Method for generating immunomodulatory cells in a blood-derived sample

Abstract

The present invention relates to a method comprising the steps of providing a sample derived from a blood sample of a subject receiving checkpoint inhibitor therapy and suspected of developing or having developed symptoms of an immune-related adverse event (irAE), adding a photosensitizer to the sample, and subjecting the sample to irradiation, preferably to generate immunoregulatory NK cells in the sample. In embodiments, the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation. In another aspect, the present invention relates to immunoregulatory NK cells obtained from a method comprising the steps of providing a sample derived from an isolated blood sample of the subject, adding a photosensitizer to the sample, and subjecting the sample to irradiation. Furthermore, the present invention encompasses immunoregulatory NK cells for use in treating and / or preventing irAEs in subjects receiving checkpoint inhibitor therapy.

Description

Technical Field

[0001] The present invention relates to a method comprising the steps of providing a sample derived from a blood sample of a subject who has received checkpoint inhibitor therapy and who is suspected of having or has developed symptoms of immune-related adverse events (irAE), adding a photosensitizer to the sample, and subjecting the sample to irradiation, preferably generating immunomodulatory NK cells in the sample. In embodiments, the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation. In another aspect, the present invention relates to immunomodulatory NK cells obtained from a method comprising the steps of providing a sample derived from an isolated blood sample of a subject, adding a photosensitizer to the sample, and subjecting the sample to irradiation. Furthermore, the present invention encompasses immunomodulatory NK cells for use in the treatment and / or prevention of irAE in a subject who has received checkpoint inhibitor therapy.

Background Art

[0002] Immune checkpoints are regulatory molecules of the immune system and play important roles in maintaining immune homeostasis and self-tolerance. The first identified immune checkpoints include cytotoxic T lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1). CTLA-4 is expressed on the surface of T cells, binds to B7-1 (CD80) or B7-2 (CD86) molecules on antigen-presenting cells, and functions as a negative regulator of T cells. PD-1 also adversely affects T cell activity through its interaction with its ligands, including programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2). Unlike CTLA-4, PD-1 is found not only on T cells but also widely expressed on many immune cells including B cells and natural killer cells. In healthy individuals, the surface expression of both CTLA-4 and PD-1 is tightly and dynamically regulated.

[0003] During cancer development, malignant cells inhibit the immune response by activating immune checkpoints. Previous studies have shown that PD-L1 is expressed in a wide range of cancers. In the tumor microenvironment, PD-L1 expressed by cancer cells interacts with PD-1 on the surface of T cells to inhibit the effector function of T cells. Furthermore, several studies have shown that high tumor expression of PD-L1 is significantly correlated with poor cancer prognosis. These studies suggest that there is a therapeutic effect of blocking the PD-1 signaling pathway in cancer.

[0004] Recent clinical trials have revealed that several anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors (ICIs) are effective against various cancers such as melanoma, non-small cell lung cancer, renal cell cancer, and head and neck cancer. Additional clinical trials are currently underway to expand the efficacy of ICIs. So far, the US Food and Drug Administration (FDA) has approved three anti-PD-1 antibodies, nivolumab, pembrolizumab, and cemiplimab, as well as three anti-PD-L1 inhibitors, atezolizumab, avelumab, and durvalumab, for the treatment of different types of cancer.

[0005] As the use of ICIs increases, the adverse events associated with this class of drugs have become an important issue. ICIs have a different toxicity profile from conventional cytotoxic chemotherapy. Side effects associated with increased immune system activity by ICIs, known as immune-related adverse events (irAEs), can affect multiple organs of the body, including the skin, gastrointestinal tract, endocrine system, liver, lungs, nervous system, and musculoskeletal system.

[0006] For example, the combination of immune checkpoint inhibitor therapy with anti-CTLA4 antibody and anti-PD-1 antibody is an effective first-line treatment for malignant melanoma. However, approximately 50% of patients develop severe immune-related adverse events (irAEs). 1,2 Autoimmune colitis occurs in 20% of cases and may become corticosteroid-refractory. 3 ​

[0007] In summary, checkpoint inhibitor therapy, particularly therapy with anti-CTLA4 and anti-PD-1 antibodies, is an effective treatment for multiple forms of cancer, particularly melanoma, but the treatment can be associated with substantial side effects that can result from the activation of the immune system due to checkpoint inhibitor therapy. Such side effects can lead to autoimmune reactions and can manifest in different clinical symptoms, which can be summarized as irAEs. A prominent irAE is autoimmune colitis. Autoimmune reactions, particularly side effects of treatment such as autoimmune colitis, may persist even after discontinuation of checkpoint inhibitor treatment.

[0008] Accordingly, there is a need in the art for alternative or improved means for treating autoimmune reactions, particularly immune-related adverse events such as autoimmune colitis, in patients receiving checkpoint inhibitor therapy. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] Based on the prior art, the technical problem underlying the present invention is to provide improved or alternative means for the treatment and / or prevention of autoimmune reactions, particularly immune-related adverse events such as autoimmune colitis, in patients receiving checkpoint inhibitor therapy.

