×106 cells. Dead cells were stained with 2.5 μM MM-DOTA 139La for 5 minutes at room temperature, followed by surface protein staining for 30 minutes at room temperature. Cells were fixed in PBS containing 1.6% PFA for 5 minutes at room temperature and permeabilized using a transcription factor staining buffer set (eBioscience). Intracellular proteins were labeled for 60 minutes at room temperature, followed by labeling with secondary antibodies for 30 minutes at room temperature. Cells were stained overnight with 125 nM Cell-ID Intercalator-Ir (Fluidigm) in PBS containing 4% PFA at 4°C. Data were collected on a Helios CyTOF (Fluidigm) and processed using FlowJo v10 (TreeStar) and OMIQ (Dotmatics) software. Files were manually cleaned by excluding doublets and dead cells. Cells were scaled from -5 to 12,000 using Arcsinh and pre-gated on CD45.2 receptor cells, followed by subsampling on 23,000 (colon) or 8,000 (spleen) cells. Dimensionality reduction was run to create opt-SNE plots. Different populations were generated by FlowSOM clustering with elbow and consensus meta-clustering. Clusters were identified based on marker expression. Violin plots show the median marker intensity in a given cluster. Heat maps were generated based on median values ​​with Euclidean spacing. All antibodies used for CyTOF are summarized in Table S9 (as shown below).

[0584] Table S9: CyTOF Antibodies

[0585]

[0586]

[0587]

[0588] RNA isolation, reverse transcription, and qPCR

[0589] Total RNA was isolated from tissues using QIAzol lysis reagent, and RNA was isolated from cells using the RNeasy Mini or Micro kit. RNA was isolated from formalin-fixed, paraffin-embedded patient samples (all QIAGEN) using the RNeasy FFPE kit. Up to 1 μg of total RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoScientific), and 15 ng of yeast RNA (ThermoScientific) was added to the RNA sample from FFPE tissue. Quantitative PCR (qPCR) was performed on a LightCycler 480 machine (Roche) using 10 ng–40 ng of cDNA and 5 μM of forward and reverse primers using the LightCycler 480 SYBR Green I Master Mix (Roche). Primers (Table S10) were obtained from Eurofins Genomics (Germany). 2 -ΔΔCT Methods Gene expression was calculated relative to a reference gene or relative to a control. Table S10 is shown below.

[0590] S10: Primer sequences

[0591]

[0592]

[0593] Microarray analysis

[0594] Microarray analysis was performed on colon tissue from mice with DSS-induced colitis treated with anti-PD1 alone or anti-PD1 plus ECP. Tissue was harvested, rinsed, washed in ice-cold PBS, and total RNA was homogenized using the miRNeasy Mini Kit (Qiagen). RNA quality was assessed using an Agilent 2200 TapeStation (Agilent Technologies), and the RIN for all samples was greater than 8.8. RNA was analyzed using GeneChip TM 250 ng of total RNA was processed into fragmented and labeled ds-cDNA using the WT PLUS kit (Applied Biosystems) and cloned using GeneChip TMHybridization, washing and staining kit (Applied Biosystems) was hybridized on Clariom S mouse microarray (Applied Biosystems) and scanned according to the manufacturer's instructions. Microarray analysis was performed as described in Hamarsheh, S. et al., "Immune modulatory effects of oncogenicKRAS in cancer", Nat Commun., Vol. 11: p. 5439 (2020). Briefly, the array was standardized by robust multi-chip averaging as implemented in the R / Bioconductor oligo package, and gene level annotations were retrieved from the pd.clariom.s.mouse R / Bioconductor package. The limma R / Bioconductor package, a linear model-based method, was used to identify regulatory genes between DSS / anti-PD1 and DSS / anti-PD1 / ECP. Adjusted (Benjamini-Hochberg) p values ​​below 0.05 were considered significant.

[0595] Single-cell RNA sequencing (scRNA Seq)

[0596] WT C57BL / 6 mice were treated with DSS and ICI according to the previously described schedule to induce colitis, whereas one group received ECP splenocytes (n=2 per group). On day 8, the colon was harvested and digested to isolate intestinal leukocytes. Dead cells were removed using a dead cell removal kit (Miltenyi), followed by immune cell enrichment using CD45 microbeads (Miltenyi). Cells were washed and counted in 0.04% BSA / PBS according to the 10X Genomics protocol, and 10,000 CD45+ cells were encapsulated into droplets with barcoded gel beads using the Chromium Controller single cell instrument (10X Genomics). Libraries were prepared using the Chromium Next GEM Single Cell 3' Kit version 3.1 (10X Genomics). The generated scRNA Seq libraries were pooled and sequenced on a NovaSeq 6000 (Illumina) with an average of 44,658 reads per cell. scRNA fastq files were processed with 10x Genomics Cell Ranger 6.1.2 using the command “cellranger count --no-bam-nosecondary”. The mouse reference transcriptome was downloaded from the pre-built Cell Ranger reference package (mm 10GENCODE vM23 / Ensembl 982020-A). Other parameters were set to default values. Downstream analysis was performed using the Seurat (version 4.3.0) R / Bioconductor package. Single cells were discarded if they expressed fewer than 200 genes or more than 4000 genes, or if their percentage of mitochondrial reads was higher than 10%. Genes quantified in fewer than 3 cells were filtered out. Read counts were normalized with a scaling factor of 10,000. The normalized data were scaled and centered, and the percentage of mitochondrial content was regressed out. The principal component analysis of the top 3000 variable features was then calculated. A nearest neighbor graph was constructed for the first 20 principal components, and Louvain clustering was performed with a resolution set to 0.8. Finally, the remaining cells were visualized in 2 dimensions using Uniform Manifold Approximation and Projection (UMAP). Using a customized list of immune system marker genes (Table S11), the cell types were annotated using the sctype R script. Cluster / cell type specific marker genes were found using the "FindConservedMarkers" function with the following parameters: test.use = "MAST", min.pct = 0.25, logfc.threshold = 0.25, min.cells.group = 10, only.pos = TRUE. Table S11 is shown below. Table S11: Modified scType immune system marker genes

[0597]

[0598]

[0599]

[0600]

[0601]

[0602] To quantify transcription factor and signaling pathway activities, the decoupler R package with Dorothea and Progeny annotations, respectively, was used. Regulatory activity was calculated for each single cell using wmean. Differences in activity between different cell types and / or conditions were assessed using the Wilcoxon test.

[0603] Measurement of adiponectin in mouse serum

[0604] To quantify adiponectin concentration in mouse serum samples, blood was harvested by retro-orbital bleeding and serum was separated by centrifugation. Adiponectin concentration was measured using an adiponectin mouse ELISA kit (ThermoFisher) at a 20,000-fold dilution.

[0605] Measurement of adiponectin in patient serum

[0606] Adiponectin concentrations in patient serum samples were measured at 1,000-fold dilutions using the Human Adiponectin ProQuantum Immunoassay Kit (ThermoFisher) in a 384-well format.

[0607] Isolation of immune cell populations

[0608] Immune cells were isolated from the bone marrow using a neutrophil separation kit or a monocyte separation kit for gene expression analysis, or were isolated from the spleen using a NK cell separation kit, a Pan B cell separation kit, or a Pan T cell separation kit II (all for mice; Miltenyi). The production and cultivation of bone marrow-derived macrophages (BMDM) BMDM were produced by bone marrow after erythrocyte lysis. Cells were cultured in RPMI supplemented with 10% FCS, 1% P / S, and 20ng / mL rmM-CSF (Peprotech). Culture medium was changed on the 5th and 6th days. Cells with a purity > 95% confirmed by surface CD11b and F4 / 80 expression were used on the 7th day. BMDM (M0) was polarized for 16 hours, polarized to M1 with 50ng / mL rmIFNγ (Peprotech) and 20ng / mL LPS (Sigma), or polarized to M2 with 20ng / mL rmIL-4 (Peprotech). If necessary, BMDM were treated with increasing concentrations of recombinant adiponectin / Acrp30 (GenScript) or AdipoRon (MedChemExpress) for 48 hours.