[0010] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0011] In a first aspect, the present invention relates to a method comprising: - providing a sample derived from a blood sample of a subject who has received checkpoint inhibitor therapy and who is suspected of having developed or has developed symptoms of an immune-related adverse event (irAE); - adding a photosensitizer to the sample; - subjecting the sample to irradiation.

[0012] The present invention is based on the completely surprising finding that patients who have received checkpoint inhibitor therapy due to, for example, a cancerous disease and who have developed irAE, such as autoimmune colitis, can be effectively treated by applying ECP. The ECP used in the examples basically corresponds to a method of adding a photosensitizer to a blood-derived sample of an irAE patient and subjecting the sample to irradiation. Surprisingly, it has been found that pre-forming this method on a blood sample, particularly a blood sample containing mononuclear cells (MNC), leads to the generation of immunomodulatory NK cells in the above sample. In this context, the "generation" of immunomodulatory NK cells is 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 immunomodulatory NK cells.

[0013] Samples resulting from the method of the present invention, particularly the induced immunomodulatory NK cells contained by the sample, have been found to be useful for the treatment of irAE patients. Administering such a sample or cells contained in such a sample that has undergone the method of the present invention to a subject who has received checkpoint inhibitor therapy has been surprisingly shown to be effective in preventing the occurrence of irAE and also effective in the treatment of irAE that has already been established in the subject. Importantly, the method of the present invention can be carried out on a sample of the same subject who has received checkpoint inhibitor therapy. Thus, the cells or samples resulting from the method of the present invention can be administered to the same subject who has functioned as a blood donor, and thus the resulting sample can represent autologous cell therapy.

[0014] As used herein, the term "subject who has received checkpoint inhibitor therapy" includes a subject who is currently undergoing checkpoint inhibitor therapy or a subject who has received checkpoint inhibitor therapy that has been discontinued, for example, after the occurrence of irAE symptoms.

[0015] It was quite unexpected that such cell therapies using samples or cells resulting from the method of the present invention are effective even in patients with immune-related adverse events (irAEs) who are refractory to other immunosuppressive therapies such as steroids or anti-TNF antibodies and continue to show symptoms of irAEs or still suffer from them even after checkpoint inhibitor treatment has been discontinued.

[0016] As shown in the examples, the positive effects and efficacy of the administration of samples that have undergone the method of the present invention may be at least partially due to the regulation of NK cell function of NK cells contained in the samples, and the above regulation is the result of irradiation in the presence of a photosensitizer. Accordingly, the present invention also encompasses immunomodulatory NK cells for use in the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy. Preferably, the immunomodulatory NK cells are generated by subjecting blood or MNCs or NK cells from an individual, preferably a subject who has received checkpoint inhibitor therapy and is suspected of having developed or has developed irAEs, to the method of the present invention, and are then used to treat a subject, preferably a donor, who has received checkpoint inhibitor therapy and is suspected of having developed or has developed irAEs.

[0017] In a further aspect, the present invention relates to - the step of providing a sample derived from an isolated blood sample of a subject, - the step of adding a photosensitizer to the sample, - the step of subjecting the sample to irradiation, and relates to immunomodulatory NK cells obtained from a method comprising the steps.

[0018] This aspect of the present invention adopts an advantageous immunomodulatory phenotype when using the cells obtained in the treatment and / or prevention of irAEs based on the observation that NK cells are contained in a sample derived from blood that has been irradiated after adding a photosensitizer. Such advantageous and beneficial properties have not been observed for any other NK cells heretofore in this context.

[0019] Preferably, the subject of the present invention is a human. Preferably, the blood samples and cells of the present invention are human.

[0020] In a preferred embodiment, the blood sample used to generate the immunomodulatory NK cells of the present invention is from a subject who has received checkpoint inhibitor therapy and is suspected of having developed or has developed symptoms of immune-related adverse events (irAEs). This embodiment is particularly advantageous because the cells are autologous to the patient and there are no adverse events that can occur during the transplantation of heterologous cells.

[0021] A further aspect of the present invention is directed to immunomodulatory NK cells for use in the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy. This includes subjects who are currently receiving ongoing checkpoint inhibitors or subjects who have received checkpoint inhibitor therapy that has been discontinued, for example, after the occurrence of irAE symptoms.

[0022] Preferably, the immunomodulatory NK cells for such use in treatment or prevention are generated by adding a photosensitizer to a sample derived from a blood sample of a human subject and subjecting the sample to irradiation.

[0023] Adding a photosensitizer to a blood-derived sample and subjecting the sample to irradiation generates or induces (the formation of) immunomodulatory NK cells in the sample.

[0024] In an embodiment, the photosensitizer is 8-methoxypsoralen. Further, in an embodiment, the irradiation is UVA irradiation. In a preferred embodiment, the photosensitizer is 8-methoxypsoralen and the irradiation is UVA irradiation.

[0025] In the context of the present invention, the irradiation is preferably preformed by an extracorporeal photopheresis (ECP) system. Irradiation of the blood sample can be performed using any suitable system or irradiation device known to those skilled in the art.