[0609] Generation and culture of bone marrow-derived dendritic cells (BMDCs)

[0610] BMDCs were generated and cultured from bone marrow as described in Stickel, N. et al., "MicroRNA-146a reduces MHC-II expression via targeting JAK / STAT-signaling in dendritic cells after stem cell transplantation," Leukemia, Vol. 31: 2732-2741 (2017). Cells with a purity >90% confirmed by CD11c surface expression were used on day 7. LPS (Sigma) stimulation was performed at 100 ng / mL for 16 hours.

[0611] In vitro phagocytosis assay

[0612] WT C57BL / 6 (CD45.2) bone marrow cells were differentiated into BMDM and BMDC for 7 days and cultured at 10 6 The next day, splenocytes from CD45.1 mice were treated with ECP as described above, stained with CellTraceViolet (CTV; ThermoScientific), and 2×10 6 CTV +Splenocytes were added to BMDM and BMDC cultures. The plates were centrifuged at 300 × g for 2 minutes and cultured for 48 hours before further FACS or PrimeFlow analysis. Phagocytosis was analyzed by FACS using homologous markers and CTV signals. If necessary, endocytosis was blocked by pre-incubating BMDC with 50 μM Endosidin9 (Sigma) or 50 μM PitStop2 (Sigma) for 1 hour before adding splenocytes. One hour after adding splenocytes, STAT6 inhibitor AS1517499 (MedChemExpress) or PPARγ inhibitor T0070907 (MedChemExpress) was added to the co-culture at 500 nM or 1 μM, respectively.

[0613] Flow cytometry (FACS)

[0614] The spleen was separated, sieved and lysed with red blood cells. Single cell suspensions of tissue were prepared as described above. Viability was analyzed by staining with ZombieNIR fixable dye (BioLegend) or LIVE / DEAD fixable Aqua (Invitrogen). Fc receptors were blocked for 10 minutes at 4°C with anti-mouse CD16 / CD32 (BioLegend), followed by surface staining with fluorophore-conjugated antibodies at 4°C for 30 minutes. Cells were fixed and permeabilized for 40-60 minutes at 4°C using a fixation / permeabilization kit (BD Biosciences) or transcription factor staining buffer set (eBioscience) for staining of cytoplasmic proteins and cytokines or intranuclear proteins, respectively. For intracellular cytokine staining, cells were stimulated in the presence of a cell stimulation mixture (eBioscience) with protein transport inhibitors for 4 hours before surface and intracellular cytokine staining. Antibodies against CD107α were added during stimulation. Calreticulin was stained with unconjugated antibody for 20 minutes, followed by staining with fluorophore-conjugated anti-rabbit secondary antibody for 20 minutes. 257-264Antigen-specific CD8 T cells were analyzed after dextramer staining (Immudex). In order to determine protein phosphorylation, cells were surface stained, incubated with Lyse / Fix (BD Biosciences) at 37°C for 10 minutes, and permeabilized with ice-cold Phosflow Perm buffer III (BD Biosciences) at 4°C for 30 minutes. Phosphorylated STAT6 (pY641) was stained at 4°C for 30 minutes. If total cell counts are required, 123count eBeads counting beads (Invitrogen) are added directly before collection. All data were collected on BD LSRFortessa (BD Biosciences) and analyzed with FlowJo 10 version (TreeStar). In order to analyze immune cells in patient blood, 50 μL EDTA blood was incubated with 5 μL of corresponding antibodies for 30 minutes, followed by the addition of 2.5 mL Lyse / Fix (BD Biosciences). The sample was incubated for 10 minutes and then resuspended in FACS Flow (BD Biosciences). For some patients, analysis was performed using a BD Multitest 6-Color TBNK and BD Trucount tubes (BD Biosciences) according to the manufacturer's instructions. Data were collected using a Canto II flow cytometer (BD Biosciences) and analyzed using FACSDiva version 6 software (BD Biosciences). Antibodies used for flow cytometry are summarized in Table S12 (as shown below).

[0615] Table S12: Flow cytometry antibodies

[0616]

[0617]

[0618]

[0619] PrimeFlow TM RNA assay

[0620] PrimeFlow was performed according to the manufacturer's instructions. TM RNA assay (Invitrogen) was used to detect gene expression at the single-cell level by FACS. Hybridization was performed using an Adipoq-specific AlexaFluor 647-labeled probe (assay ID: VB1-17726-PF, type 1; Invitrogen).

[0621] Confocal imaging of phagocytosis

[0622] BMDM were cultured from C57BL / 6 mice as described above and 10 5 The next day, splenocytes from C57BL / 6 mice were treated with ECP, stained with 5 μM cell proliferation dye eFluor670 (AxF647; eBioscience), and 10 6 Splenocytes were added to BMDM. The co-cultures were incubated for 24 hours, washed with PBS, stained for CD11b surface expression (Table S13), and fixed with 4% PFA (Merck). Slides were mounted with ProLong Diamond mounting medium (Invitrogen) containing DAPI. Immunofluorescence images were taken on an LSM710 confocal microscope (Zeiss). For quantification, slides were scanned on a ScanR (Olympus) microscope, and AxF488+AxF647+ cells were counted using ScanR analysis software (Olympus). Table S13 is shown below.

[0623] Table S13: Antibodies used for confocal imaging

[0624] antigen Fluorophore Isotype clone dilution CD11b Alexa Fluor 488 Rat IgG2b, kappa M1 / 70 1:100

[0625] Western blotting

[0626] Cells were lysed with RIPA lysis buffer (Santa Cruz Biotechnology) supplemented with phosphatase inhibitor cocktail 2 (Sigma). Protein concentration was determined using a Pierce BCA protein assay kit (ThermoScientific). Proteins were separated on SDS-PAGE gradient gels (Bis-Tris, 4%-12%; ThermoScientific) and then transferred to a nitrocellulose membrane (Merck). The membrane was western blotted with specific antibodies and imaged using an INTAS ECL Chemocam imager after incubation with a chemiluminescent substrate (Advansta). Western blots were quantified using Lab Image ID software. Antibodies and dilutions are summarized in Table S14 (as shown below).

[0627] Table S14: Western Blot Antibodies

[0628] antigen Isotype / Source clone dilution Mouse caspase 3 rabbit Polyclonal 1:1000 Mouse caspase 8 Rabbit IgG D35G2 1:1000 Mouse caspase 9 Mouse IgG1 C9 1:1000 Mouse p53 Rabbit IgG D2H9O 1:1000 Mouse PARP rabbit Polyclonal 1:1000

[0629] Viability and caspase 3 / 7 activity assays

[0630] To analyze cell viability and caspase 3 / 7 activity after ECP treatment, WT C57BL / 6 splenocytes were treated with 8-MOP or UVA light alone, without treatment, or a combination thereof (ECP). Viability was analyzed using the CellTiter-Glo 2.0 cell viability assay (Promega), where viability is correlated with luminescent signal. Caspase activity was measured using the Caspase Gio 3 / 7 assay system (Promega) by increasing luminescent signal.