[0026] In an embodiment, the method of the present invention is performed 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, the term relates to a method performed on a sample derived from blood taken out of the human body, and the method of the present invention is performed on a sample derived from blood outside the human body. For this purpose, the blood of the donor is taken out of the body, which means taking out the blood of the physiological circulatory system. As used herein, an "isolated blood sample" is a sample of blood (meaning a specific amount of blood) taken out from the donor's circulatory system to a position outside the human body.

[0027] Such an isolated blood sample can be supplied or introduced into an extracorporeal photopheresis (ECP) system or an apheresis system for performing at least certain steps of the method of the present invention. Among them, such a system can be an online system that is in fluid connection with the blood circulatory system of the subject. In an alternative embodiment, the method can be performed offline, and the blood-derived sample to be irradiated is disconnected from the donor's circulatory system.

[0028] Therefore, in an embodiment, the method of the present invention is an in vitro method. In an embodiment of the method of the present invention, the blood sample is an isolated blood sample. In an embodiment, the method is an in vitro method and the blood sample is an isolated blood sample.

[0029] As used herein, the term "blood sample" includes any kind of blood-derived sample, including a blood cell sample such as an MNC sample generated from blood.

[0030] In embodiments of the present invention, a subject (blood donor and / or recipient of cells) exhibits symptoms of, or is suffering from, an irAE. In further embodiments, the subject has received checkpoint inhibitor therapy, which was discontinued after symptoms and / or signs of an irAE developed in the subject. In embodiments, symptoms and / or signs of an irAE occurred in the subject after the checkpoint inhibitor therapy was discontinued. In embodiments, symptoms and / or signs of an irAE persisted after the checkpoint inhibitor therapy was discontinued.

[0031] In embodiments of the present invention, if a subject who has received checkpoint inhibitor therapy and is suspected of having developed or has developed symptoms of an irAE functions as a blood donor, the provision and / or isolation of a blood sample can be performed at any of the times described in the above embodiments. For example, sample isolation can occur before or after the onset of irAE symptoms. Further, sample isolation can occur during checkpoint inhibitor therapy or after discontinuation of the therapy.

[0032] In a preferred embodiment of the present invention, a sample resulting from the method of the present invention or NK cells for use according to the present invention are administered to a subject while checkpoint inhibitor therapy is ongoing.

[0033] In a preferred embodiment, sample isolation occurs during checkpoint inhibitor therapy, either before or after the onset of irAE symptoms. Samples or cells resulting from the methods of the examples can be used prophylactically or therapeutically by administering the sample / cells to a subject during ongoing checkpoint inhibitor therapy. Thus, in a preferred embodiment of the invention, a subject receiving checkpoint inhibitor therapy can receive immunomodulatory NK cells, which are preferably autologous and generated by irradiating a blood-derived sample according to the methods described herein, while the checkpoint inhibitor therapy is ongoing. Such embodiments are particularly advantageous since the patient receives the cells of the invention and / or cells resulting from the methods of the invention while a checkpoint inhibitor therapy such as an anti-cancer checkpoint inhibitor therapy can be maintained.

[0034] Thus, in such embodiments, administration of the irradiated cells either prevents the occurrence of irAE or remits the irAE so that the checkpoint inhibitor therapy can be continued, and thus the checkpoint inhibitor therapy can be administered to the subject for a longer period. It can be surprisingly shown that the methods of the invention, particularly the administration of such cells arising from the immunomodulatory NK cells of the invention, do not interfere with the anti-cancer response of subjects to checkpoint inhibitor therapy. This represents an important advantage compared to known therapeutic / preventive measures for irAE, particularly the administration of immunosuppressive drugs such as corticosteroids. Surprisingly, performing ECP does not significantly alter the anti-cancer effect of checkpoint inhibitor therapy, while co-administration of glucocorticoids (a particular class of corticosteroids), particularly prednisolone, results in a worse outcome, showing a decrease in the effectiveness of checkpoint inhibitor treatment.

[0035] Furthermore, in embodiments where a method of adding a photosensitizer to a sample and subjecting the sample to irradiation is received, a sample (human) derived from blood, the administration of a sample or cells resulting from such a method, regardless of the source of the blood-derived sample (autologous or heterologous), can occur, for example, before or after the onset of irAE symptoms and / or during checkpoint inhibitor therapy or after discontinuation of checkpoint inhibitor therapy.

[0036] In embodiments of the present invention, irAE (blood donor and / or recipient of cells) includes symptoms of an autoimmune disease and / or is caused by an autoimmune reaction. In preferred embodiments, irAE includes or is autoimmune colitis.

[0037] In embodiments, irAE includes at least one irAE selected from the group consisting of autoimmune colitis, autoimmune hepatitis, autoimmune thyroiditis, and autoimmune dermatitis.

[0038] In embodiments, irAE includes at least one irAE selected from the group consisting of immune checkpoint inhibitor-related colitis, immune checkpoint inhibitor-related hepatitis, immune checkpoint inhibitor-related thyroiditis, and immune checkpoint inhibitor-related dermatitis.

[0039] In embodiments, the subject (blood donor and / or recipient of cells) has a cancer such as melanoma or another cancer treatable by checkpoint inhibitor therapy.

[0040] In embodiments, the subject is receiving and / or is refractory to immunosuppressive drugs such as steroids, corticosteroids, cyclosporine, and / or anti-TNF antibodies (e.g., infliximab).

[0041] It was quite surprising that the administration of cells resulting from the methods described herein, such as immunomodulatory NK cells, has a therapeutic effect on irAE patients who are refractory to immunosuppressive drugs administered to treat irAE symptoms. For such patients, there is no effective treatment available to remit the symptoms of irAE, and thus, the present invention represents a completely unexpected possibility for the treatment of irAE in these patients.

[0042] In an embodiment, the checkpoint inhibitor therapy comprises administration of at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody.

[0043] In an embodiment, the immunomodulatory NK cells for use in the treatment and / or prevention of irAE in a subject who has received checkpoint inhibitor therapy are autologous to the subject. In an alternative embodiment, the NK cells can be allogeneic to the subject.

[0044] In an embodiment, the immunomodulatory NK cells according to the present invention are administered at the onset of irAE symptoms. In an embodiment, the cells are administered 1, 2, 3, 4, 5, 6 or 7 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45 or 50 weeks after the onset of irAE symptoms. In an embodiment, the immunomodulatory NK cells are administered during the progression of the checkpoint inhibitor therapy or after the checkpoint inhibitor therapy has been discontinued.

[0045] In an embodiment, the immunomodulatory NK cells are administered to the subject at least once, preferably at least twice, preferably daily. Thus, the subject can receive more than one dose of NK cells, but preferably, the administration does not exceed one dose per day. In a further embodiment, the subject receives at least 2, 3, 4, or 5 doses of the NK cells of the present invention. In an embodiment, the immunomodulatory NK cells of the present invention are administered to the subject at least every 8 weeks, preferably every 2 to 4 weeks.

[0046] Each optional or preferred feature of the 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.

[0047] Furthermore, the invention also relates to the following embodiments.

[0048] 1. An extracorporeal photopheresis (ECP) system for use in the treatment and / or prevention of immune-related adverse events (irAEs) in a subject receiving checkpoint inhibitor therapy, wherein the ECP is preferably carried out in conjunction with the administration of 8-methoxypsoralen.

[0049] The present invention is based on the completely surprising finding that patients who have received checkpoint inhibitor therapy due to, for example, a cancerous disease and who have developed irAEs, particularly autoimmune colitis, can be effectively treated by applying ECP. This also applies to patients who are refractory to other immunosuppressive therapies such as steroids or anti-TNF antibodies and who continue to show symptoms of irAEs or remain affected even after discontinuation of checkpoint inhibitor treatment.

[0050] Surprisingly, it has been found that the positive effects and efficacy of ECP treatment result, in particular, from the modulation of NK cell function after ECP. Accordingly, the invention also relates in particular to immunomodulatory NK cells for use in the treatment and / or prevention of immune-related adverse events (irAEs) in a subject receiving checkpoint inhibitor therapy, wherein the immunomodulatory NK cells are generated by subjecting blood or MNCs or NK cells from an individual, preferably a subject of the invention, to ECP treatment.

[0051] 2. An ECP system for use according to the invention, wherein the ECP system is an online ECP system.

[0052] 3. An ECP system for use according to the present invention, wherein the ECP system is an offline ECP system.

[0053] 4. An ECP system for use according to the present invention, wherein the subject exhibits symptoms of irAE or is suffering from irAE.

[0054] 5. An ECP system for use according to the present invention, wherein the checkpoint inhibitor therapy was discontinued after symptoms and / or signs of irAE occurred in the subject.

[0055] 6. An ECP system for use according to the present invention, wherein the symptoms and / or signs of irAE occurred after the checkpoint inhibitor therapy was discontinued.

[0056] 7. An ECP system for use according to the present invention, wherein the symptoms and / or signs of irAE persisted after the checkpoint inhibitor therapy was discontinued.

[0057] 8. An ECP system for use according to any of the present inventions, wherein ECP treatment is initiated upon the occurrence of irAE symptoms.

[0058] 9. An ECP system for use according to the present invention, wherein ECP treatment is initiated 1, 2, 3, 4, 5, 6, or 7 days after the occurrence of irAE symptoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50 weeks after the occurrence of irAE symptoms.

[0059] 10. An ECP system for use according to any of the present inventions, wherein ECP treatment is initiated during the course of checkpoint inhibitor therapy or after the checkpoint inhibitor therapy has been discontinued.

[0060] 11. An ECP system for use according to the present invention, wherein the ECP treatment comprises at least 1, preferably at least 2 cycles of ECP, preferably on consecutive days.

[0061] 12. An ECP system for use according to the present invention, wherein the ECP treatment is performed at least 2, 3, 4, or 5 times.

[0062] 13. An ECP system for use according to the present invention, wherein the ECP treatment is performed at least every 8 weeks, preferably every 2 - 4 weeks.

[0063] 14. An ECP system for use according to the present invention, wherein the irAE comprises symptoms of an autoimmune disease.

[0064] 15. An ECP system for use according to the present invention, wherein the irAE is caused by an autoimmune reaction.