[0631] DSS colitis model

[0632] To induce colitis, Adipoq - / - C57BL / 6WT mice deficient in Arg1 or Arg1 received drinking water containing 3% dextran sulfate sodium (DSS colitis grade, 36 kDa to 50 kDa; MP Biomedicals) from day 0 to day 3, and then received normal drinking water. On days 0, 3, and 6 of the experiment, mice were injected (intraperitoneally) with 0.2 mg of immune checkpoint blocker (ICB) or corresponding isotype control. On days 3 and 6, 10 7 ECP-treated donor splenocytes were transplanted intravenously into recipients. In some experiments, steroids, adiponectin, and AdipoRon were administered daily from day 3 to day 7. Arginase-1 inhibitors were administered twice daily from day 4 to day 7. Serum was harvested by retroorbital bleeding under ketamine / xylazine anesthesia on day 8, followed by harvest of the large intestine and spleen.

[0633] Immunohistochemistry and evaluation of adiponectin staining

[0634] For immunohistochemistry (IHC), 2 μm FFPE tissue sections were produced, dewaxed, rehydrated and subjected to mature heat-induced antigen retrieval (HIAR-steamer, 20 minutes, pH 9). After HIAR, 1% H2O2 was used to block peroxidase for 15 minutes. The sections were blocked in PBS containing 5% NGS for 30 minutes, followed by incubation of the primary antibody in blocking solution for 1 hour (primary antibody adiponectin, ThermoFisher, #710179, lot number: 2052429, 1:400 dilution). The secondary antibody (Dako) connected to HRP was applied in blocking solution for 30 minutes. Visualization and staining were performed using the DAB+ substrate chromogen system (Dako), including counterstaining with hematoxylin. After dehydration, slides were sealed using Entellan.

[0635] Microscopic examination and scoring of adiponectin staining

[0636] For analysis of immunohistochemistry (IHC) and histology, an inverted Zeiss Axio imager microscope (Zeiss Imager.M1) equipped with an Axiocam 506 color camera and equipped with 10x, 20x, and 40x objectives was used. Quantification of adiponectin-positive cells in the lamina propria was performed on digitized images (randomly captured at 40x magnification). Positive and negative cells in hotspot areas were assigned using Fiji ImageJ version 1.52. Adiponectin-positive cells were expressed as density (in patient samples) or positive cells per hotspot area (in mouse samples).

[0637] MC38 OVA Subcutaneous tumor model

[0638] 1×10 6 MC38 OVA or 0.5×10 6 MC38 (control only) cells were transplanted subcutaneously into the shaved right flank of WT C57BL / 6 recipients. Recipients were treated with 0.2 mg ICB on days 1, 4, 8, 11, and 15. ECP-treated splenocytes (10 7 ; intravenous), steroids were administered daily (intraperitoneally) from day 6 to day 15. On day 16, serum samples were harvested by retro-orbital bleeding, mice were euthanized, and tumors and spleens were removed for further studies.

[0639] B16.F10 with adoptive T cell transfer OVA Subcutaneous model

[0640] 1×10 6 B16.F10 OVA The cells were transplanted subcutaneously into the shaved right flank of CD45.1 recipients. Recipients were treated with 0.2 mg ICB on days 1, 4, 8, 11, and 15. ECP-treated splenocytes (10 7 ; intravenous), steroids were given daily (intraperitoneally) from day 6 to day 15. On day 2, 100 U / mL rmIL-2 (Peprotech) and 0.1 μg / mL OVA were added. 257-264 In the presence of peptide (Sigma), 2×10 6 OT-I splenocytes (CD45.2) were activated at a density of 10 cells / mL. On day 4, the splenocytes were washed and incubated with 100 U / mL rmIL-2 at a concentration of 2 × 10 6On day 5, OT-I splenocytes were washed and 5×10 6 The cells were injected intravenously into the tail vein of tumor-bearing mice. On day 16, serum samples were harvested by retro-orbital bleeding, the mice were euthanized, and the tumors and spleens were removed for further study.

[0641] ALT and AST activity assay

[0642] ALT and AST activities were assayed in sera harvested from mice with xenograft-induced irAEs according to the manufacturer's instructions (Sigma).

[0643] Results: ECP reduces immune-related colitis through adiponectin expression

[0644] To test ECP in a controlled in vivo irAE colitis model, mice were treated with 3% DSS and anti-PD1 to induce colitis ( Figure S1a ). Anti-PD1 treatment increased colitis severity compared to isotype (Figures S1b and S1c), indicating ICI-induced colitis. DSS and anti-PD1 treatment reduced the body weight of mice, which is consistent with the development of colitis ( Figure 1a Transfer of ECP-treated donor splenocytes reduced body weight loss compared to the group treated with anti-PD1 alone ( Figure 1a ) and prevented inflammation-induced colon shortening ( Figure 1b and Figure 1c Colon length was reported as a surrogate parameter for the severity of ICI-induced colitis, and colon length and body weight were found to be correlated ( Figure S1d Consistent with the reduced severity of colitis, a decreased infiltration of neutrophils (neutrophils) and lymphocytes in the colon wall of mice treated with ECP was observed compared to the group treated with anti-PD1 ( Figure 1d to Figure 1f Transfer of untreated splenocytes had no protective effect ( Figure S1e to Figure S1h), and the improvement of colitis by ECP was comparable to that by steroid treatment ( Figure S1i As with splenocytes, PBMCs were protected against colitis induced by the ECP procedure ( Figure S1m Figure S1p), indicating that the application of ECP is comparable to that in patients using PBMCs. Transplantation of ECP-treated splenocytes in DSS / anti-CTLA4-induced colitis showed similar results ( Figure S2a (Figure S2d), anti-CTLA4 is often administered in combination with anti-PD1 as therapy for metastatic melanoma.

[0645] Studying transcriptional changes in cells lining the intestinal wall, they found that Adipoq (adiponectin) was the most significantly upregulated gene in colitis mice treated with anti-PD1 / ECP compared to anti-PD1 alone ( Figure 1g and Figure 1h Furthermore, in DSS / anti-PD1 colitis mice, adiponectin serum concentrations increased after ECP treatment ( Figure 1i The same pattern was observed in a colitis model using anti-CTLA4 ( Figure S2e ). Adipoq - / - Receptors are not protected by ECP ( Figures 1j to 1m ), which is different from when the ECP donor and acceptor are Adipoq - / - The situation is quite similar ( Figure S3a To Figure S3d). Adiponectin deficiency in ECP-treated splenocytes was not associated with the immunosuppressive ECP effect ( Figure S3e These findings suggest that ECP reduces ICI-induced colitis in mice by triggering adiponectin production in recipient cells.

[0646] Adiponectin is induced by apoptotic leukocytes that accumulate in inflamed colonic tissue.