[0065] 16. An ECP system for use according to the present invention, wherein the irAE comprises at least one irAE selected from the group consisting of autoimmune colitis, autoimmune hepatitis, autoimmune thyroiditis, and autoimmune dermatitis.

[0066] 17. An ECP system for use according to the present invention, wherein the irAE comprises at least one irAE selected from the group consisting of immune checkpoint inhibitor - related colitis, immune checkpoint inhibitor - related hepatitis, immune checkpoint inhibitor - related thyroiditis, and immune checkpoint inhibitor - related dermatitis.

[0067] 18. An ECP system for use according to the present invention, wherein the irAE comprises autoimmune colitis.

[0068] 19. An ECP system for use according to the present invention, wherein the IrAE is autoimmune colitis.

[0069] 20. An ECP system for use according to the present invention, wherein the subject is a human, the ECP system.

[0070] 21. An ECP system for use according to the present invention, wherein the subject has cancer such as melanoma or another cancer treatable by checkpoint inhibitor therapy, the ECP system.

[0071] 22. An ECP system for use according to the present invention, wherein the subject is receiving an immunosuppressive drug such as a steroid, corticosteroid, cyclosporine, and / or an anti-TNF antibody (e.g., infliximab), and / or is refractory to an immunosuppressive drug, the ECP system.

[0072] 23. An ECP system for use according to the present invention, wherein the subject is refractory to an immunosuppressive drug such as a steroid, corticosteroid, cyclosporine and / or an anti-TNF antibody (e.g., infliximab), the ECP system.

[0073] 24. An ECP system for use according to the present invention, wherein the checkpoint inhibitor therapy includes administration of at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody, the ECP system.

[0074] 25. An ECP system for use according to the present invention, wherein the ECP system is used for performing blood irradiation therapy, the ECP system.

[0075] 26. An ECP system for use according to the present invention, wherein the ECP system includes an immunomodulatory molecule, preferably an immunomodulatory property bound to the membrane of the ECP system, and the immunomodulatory molecule contacts the immune cells of the subject, the ECP system.

[0076] 27. An in vitro method, - A step of providing a sample derived from an isolated blood sample of a subject who has received checkpoint inhibitor therapy and who is suspected of having developed or has developed symptoms of immune-related adverse events (irAE), and - A step of subjecting the above sample to extracorporeal photopheresis, an in vitro method comprising.

[0077] 28. A method of treating a subject who has received checkpoint inhibitor therapy and who is suspected of having developed or has developed symptoms of immune-related adverse events (irAE), the method comprising subjecting the subject to extracorporeal photopheresis (ECP) therapy, such as blood irradiation therapy by an ECP system.

[0078] 29. Immunomodulatory NK cells for use in the treatment and / or prevention of immune-related adverse events (irAE) in subjects who have received checkpoint inhibitor therapy.

[0079] 30. Immunomodulatory NK cells for use according to the present invention, wherein the immunomodulatory NK cells are generated by subjecting a human blood sample or cells contained in a human blood sample to extracorporeal photopheresis.

[0080] 31. Immunomodulatory NK cells for use according to the present invention, wherein the NK cells are autologous or heterologous to the subject.

[0081] 32. Immunomodulatory NK cells for use according to the present invention, wherein the NK cells are intravenously administered to the subject.

[0082] 33. Immunomodulatory NK cells for use according to the present invention, wherein the NK cells are isolated from a human blood sample before or after being subjected to extracorporeal photopheresis.

[0083] All features disclosed in the context of an ECP system for use in the treatment and / or prevention of immune-related adverse events (irAEs) in a subject undergoing a checkpoint inhibitor therapy of the invention are also disclosed herein in the context of the in vitro method of the invention, the method of treatment of the invention, and NK cells for use in the treatment and / or prevention of immune-related adverse events (irAEs) in a subject undergoing a checkpoint inhibitor therapy of the invention, and vice versa. (Mode for Carrying Out the Invention)

[0084] All cited patent and non-patent documents are hereby incorporated by reference in their entirety.

[0085] Disclosed herein is a method comprising the steps of providing a sample derived from a blood sample of a subject, preferably a subject undergoing a checkpoint inhibitor therapy and suspected of having developed or having developed symptoms of an immune-related adverse event (irAE), adding a photosensitizer to the sample, and subjecting the sample to irradiation. Irradiation of the blood sample can be performed using any suitable system or irradiation device known to those skilled in the art. Preferably, the irradiation is performed by an extracorporeal photochemotherapy (ECP) system.

[0086] The invention also relates to an extracorporeal photochemotherapy (ECP) system for use in the treatment and / or prevention of immune-related adverse events (irAEs) in a subject undergoing a checkpoint inhibitor therapy, wherein the ECP is preferably performed in conjunction with the administration of 8-methoxypsoralen.

[0087] Irradiation and extracorporeal photochemotherapy (ECP) Photopheresis, or extracorporeal photochemotherapy (ECP), is a form of apheresis and photodynamic therapy that involves treating blood with a photosensitizer and then irradiating it with light of a specific wavelength to achieve an effect. For example, the buffy coat (WBC + platelets) can be separated from whole blood, chemically treated with 8-methoxypsoralen (either by dropping it into the collection bag or administering it several times in advance), exposed to ultraviolet light (UVA), and then returned to the patient. The activated 8-methoxypsoralen cross-links DNA within the exposed cells, ultimately leading to apoptosis of nucleated cells. The photochemically damaged T cells returned to the patient appear to induce a cytotoxic effect on T cell formation.

[0088] Photopheresis photochemotherapy with 8-methoxypsoralen was first described in the 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 US Food and Drug Administration (FDA) for cutaneous T-cell lymphoma. This treatment has been suggested to be potentially effective in the treatment of graft-versus-host disease. Photopheresis has also been successful in the treatment of epidermolysis bullosa when all other treatments have been ineffective.

[0089] 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 excluding blood irradiation therapy.

[0090] Blood irradiation therapy is a procedure in which blood is exposed to low-level red light (usually laser light) for therapeutic reasons. Blood irradiation therapy can be administered in three ways. Outside the body, the blood is drawn out and irradiated in a special cuvette. This method is used for ultraviolet (UV) blood irradiation (UVBI) with a UV lamp. Laser light is monochromatic, that is, it has a wavelength such that the light can be taken into an optical fiber and intravenously irradiated through an intravenous catheter. This method is simpler and more effective. Blood irradiation therapy is also administered externally through the skin over a prominence of a large blood vessel.

[0091] Intravenous or intravascular laser blood irradiation (ILBI) involves in vivo illumination of blood by delivering low-level laser light generated by a helium-neon laser at a wavelength of 632.8 nm and a power of 1 - 3 mW into a blood vessel channel, usually a vein in the forearm, under the assumption that any therapeutic effects will circulate through the circulatory system. In most cases, wavelengths of 365, 405, 525, 635 nm and a power of 2.3 mW are used. This technique is currently widely used in Russia but less so in Asia and not widely used in other regions of the world. ILBI has been shown to improve blood flow and its transport activity, and thus tissue trophism has a positive effect on the immune system and cell metabolism. This issue is the subject of doubt. There have been some needs to increase research on this topic. Transdermal therapy applies laser light to intact skin in an area with numerous blood vessels (such as the forearm). Since the skin acts as a barrier to the blood and absorbs low-level laser energy, the power of the laser is often enhanced to compensate. The problem can be solved by using a pulsed matrix laser light source. Extracorporeal irradiation is only used for ultraviolet blood irradiation which involves drawing blood out through a vein and irradiating it outside the body. It was promoted as a cancer treatment, but in a 1952 review in the Journal of the American Medical Association and another review by the American Cancer Society in 1970, this treatment was concluded to be ineffective.

[0092] Extracorporeal photochemotherapy (ECP), also known as extracorporeal photoimmunotherapy or photochemotherapy, is a therapy based on leukapheresis that was first used in patients with cutaneous T-cell lymphoma (CTCL). Specifically, for the treatment of refractory CTCL patients suffering from the leukemic variant, Sézary syndrome, ECP received FDA (U.S. Food and Drug Administration) approval in 1988. During ECP, a patient's whole blood is collected via an elbow vein or a permanently implanted catheter in order to separate white blood cells from plasma and non-nucleated cells. In a specially constructed device for this procedure, the collected white blood cells, so-called buffy coat, are exposed to ultraviolet A (UVA) irradiation in the presence of a photosensitizer, 8-methoxypsoralen, prior to reinfusion into the patient.

[0093] Two fundamentally different methods for performing the ECP procedure are described and encompassed by the present invention. These differ in the devices used for leukocyte collection and UVA irradiation: the "closed system" and the so-called "open system". The closed system is based on the original design by Edelson and colleagues and is the only system approved by the FDA. The open system is a system incorporating various separation instruments and is mostly used outside the United States. ECP has been an effective treatment for 30 years and more than 2 million treatments have been performed, but there are no reports of negative cytogenetic effects.

[0094] The indications for initiating ECP have been continuously extended since its introduction. ECP treatment is generally well tolerated by patients and has few significantly undesirable side effects. Overall, ECP combines an excellent safety profile with effectiveness.

[0095] Extracorporeal photochemotherapy (sometimes also referred to as extracorporeal photopheresis) is a process that includes the following: (1) collection of patient-derived mononuclear cells (MNCs), (2) photoactivation treatment of the collected MNC cells, and (3) reinfusion of the treated cells (MNCs) into the patient. More specifically, ECP involves the ex vivo exposure of peripheral blood mononuclear cells in combination with a photoactive compound such as 8-methoxypsoralen or "8-MOP", followed by photoactivation with ultraviolet light and subsequent reinfusion of the treated mononuclear cells. The combination of 8-MOP and UV radiation is thought to cause apoptosis or programmed cell death of T cells treated with ECP.