[0647] ECP-treated leukocytes undergo rapid apoptosis in vitro, and only the combination of ultraviolet A (UVA) and 8-methoxypsoralen (8-MOP), i.e., ECP, induces cell death ( Figure S4a and Figure S4b p53 stabilization signals apoptosis through enhanced caspase activity and cleavage of caspases 3 / 8 / 9 and PARP ( Figures S4c to S4n ). By transferring luciferase-transgenic splenocytes from melanoma-bearing donors into Rag2 - / - γc - / - In mice, only living cells converted luciferin and emitted light, confirming these findings in vivo. The BLI signal was reduced when ECP-treated splenocytes were transferred ( Figure S5a and Figure S5b To understand how ECP mediates adiponectin induction, ECP-treated leukocytes (CD45.1 + ) were exposed to bone marrow-derived macrophages (BMDM, CD45.2 + ), these macrophages have been shown to regulate intestinal inflammation. ECP-treated leukocytes are internalized by BMDM ( Figures 2a to 2d ), which is consistent with high surface levels of calreticulin acting as a phagocytic signal and stimulating phagocytes to clear dying cells ( Figure S6a and S6b Results were reproducible when bone marrow-derived dendritic cells (BMDCs) were used as phagocytic cells ( Figure S6c and Figure S6d Phagocytosis of the cells was confirmed when SIINFEKL peptide-loaded leukocytes were co-cultured with BMDM, where the cells were cytosed after co-culture on the CellTrace Violet + (CTV + ) SIINFEKL was found in BMDM ( Figure S6e Mechanistically, the phagocytic activity of ECP-treated splenocytes is consistent with STAT6 activation in macrophages ( Figure 2e ), STAT6 activation is known to drive macrophage polarization toward an immunosuppressive phenotype (M2-like) and may explain the protective effects of ECP in the intestine. Consistent with these results, co-culture with ECP-treated splenocytes induced Arg1, CD206, and CD301 in macrophages in vitro ( Figure 2f to Figure 2h ), indicating an anti-inflammatory phenotype. In addition, Adipoq gene expression was increased in BMDMs when co-cultured with ECP-treated splenocytes ( Figure 2i and Figure 2j To understand whether ECP-treated leukocytes reach the intestine, we used cells transplanted into CD45.2 + VivoTrack680-stained (VT680) CD45.1 in recipients treated with ECP + Donor cells. High fluorescence signals were observed in colitis 12 hours after transplantation, but not in B16.F10 melanoma tissues ( Figures 2k to 2m ). In ECP-treated CD45.1 + VT680 + After leukocyte transfer, VT680 was detected primarily in the colon of colitis / melanoma mice + CD45.2 + Receptor cells ( Figure 2n ), indicating that VT680 was engulfed in the inflamed intestine + Labeled donor cell phagocytosis. VT680 + Recipient myeloid cells showed high arginase-1 expression 12 hours after ECP transplantation, indicating their M2-like polarization after uptake of ECP-treated leukocytes ( Figure 2o and Figure 2p ). In vitro blocking of ECP-treated leukocyte uptake ( Figure S6f ) reduced STAT6 phosphorylation, M2-like polarization, and adiponectin induction in BMDM ( Figure 2q to Figure 2u Depletion of colonic phagocytes in vivo using clodronate-loaded liposomes in DSS / anti-PD1-induced colitis ( Figure 2EA 、 Figure S6g and Figure S6h ) eliminated the protective effect of ECP ( Figures 2EB to 2EE), increased infiltration of inflammatory neutrophils ( Figure 2EF and Figure 2EG ), and reduced protective adiponectin production and Arg1 expression ( Figure 2EH to Figure 2EJ ).

[0648] To test whether protection against ICI colitis depends on the ability of cells exposed to ECP prior to apoptosis to migrate to inflamed tissue, we compared myeloid cells (CD45 + CD11b + ), T cells (CD45 + CD3 + ) and epithelial cells. Epithelial cells were used as a control because they lack most molecules important for transendothelial migration ( Figure S7a After ECP treatment, epithelial cells also underwent apoptosis and upregulated surface calreticulin ( Figure S7b and Figure S7c While immune cells exposed to ECP had a protective effect, epithelial cells treated with ECP had no protective effect ( Figure S8a to Figure S8l), indicating that the ability to migrate into the inflamed colon is required for the anti-inflammatory effects of ECP ( Figure 2EK ).

[0649] These findings suggest that ECP-induced apoptotic leukocytes accumulate in colitis tissue, where they are phagocytosed by intestinal macrophages. This induces local anti-inflammatory polarization and adiponectin production. In contrast, no CD45.1 ECP-treated cells were detected in the tumor microenvironment. + VT680 + Phagocytosis of leukocytes.

[0650] ECP enhances STAT6 and PPARγ activities in colonic macrophages

[0651] Using scRNA-seq, we profiled intestinal immune cells from anti-PD1-treated and anti-PD1 / ECP-treated colitis mice and identified major changes in colonic myeloid cells after ECP treatment ( Figure 3a and Figure 3b In ECP-treated mice, macrophages were enriched and neutrophils were decreased ( Figure 3a and Figure 3b ), which is consistent with the known role of neutrophils in colitis. When analyzing transcription factor activity, an increase in cells with higher STAT6 and PPARγ activity was observed in colonic macrophages after ECP treatment ( Figures 3c to 3e ), both of which are important in polarizing macrophages toward an anti-inflammatory phenotype. In addition, KLF4 and PPARα (both of which promote an anti-inflammatory state) showed increased activity in macrophages after ECP treatment ( Figure S9aSupporting the importance of both PPARγ and STAT6 for the immunosuppressive effects of ECP, inhibition of either abolished the protective ECP effect in colitis mice ( Figures 3f to 3i ), and beneficial increases in adiponectin and Arg1 ( Figures 3j to 3l Consistent with these results, upon PPARγ and STAT6 inhibition, reduced anti-inflammatory polarization and decreased Adipoq expression were observed in BMDMs co-cultured with ECP-treated splenocytes ( Figures 3EA to 3ED Further scRNA-seq analysis confirmed the shift from pro-inflammatory macrophages to M2-like macrophages ( Figures 3EA to 3EE , Table S1), whereas neutrophils expressed lower levels of tissue-damaging matrix metalloproteinases in ECP-treated mice ( Figure S9b , Table S1). Table S1 is shown below.

[0652] Table S1. Selected genes from scRNA-Seq analysis clustering

[0653]

[0654]

[0655] scRNA-seq-based analysis and functional inhibitor-based studies revealed that ECP induces a regulatory PPARγ- and STAT6-dependent program in macrophages, leading to an alternative activated macrophage phenotype and adiponectin production.

[0656] Adiponectin reduces intestinal inflammation

[0657] The importance of adiponectin in the resolution of ICI therapy-induced colitis was confirmed by administering Adiponectin receptor agonist (ARA) or recombinant adiponectin to mice with DSS / anti-PD1-induced colitis ( Figures 4a to 4h Both treatments synergized with ECP to alleviate ICI-induced colonic inflammation ( Figure 4i to Figure 4p ARA treatment is effective not only in anti-PD1-mediated colitis but also in DSS / anti-CTLA4-induced intestinal inflammation ( Figures S10a to S10d ). These data show that adiponectin signaling can reduce the severity of colitis induced by ICI therapy and that it acts synergistically with ECP.

[0658] Adiponectin and ECP elevate Arg1 in myeloid cells

[0659] Profiling of various immune cells isolated from untreated mice revealed particularly high Adipoq expression in monocytes and M2-polarized macrophages ( Figure 5a), whereas Adipor1 and Adipor2 receptors are expressed by multiple cells, and particularly high expression is observed in neutrophils ( Figure 5b and Figure 5c The effects of adiponectin on BMDM were evaluated in vitro and upregulation of the immunosuppressive Arg1 was found upon exposure to adiponectin or ARA ( Figure 5d and Figure 5e ), however, the expression of Adipoq and Arg1 is highly correlated in BMDM ( Figure 5f ).

[0660] Using CyTOF-based analysis of immune cells isolated from colitis mice, we demonstrated major changes in receptors derived from myeloid cells of the lamina propria and spleen upon ECP treatment ( Figure 5g 、 Figure 5h 、 Figure S11a and Figure S11b Arginase-1 was one of the most significantly upregulated markers in ECP-treated colitis mice ( Figure 5i and Figure S11c Consistently, Arg1 expression was upregulated in colitis tissues during ECP therapy ( Figure 5j Furthermore, after ECP treatment, the expression of immunosuppressive molecules CD39, PD-L1, Tim3, and B7-H4 increased in the spleen and bone marrow subsets of colitis mice ( Figures S12a to S12e Higher expression of intestinal stem cell-related genes Ascl2 and Axin2 indicated increased intestinal repair ( Figure S13a and Figure S13b Further investigation of the connection between adiponectin and arginase-1 revealed that M2-polarized Adipoq - / - -Arg1 expression is reduced in SMDM ( Figures 5k to 5m This is consistent with lower levels of M2-like markers CD206 and CD301 ( Figure 5n and Figure 5o ).

[0661] These findings suggest a direct link between adiponectin and higher Arg1 expression and anti-inflammatory polarization in mice with ICI-induced colitis.

[0662] Arg1 blockade abolishes the protective ECP effect. Comparison of Arg1 and Adipoq expression in the colon of mice with DSS-induced colitis revealed a positive correlation when mice received anti-PD1 / ECP treatment but not anti-PD1 alone ( Figure 6a and Figure 6b Consistent with a functional role for Arg1 after ECP, Arg1 inhibition reduced the protective ECP effect in DSS / anti-PD1-induced colitis ( Figures 6c to 6f). Using Arg1 - / - mice, its functional role in alleviating ICI-induced colitis was demonstrated only in ECP recipients but not in the donor compartment ( Figure 6g to Figure 6m These findings indicate that Arg1 is required in recipient tissues to allow for a fully protective ECP effect, whereas it is not required in leukocytes undergoing ECP.

[0663] Adiponectin and ECP reduce T cell activation

[0664] The effects of adiponectin and ECP on T cells were investigated by activating T cells in vitro with anti-CD3 / CD28 beads in the presence of adiponectin. This reduced CD4 + and CD8 + The expression of CD25, the most prominent T cell activation marker on T cells ( Figures 7a to 7c ), but no effect on FoxP3 expression and CD4 + / CD8 + Effect of T cell frequency ( Figures S14a to S14c The functional role of CD25 reduction upon adiponectin treatment was demonstrated by adoptive transfer of vehicle-treated and adiponectin-treated T cells into DSS / anti-PD1-treated mice. Colitis was less severe in mice receiving adiponectin-treated T cells compared to vehicle ( Figure 7d and 7e CyTOF profiling of splenic T cells from DSS / anti-PD1 colitis mice revealed decreased TOX and enhanced expression of B and T lymphocyte attenuator (BTLA), killer cell lectin-like receptor G1 (KLRG1), and CD47 on T cells upon ECP treatment ( Figures 7f to 7i The results were confirmed in anti-CTLA4-induced colitis ( Figures S14d to S14f The findings suggest that T cell exhaustion and cytotoxicity were reduced upon ECP therapy, as these markers were associated with reduced proliferation capacity and decreased cytokine production. Transfer of splenic T cells from DSS / anti-PD1 or DSS / anti-PD1 / ECP donors (Figures S14g and S14h) into colitis secondary recipients confirmed their tolerogenic phenotype upon ECP treatment ( Figure 7j and Figure 7k ). The transferred T cells are rich in CD4 + KLRG1 + CD47 + BTLA + TOX 低 T cells, but in Treg, CD4 + and CD8 + There is no difference in frequency ( Figures S14i to S14qThese findings support the concept that ECP and adiponectin influence recipient T cells, leading to tolerogenic CD4 + CD25 低 T cells and induce CD4 + KLRG1 + CD47 + BTLA + TOX 低 Consistently, scRNA-seq confirmed that T cell activation was reduced in colonic lamina propria-derived effector T cells after ECP treatment ( Figure 7l , Table S1 ).

[0665] Immunosuppressive ECP effects against irAEs allowing anti-tumor immunity

[0666] Untreated mice were treated with either daily steroids or weekly ECP transplants for 50 days to understand whether the immunomodulatory ECP effects affect recipient immunity. Long-term steroids are the standard first-line therapy for patients with severe ICI-induced colitis. Steroids reduced all major and effector spleen immune cell populations, while ECP treatment showed no reduction compared to vehicle ( Figures 8a to 8i These results were confirmed using a metastatic B16.F10 melanoma model, in which additional steroid administration, but not ECP treatment, reduced survival compared with anti-PD1 therapy alone (Figures S16 and Figure 8j Transfer of untreated splenocytes did not affect the survival rate of tumor-bearing mice ( Figure S16b ). Using 4344-BRAF V600E Mutant melanoma cells served as a second model to reproduce these findings ( Figure 8k The results were supported by an additional model in which DSS / anti-PD1-mediated colitis was induced in melanoma-bearing mice. Although colitis was similarly reduced between steroid-treated and ECP-treated groups (Figures S16c and S16d), steroid-treated mice showed a higher tumor burden, as demonstrated by luminescence imaging and expression of melanoma-specific transcripts in the lung (Figures S16e and S16f). Figures 81 to 8o ) were used to identify the control of tumor growth. The control of tumor growth was comparable between the anti-PD1 and ECP treatment groups ( Figures 81 to 8o Consistent with functional anti-melanoma immunity, cytotoxic T cell function was not diminished upon ECP treatment in melanoma-bearing colitis mice ( Figures S16g to S16j Using OVA transgenic tumors, we demonstrated that B16.F10-OVA melanoma and MC38-OVA dim Adoptively transferred OT-I T cells and endogenous CD8 in the subcutaneous microenvironment of colorectal tumors +OVA-Dextramer + In contrast, ECP treatment did not affect tumor-specific T cells ( Figure 8p 、 Figure 8q and Figure S16k In support of these findings, T cells isolated from anti-PD1-treated and anti-PD1 / ECP-treated mice bearing B16.F10 melanomas showed recall immunity when transplanted into secondary B16.F10 melanoma-bearing recipients, whereas this was not the case when T cells from anti-PD1 / steroid-treated donors were used ( Figure 8r 、 Figure S16l and Figure S16m To understand why antitumor immunity remained unchanged during ECP, we analyzed Adipoq and Arg1 in the tumor microenvironment. Neither the local expression nor the systemic level of adiponectin increased during ECP treatment ( Figure 8s to Figure 8v Furthermore, analysis of tumor-specific T cells revealed no increase in the frequency of tolerogenic T cells in the tumor microenvironment ( Figure S17a to Figure S17h).

[0667] These findings suggest that ECP does not interfere with anti-PD1-induced antitumor immunity. The immune response remains intact due to the tissue-specific effects of ECP induced by phagocytosis of apoptotic leukocytes accumulated in the inflamed colon, but is ineffective in the tumor microenvironment ( Figures 2k to 2n ).

[0668] Example 2

[0669] patient

[0670] Key eligibility criteria included a minimum age of 18 years and steroid-refractory irAEs affecting the gut, liver, skin, or lungs. Patients were eligible if they had received treatment with anti-PD1, anti-PD-L1, anti-CTLA4 antibodies, or any combination of these antibodies for any type of malignancy within 24 months prior to screening. Patients should have clinical and / or histological evidence of the following immune-related adverse events:

[0671] Colitis: Diarrhea, an increase in the frequency of stools by ≥4 times over baseline; no improvement after 72 hours of treatment with at least 1 mg / kg BW / day prednisolone equivalents;

[0672] Hepatitis: Alanine aminotransferase and / or aspartate aminotransferase ≥3× ULN (if baseline is normal); or ≥3× baseline (if baseline is abnormal); no improvement after 72 hours of treatment with at least 1 mg / kg BW / day prednisolone equivalents;

[0673] Pneumonia: Radiographic changes and new symptoms such as cough, dyspnea, or chest pain that do not improve after 72 hours of treatment with 1 mg / kg BW / day prednisolone equivalents;

[0674] Dermatitis: skin erythema, maculopapular or pustular papule covering ≥30% of body surface area; no improvement after 72 hours of treatment with 1 mg / kg BW / day prednisolone equivalents;

[0675] Prior to initiation of ECP, up to one additional (second-line) therapy after steroid therapy (eg, infliximab for colitis).