[0096] The exact mechanism of action in ECP treatment (in different disease states) is not fully understood, but according to an early theory, photoactivation was thought to irreversibly covalently bind 8-MOP to the DNA strands contained in the T cell nucleus. When photochemically damaged T cells are reinfused, a cytotoxic effect is induced. For example, cytotoxic T cells or "CD8+ cells" release cytotoxins when exposed to infected or damaged cells or, alternatively, attack cells that carry specific foreign or abnormal molecules on their surface. The cytotoxins target the membrane of the damaged cells, enter the target cells, and ultimately lead to apoptosis or programmed cell death of the target cells. That is, after the treated mononuclear cells are returned to the body, the immune system recognizes dying abnormal cells and begins to generate healthy lymphocytes (T cells) to combat those cells.

[0097] In addition to the above, it is also theorized that extracorporeal photochemotherapy can induce 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 treated apoptotic T cell antigens as described above. Other possible mechanisms of action will be understood to potentially be involved in the benefits observed from the ECP treatment of mononuclear cells and the subsequent achievement of benefits for patients receiving ECP-based therapies.

[0098] More recently, ECP has been hypothesized to potentially induce an immunotolerant response in patients. For example, in the case of graft-versus-host disease, injection of apoptotic cells can stimulate the generation of regulatory T cells, inhibit inflammatory cytokine production, cause the deletion of effector T cells, and result in other responses. See Peritt, “Potential Mechanisms of Photopheresis in Hematopoietic Stem Cell Transplantation,” Biology of Blood and Marrow Transplantation 12:7-12 (2006). Currently, the theory of an immunotolerant response appears to be among the leading explanations, but other theories exist regarding the mechanism of action of ECP in graft-versus-host disease, as well as other disease states.

[0099] Examples of systems for performing ECP include, for example, the UVAR XTS Photopheresis System and the CellEx Photopheresis System available from Therakos, Inc., of Exton, Pa. Further details for performing ECP with the Therakos system can be found, for example, in U.S. Patent No. 5,984,887.

[0100] Currently, there are two methods commonly used to implement photochemotherapy - online and offline systems and methods.

[0101] In the online method, a dedicated photochemotherapy device such as the above-described Therakos device is used to perform the entire treatment, including reinfusion of the processed MNCs. Such devices are “dedicated” photochemotherapy devices designed only for performing photochemotherapy and cannot perform other collection protocols required in a hospital or blood processing setting, including, for example, platelet, plasma, RBC, granulocyte collection and / or plasma / RBC exchange protocols, or multifunctional apheresis protocols.

[0102] In the method of offline photochemotherapy, a multifunctional apheresis device may be used to collect mononuclear cells. The collected MNCs, typically contained in one or more collection containers, are detached from the tubing set used during collection or otherwise separated, and they are subsequently processed with a separate irradiation or UVA light device, followed by the manually reinfusion of the processed cells into the patient. However, during such an offline method, when the cells are transferred from the apheresis device to the irradiation device (the device may be located in a separate room or laboratory), communication with the donor must be severed, and thus the cells must be detached from the donor. Therefore, additional traceability procedures are required to ensure that the processed MNC product is ultimately reinfused into the correct donor.

[0103] Device and immobilization of enzymes within the device: In embodiments of the present invention, the ECP system may include a device that includes a matrix having immobilized immunomodulatory molecules or other biomolecules, such as enzymes. Preferably, the matrix having the bound molecules is exposed to blood or MNCs in the context of the present invention.

[0104] Accordingly, as used herein, "matrix" refers to the material inside a blood treatment device that provides an internal material or surface through which blood or plasma passes. The matrix used in the context of the present invention preferably includes a support to which immobilized immunomodulatory molecules or other biomolecules are bound. Accordingly, the support functions as a carrier for the immobilized immunomodulatory molecules or other biomolecules, although it may perform other functions.

[0105] As used herein, "support" refers to a portion of a matrix that functions as a "substrate" or "support material" to which an immobilized immunomodulatory molecule or other biomolecule according to the present invention is bound. Such a support or support material may also be referred to as an "adsorbent material" or "adsorbent" when used in an "adsorption column" or "column" or "adsorption cartridge". Preferred supports according to the present invention should be uniform, hydrophilic, mechanically and chemically stable over the relevant pH range and temperature, with no or negligible leaching of the enzyme during use, have good flow characteristics for whole blood and / or plasma, and provide a large surface area for enzyme binding.

[0106] The support can be, for example, a resin, a membrane, or a non-woven material. A "non-woven" material is broadly defined as a sheet, fabric or web structure joined together mechanically, thermally, or chemically, but not by weaving or knitting, by winding fibers or filaments (and by perforating films). A "resin" refers to an insoluble material that can take the form of a gel or gel beads or microporous beads, or a sponge. Such resins can be natural or biopolymers, synthetic polymers and inorganic materials. Agarose, dextran and cellulose beads are commonly used natural supports. Synthetic polymer or organic supports are mainly based on acrylamide, polystyrene and polymethacrylate derivatives, while porous silica and glass are some of the frequently used inorganic supports.