[0676] Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 2.

[0677] Clinical EC treatment in patients treated in a prospective phase Ib / II trial

[0678] ECP was performed on a Therakos CellEx photopheresis system. It is administered as a photosensitizer, and 1500 mL of blood is processed during each procedure. For the first four weeks, the procedure is performed twice a week on consecutive days. After the fourth week, the interval is increased, and ECP is performed for two days every other week. Response is assessed at six and 12 weeks after the start of ECP.

[0679] Mitigation and safety assessment

[0680] Definitions of irAE severity and remission were established based on ASCO guidelines. Patients were monitored for clinical or laboratory signs of irAEs at each visit. Adverse events (AEs) and serious adverse events (SAEs) were scored according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. The relationship of each AE / SAE to the ECP was assessed by the investigator.

[0681] end

[0682] The primary endpoint was the rate of treatment-related adverse events (AEs) and serious adverse events (SAEs) in patients with immune checkpoint inhibitor-induced colitis, pneumonitis, hepatitis, or dermatitis treated with ECP. A positive outcome for the study was defined as ≤50% of patients experiencing a treatment-related SAE. The key secondary endpoint was the objective response rate (ORR) to treatment after 6 and 12 weeks of ECP therapy. The response criteria for each organ were described in the study protocol. Additional secondary endpoints were duration of response, discontinuation of other immunosuppressive therapy, and the incidence of tumor progression or recurrence.

[0683] Patient Cohort Analysis The retrospective irAE patient cohort analysis included 11 patients who were treated with ECP for irAEs after informed consent. Inclusion criteria were: previous treatment with anti-CTLA4, anti-PD1, anti-PD-L1 antibodies, or a combination of these antibodies; clinical diagnosis of irAE; and at least one prior therapy for irAE before off-label treatment with ECP. All patients were informed of the off-label use of ECP as part of the pilot study and gave written informed consent before the first ECP procedure. Analysis of clinical data was approved by the institutional review board. All procedures involving peripheral blood analysis were approved by the local ethics committee. Patients gave written informed consent for the use of biological materials for research.

[0684] Clinical ECP treatment of patients in a real-world patient cohort

[0685] At all four centers, ECP was performed on the Therakos CellEx photopheresis system. It is administered as a photosensitizer, and 1500 mL of blood is processed during each procedure. At three centers, two procedures were typically performed on consecutive days. The frequency and total number of ECP procedures were determined on an individual basis for each patient. At one center, ECP procedures were performed every 2-3 days for a total of 14 days.

[0686] mice

[0687] Mice between 6 and 14 weeks of age, weighing 15-25 mg, and only female or male donor / recipient pairs were used.

[0688] Statistical analysis of clinical data

[0689] Clinical data were managed and analyzed using Prism 9 (GraphPad) and SAS 9.4. Descriptive statistics were used to report the frequencies and percentages of continuous and discrete variables. P values ​​for comparisons of paired samples were calculated using a two-tailed paired Student's t-test.

[0690] Statistical analysis of mouse and in vitro experiments

[0691] For the sample size in the mouse survival experiment, power analysis was performed. A sample size of at least n=10 per group was determined by achieving a statistical significance of 0.05 with 80% power to detect an effect size of at least 1.06. Animal survival differences (Kaplan-Meier survival curves) were analyzed by the Mantel Cox test. The experiment was performed in an open-label manner. All data were tested for normality using the Kolmogorov-Smirnov test. For statistical analysis of two groups, unpaired or paired two-tailed Student's t-test was applied, depending on the study design. If the data did not meet the normality criteria, the Mann-Whitney test or Wilcoxon test was applied for unpaired or paired analysis, respectively. For comparison of more than 2 groups, the Kruskal-Wallis test (no matching or pairing) or Friedman test (pairing) with Dumi's multiple test (if a non-parametric test is recommended) was used. In the case of normal distribution of data, ordinary (no matching or pairing) or repeated measures (pairing) one-way analysis of variance with Tukey's multiple comparisons was performed. Statistical analysis was performed using GraphPad Prism 9 (GraphPad). Data are presented as mean ± standard error of the mean (SEM). P values ​​< 0.05 were considered significant.

[0692] The clinical activity of ECP in patients with irAEs was based on preclinical findings, and the clinical efficacy and safety of ECP were evaluated in a prospective, multicenter, phase Ib / II trial. As predefined in the study protocol, 14 patients were enrolled, and their demographics and clinical characteristics are summarized in Table S2. All patients had received one or two prior therapies for their irAEs and were corticosteroid-resistant. This was defined as the lack of symptom improvement after at least one week of 1 mg / kg BW steroid equivalents or symptom rebound after immunosuppression was reduced. Patients received corticosteroid therapy before ECP for a median of 5 weeks (range, 2 to 15 weeks) (Figure S18a), and 57% of patients had irAEs of grade 3 to 4 severity before the start of ECP (Table S2). At study entry, patients presented with colitis, hepatitis, dermatitis, or a combination of these. Underlying diseases included cutaneous melanoma, uveal melanoma, non-small cell lung cancer (NSCLC), or thyroid cancer (Table S2). Patients underwent a median of 16 ECP procedures (Fig. S18b), and AEs were monitored during ECP treatment (Table S3). SAEs were not considered directly related to ECP. Tumor staging before and after ECP showed no evidence of an increased rate of disease progression (PD) after ECP treatment (Fig. S18c). At the time of analysis, 2 of the 14 patients had died due to tumor progression. Both patients had PD of their tumors at study entry. Median survival was not reached, and the median follow-up was 8 months, determined by the inverse Kaplan Meier method ( Figure S18d Response to ECP treatment was defined as complete resolution (CR) or improvement of irAEs according to CTCAE grade (PR). The overall response rate (ORR) of irAEs was 93% at week 6 (all 14 patients reached this time point) and 92% at week 12 (13 patients reached this time point, and 1 patient died of tumor progression before week 12). Figure 9a Individual patients demonstrated remissions in severity of hepatitis, colitis, and dermatitis ( Figures 9b to 9g and Figure S18e One patient had a relapse of colitis after week 12 but again responded to ECP without requiring corticosteroid doses >0.5 mg / kg BW prednisone equivalents ( Figure 9c At week 12, the CR rate in patients with ICI colitis was 100% ( Figure 9d In all patients, steroids could be reduced after ECP treatment ( Figure 9h Two patients who received ECP were re-challenged with ICI after resolution of the irAE with no signs of irAE recurrence. The frequencies of major immune cell populations remained stable when analyzed before, during, or after ECP therapy (Figures S18f to Figure S18m ). Confirming preclinical data, adiponectin serum levels and colonic ADIPOQ expression increased after ECP treatment ( Figure 9i and Figure 9j ). Tables S2 and S3 are shown below.

[0693] Table S2. Demographics and Clinical Characteristics of Patients Treated in the Prospective Phase Ib / II Trial

[0694]

[0695]

[0696] Table S3: Adverse Events in Patients Treated in the Prospective Phase Ib / II Trial

[0697]

[0698]

[0699] In addition to the prospective trial, real-world data were collected from 11 patients with irAEs who received corticosteroids as monotherapy or in combination with mycophenolate mofetil (MMF), cyclosporine A (CSA), or infliximab (Table S4; Figure S19aAt the start of ECP, patients presented with colitis, hepatitis, dermatitis, arthritis, or a combination thereof. Underlying diseases included cutaneous melanoma, uveal melanoma, renal cell carcinoma, and NSCLC (Table S4). Patients underwent a median of 21 ECP procedures until best response was achieved ( Figure S19b ), AEs were monitored during ECP treatment, and SAEs may not be directly related to ECP (Table S5). Nine of 11 patients (81.8%) achieved CR, which was defined as the absence of any irAE symptoms, while one patient achieved PR (≥1 grade reduction in ≥1 organ manifestation) and one patient had SD ( Figure 9EA The clinical course of a representative hepatitis patient is shown in Figure S19c After ECP treatment, steroids were reduced in all patients ( Figure 9EB Five patients were rechallenged with ICI after resolution of the irAE. Two patients did not show recurrence of the irAE, however, three patients had recurrence from the initial irAE, leading to ICI discontinuation or switching from dual ICI therapy to single ICI therapy. Tumor staging showed no increase in disease progression rate after ECP therapy ( Figure S19d In summary, ECP appears to be safe and effective for ICI-induced irAEs, without unexpected toxicities. Tables S4 and S5 are shown below.

[0700] Table S4. Demographics and clinical characteristics of the real-world patient cohort

[0701]

[0702]

[0703] Table S5: Adverse events in a real-world patient cohort

[0704]

[0705]

[0706]

[0707] In humanized irAE models, Rag2 - / - γc - / - Mice received PBMC from healthy donors and anti-PD1 ( Figure S20a Administration of ECP alleviated histopathological signs of irAEs in major target organs, comparable to steroid treatment ( Figure 9EC 、 Figure 9ED In mice that received PBMCs alone, irAEs were minimal to absent, confirming ICI-mediated inflammation (Fig. S20h to S20o).

[0708] These findings demonstrate that ECP is active in patients with ICI-induced colitis who are refractory to steroids or other immunosuppressants in prospective clinical trials and real-world settings, and this was confirmed in a humanized irAE mouse model.

[0709] discuss

[0710] Patients who develop severe grade 3 or 4 irAEs after ICB are currently treated with glucocorticoids and have immunotherapy discontinued for a long time. As shown in retrospective analyses, both interventions may have a negative impact on anti-tumor immunity. In addition, glucocorticoids are expected to be avoided due to systemic side effects (including hyperglycemia, fluid retention, psychological disorders, and infectious complications). Currently, there are no prospective randomized trials supporting the use of glucocorticoids or second-line therapies such as mycophenolate mofetil (MMF), TNF antagonists, or cyclosporine A. There is a lack of new approaches to combat irAEs without producing strong systemic immunosuppression. ECP activity has been previously reported in patients with severe chronic graft-versus-host disease (cGVHD) refractory to ruxolitinib. In addition, patients with irAE colitis successfully treated with ECP have also been reported previously. However, the molecular mechanisms of ECP-induced immunomodulation remain unclear.

[0711] A DSS / ICI-induced colitis model was used to investigate the mechanism of resolution of ECP-induced irAE colitis. An established ICI-sensitive melanoma model was used to understand the effect of ECP on the anti-tumor response following anti-PD1-based immunotherapy. Using an unbiased microarray approach, ECP-induced adiponectin expression was found in the intestine, which had never been previously associated with ECP's mode of action. Adiponectin was originally identified in adipocytes, but subsequent studies also revealed its expression in non-adipocytes. Increased adipose tissue mass is associated with the development of autoimmune diseases and certain adipokines, such as adiponectin, whose production may serve to balance adipose tissue-associated inflammation. Obese patients have lower serum levels of adiponectin, indicating that this deficiency may contribute to the body's inability to fight inflammation in obesity. Consistently, adiponectin was observed to be reduced in patients with type 2 diabetes, highlighting its role as an anti-inflammatory molecule. Adiponectin binds to its two receptors, AdipoR1 and AdipoR2, which are highly expressed on myeloid cells. Adiponectin can polarize macrophages toward a tolerogenic phenotype, as observed by increased IL-10 production and decreased IFNγ and TNF expression. Overexpression of adiponectin in macrophages of mice fed a high-fat diet caused a decrease in serum levels of pro-inflammatory MCP-1 and TNF. Similarly, Adipoq - / -Mice display increased TNF levels in adipose tissue and blood. Furthermore, adiponectin treatment reduces TNF production and disease activity in mice with nonalcoholic fatty liver disease. Consistent with its anti-inflammatory effects, adiponectin negatively regulates pro-inflammatory IL-33 signaling in ILC2s located in adipose tissue. Adiponectin itself is negatively regulated by inflammatory mediators, and anti-IL-6R inhibition increases systemic adiponectin levels in RA patients.

[0712] Studies have shown that adiponectin is induced by phagocytic uptake of apoptotic leukocytes in inflamed tissues. ECP treatment induces apoptosis of leukocytes, which are then cleared by phagocytes located in the intestinal wall of colitis mice. Mechanistically, uptake of apoptotic cells leads to STAT6 phosphorylation and PPARγ activation. This in turn leads to anti-inflammatory polarization in macrophages, as observed by increased production of Arg1. Treatment with an arginase-1 inhibitor and Arg1 - / - Its important role in mediating ECP-mediated immunosuppression was demonstrated in mice. Arg1 depletes arginine, which is essential for the function of activated T cells. In addition to indirect T cell inhibition through Arg1 induction, adiponectin can also directly inhibit T cell activation, as confirmed by reduced expression of pathogenic CD25. Adoptive transfer of activated T cells enhanced the severity of colitis, while T cells pre-treated with adiponectin did not. ECP increased the frequency of low-exhaustion spleen tolerogenic T cells in mice with colitis, indicating that the inflammatory activity of these cells was reduced because KLRG1 identifies T cells after antigen exposure that lack the ability to proliferate. BTLA and CD47 are known inhibitory molecules that reduce proinflammatory T cell responses. The complex mechanism of action of ECP is summarized in the graphical abstract ( Figure S21 ).

[0713] Importantly, antitumor immunity remained intact in melanoma-bearing mice treated with ECP, whereas corticosteroids abolished ICI-induced antitumor immunity. Consistent with the reduced antitumor activity, prednisolone reduced all major immune cell populations in mice. Notably, the frequency of antigen-specific immune cells decreased after steroid administration, thereby limiting antitumor immunity. Further investigation examined how ECP inhibited ICI-induced colitis without affecting anti-melanoma immunity. It was found that ECP-treated apoptotic cells were phagocytosed locally in inflamed colonic tissue but not within the tumor microenvironment. Furthermore, upregulation of adiponectin and Arg1 was observed only in colitis but not in tumor tissue. Similarly, the frequency of tolerogenic antigen-specific T cells in the tumor microenvironment was not increased after ECP treatment. The tissue tropism of ECP-treated apoptotic cells could be explained by the potent cell-attracting chemokine release in the inflamed intestine, a chemokine milieu that may not be present in tumor tissue. Interestingly, ECP allows recall immunity if T cells from ECP-treated donors are adoptively transferred into secondary recipients.

[0714] Similar to the findings in the mouse model, ECP treatment was observed to result in clinical remission in 92% and 90.9% of patients with irAEs in a multicenter prospective clinical trial and retrospective study, respectively. Comparable to preclinical observations, ECP induced adiponectin in the blood and intestinal tissue of patients with irAEs without producing toxic effects. All patients receiving ECP were able to reduce their immunosuppressive regimen. In this study, the favorable safety profile of ECP observed in patients with irAEs was consistent with the safety profile reported in patients treated with ECP for acute or cGVHD. In our prospective Phase Ib / II trial, treatment of patients with irAEs with ECP revealed a 100% complete remission rate in patients with ICI colitis at week 12 after the start of ECP, which is higher than all remission rates reported to date for patients with ICI colitis. At week 12 after the start of ECP, the ORR for all irAEs was 92%. The high overall remission rate was confirmed in a real-world cohort, showing a 90.9% clinical remission rate (81.8% CR rate). The phase Ib / II study was the first prospective, interventional, controlled trial to define analysis time points (weeks 6 and 12) for patients with ICI-induced colitis, hepatitis, and dermatitis, whereas no other published reports met these criteria (Tables S6 and S7). Tables S6 and S7 are shown below.

[0715] Table S6: Clinical trials investigating ICI-targeted colitis treatments

[0716]

[0717]

[0718] Table S7: Clinical trials investigating ICI-targeted hepatitis treatments

[0719]

[0720]

[0721]

[0722]

[0723] A prospective trial using interleukin-6 blockade with tocilizumab for the treatment of ICI-induced colitis reported best remission at any time point within an 8-week period after the start of treatment. Symptoms were alleviated in 8 of 10 patients, but no CR was reported. A 100% CR rate was observed for ECP-induced ICI colitis at week 12. A retrospective observational cohort study of patients with ICI colitis reported remission rates of 89% and 88% with vedolizumab and infliximab, respectively. Counting remission as best remission at any time point during the observation period means that even patients with transient remissions that subsequently resolved were counted as responders, in contrast to prospective trials in which remission was determined at predefined time points. Furthermore, the study did not provide information on CR rates. A retrospective case series of 28 patients with ICI colitis refractory to steroids and / or infliximab who received vedolizumab reported an endoscopic remission rate of 29%. A retrospective analysis of 13 patients treated with infliximab for ICI colitis reported an ORR of 45% and a CR of 31%. A retrospective analysis of 8 patients treated with infliximab for ICI colitis reported an ORR of 50% and a CR rate of 38%. In summary, in a prospective phase Ib / II trial, the treatment of patients with irAEs with ECP revealed that at week 12 after the start of ECP, the complete remission rate in patients with ICI colitis was 100%, and the ORR for all irAEs was 92%. The high ORR was confirmed in a real-world cohort, showing a clinical remission rate of 90.9% (81.8% CR rate).

[0724] In summary, ECP was identified as a novel immunomodulatory approach to control ICI-induced colitis and irAEs in mice and humans. This is the first study to elucidate the underlying molecular mechanisms of ECP treatment in a controlled in vivo model. The study showed that in mice, ECP-treated leukocytes underwent apoptosis and were phagocytosed by intestinal macrophages, leading to activation of STAT6 and PPARγ. This increased adiponectin production in colitis tissue. Adiponectin was found to increase arginase-1 production in myeloid cells, and both ECP and adiponectin reduced the frequency of proinflammatory T cells in mice. Tolerogenic T cells increased in the inflamed intestine but not in melanoma tissue, leaving antitumor immunity unaffected. Corticosteroids reduced antitumor immunity.

[0725] These findings suggest that ECP may address the unmet clinical need for novel therapeutic approaches to treat ICI-induced irAEs without negatively impacting anti-tumor immunity.

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Claims

1. Adiponectin and / or adiponectin receptor agonists (ARAs) for the treatment or prevention of immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in subjects.

2. Adiponectin and / or ARA for use according to claim 1, wherein the subject is receiving checkpoint blockade therapy.

3. Adiponectin and / or ARA for use according to any one of the preceding claims, wherein the adiponectin and / or ARA is administered at least 1 hour before, during and / or at least one hour after administration of the checkpoint blockade therapy.

4. Adiponectin and / or ARA for use according to any one of the preceding claims, wherein the checkpoint blockade therapy comprises the administration of at least one antibody, preferably at least two antibodies.

5. Adiponectin and / or ARA for use according to any one of the preceding claims, wherein the at least one antibody is selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3).

6. Adiponectin and / or ARA for use according to any of the preceding claims, wherein the irAE may involve any organ, preferably the gastrointestinal tract, lungs, endocrine glands, skin and liver.

7. Adiponectin and / or ARA for use according to any one of the preceding claims, wherein the irAE comprises ICB-induced colitis.

8. Adiponectin and / or ARA for use according to any one of the preceding claims, wherein said administration of adiponectin and / or ARA reduces ICB-induced colitis.

9. Adiponectin and / or ARA for use according to any of the preceding claims, wherein said administration of adiponectin and / or ARA does not interfere with the anti-tumor response induced by said checkpoint blockade therapy, preferably anti-PD1 treatment, due to tissue-specific adiponectin induction.

10. A combination drug for treating immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB) in a subject, the combination drug comprising at least two of the following components: a. Photosensitizers and b. adiponectin and / or adiponectin receptor agonist (ARA), And wherein the blood sample of the subject is subjected to ultraviolet A (UVA) irradiation in vitro, preferably by extracorporeal photopheresis (ECP) therapy.

11. The combination for use according to claim 10, wherein the photosensitizer is a psoralen agent, preferably 8-methoxypsoralen.

12. The combination for use according to claims 10 to 11, wherein the wavelength of the UVA irradiation is a wavelength of 3200 angstroms to 4000 angstroms.

13. The combination for use according to claims 10 to 12, wherein the subject suffers from cancer.

14. Combination medicament for use according to claims 10 to 13, wherein the checkpoint blockade therapy comprises the administration of at least one antibody, preferably at least two antibodies.

15. The combination for use according to claims 10 to 14, wherein the at least one antibody is selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1) and / or programmed cell death ligand-1 (PD-L1) or lymphocyte activation gene 3 (LAG-3).

16. The combination drug for use according to claims 10 to 15, wherein the irAE may involve any organ, preferably the gastrointestinal tract, lungs, endocrine glands, skin and liver.

17. The combination for use according to claims 10 to 16, wherein the irAE comprises ICB-induced colitis.

18. The combination for use according to claims 10 to 17, wherein the combination induces the expression of adiponectin, arginase-1 (Arg1) and tolerogenic KLRG1 in the inflamed intestine of the subject. + BTLA + CD47 + TOX 低 Increase of T cells but no induction of adiponectin, arginase-1 (Arg1), and tolerogenic KLRG1 in cancer tissues + BTLA + CD47 + TOX 低 Increase in T cells.

19. Adiponectin and / or adiponectin receptor agonist (ARA) for use in treating a tumor patient (or: a subject with cancer), wherein the patient has experienced immune-related adverse events (irAEs) induced by immune checkpoint blockade (ICB), wherein the treatment comprises the combined administration of adiponectin and / or ARA and anti-tumor immunotherapy, wherein the immunotherapy comprises the administration of immunoregulatory T cells.

20. Adiponectin and / or ARA for use in treating tumor patients according to the preceding claims, wherein the immunoregulatory T cells. a. Obtained from a method comprising the steps of: i. providing a sample derived from an isolated blood sample of said patient, ii. adding a photosensitizer to the sample, and subjecting the sample to irradiation.

21. Adiponectin and / or ARA for use in treating tumor patients according to claim 19 or 20, wherein the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation.

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