[0107] According to an embodiment of the present invention, the resin is composed of a polymer selected from the group consisting of alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin, and sepharose, an inorganic material selected from the group consisting of zeolite, ceramic, celite, silica, glass, activated carbon, and carbon, or a synthetic polymer selected from the group consisting of polyethylene (PE), polyoxymethylene (POM), polypropylene (PP), polyvinyl chloride (PVC), polyvinyl acetate (PVA), polyvinylidene chloride (PVDC), polystyrene (PS), polytetrafluoroethylene (PTFE), polyacrylate (PAA), polymethyl methacrylate (PMMA), polyacrylamide, polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN), polyester, polycarbonate, polyethylene terephthalate (PET), polyamide, polyaramide, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polysulfone (PS), polyether sulfone (PES), polyaryl ether sulfone (PEAS), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), polyamideimide, polyaryl ether ketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyhydroxyalkanoate, polyether ether ketone (PEEK), polyether ketone ketone (PEKK), polyether imide (PEI), polyimide, polylactic acid (PLA), polymethylpentene (PMP), poly(p-phenylene ether) (PPE), polyurethane (PU), styrene acrylonitrile (SAN), polybutenoic acid, poly(4-allyl-benzoic acid), poly(glycidyl acrylate), polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polydivinylbenzene (PDVB), poly(allyl glycidyl ether), poly(vinyl glycidyl ether), poly(vinyl glycidyl urethane), polyallylamine, polyvinylamine, copolymers of the above polymers, and these polymers modified by the introduction of functional groups.

[0108] For immobilizing an immobilized immunomodulatory molecule or other biomolecule to a support and / or matrix according to the present invention, various known methods can be used. Such immobilization is preferably specific or selective in terms of immobilizing an enzyme, while on the other hand, other proteins and components (in vitro) present in blood or plasma or a sample thereof are not immobilized to a significant extent.

[0109] "Immobilization" of an immobilized immunomodulatory molecule or other biomolecule to a support for providing a matrix that can be used in a device according to the present invention refers to non-covalent or covalent interaction that holds two molecules together. According to one embodiment of the present invention, expression refers to covalent interaction, i.e., a covalently bound immobilized immunomodulatory molecule or other biomolecule. Non-covalent interactions include, but are not limited to, hydrogen bonds, ionic interactions between charged groups, van der Waals interactions, and hydrophobic interactions between non-polar groups. One or more of these interactions can mediate the binding of two molecules to each other. The binding may otherwise be specific, selective, or non-specific.

[0110] According to one embodiment, an immobilized immunomodulatory molecule or other biomolecule includes an affinity tag for immobilizing it on a support. The affinity tag can be used for purifying the protein during production and / or for immobilizing them on a support of the matrix of the present invention. The affinity tag can be a short polypeptide sequence or a whole protein co-expressed as a fusion partner with an enzyme. Different types of affinity tags are well known in the art, and polyhistidine or His6 tag, C-myc tag, and FLAG tag are particularly well described and are options for binding an enzyme according to the present invention to a support material. Non-covalent binding of biotin to streptavidin or avidin can also be used for immobilizing an immobilized immunomodulatory molecule or other biomolecule to a support.

[0111] According to another embodiment of the present invention, the immobilized immunomodulatory molecule or other biomolecule is covalently attached to the support as described in further detail below and / or as described in the prior art. Covalent attachment generally includes either non-site-directed covalent attachment of proteins or site-directed attachment of proteins. The support that forms the basis of the matrix generation must provide or facilitate chemical activation and thus enable chemical coupling of the immobilized immunomodulatory molecule or other biomolecule. Many coupling methods for immobilizing the immobilized immunomodulatory molecule or other biomolecule are well known in the art.

[0112] For example, the activating chemistry should be stable over a wide range of pH, buffer conditions, and temperatures that result in negligible leaching of the enzyme. The coupling method should avoid inappropriate orientation, multi-site binding, or steric hindrance of the immobilized immunomodulatory molecule or other biomolecule. The enzyme density per volume of the matrix can be optimized to facilitate target accessibility and reaction.

[0113] Covalent attachment can be carried out via common functional groups including amine, alcohol, carboxylic acid, aldehyde, and epoxy groups. Carbodiimide compounds can be used to activate the carboxyl groups of proteins for direct conjugation to primary amines on the support surface via amide bonds. The most commonly used carbodiimides are water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) for aqueous crosslinking and water-insoluble DCC (N’,N’-dicyclohexylcarbodiimide) for non-aqueous organic synthesis methods.

[0114] Alternatively, the support may carry a linker and / or specific functional groups for attaching an enzyme thereto. For example, functionalized resins are commercially available and are known to those skilled in the art. A wide range of coupling chemistries including primary amines, sulfhydryls, aldehydes, hydroxyls, and carboxylic acids are available in the above commercial supports. Examples of commercially available activated resins include CarboLink coupling resin, Profinity™ Epoxide resin, Affi-Gel 10 and 15, Epoxy-activated Sepharose™ 6B, tresyl chloride-activated agarose, and Purolite® Lifetech™ methacrylate polymer functionalized with Epoxy groups.

[0115] According to one embodiment of the present invention, the support material must be porous and the pore size ranges from 10 to 200 nm. According to another embodiment of the present invention, the support takes the form of beads. According to yet another embodiment of the present invention, the support according to the present invention includes magnetic beads. The magnetic beads are prepared by encapsulating magnetite within agarose or other polymeric materials on which the enzyme according to the present invention is immobilized.

[0116] According to another embodiment of the present invention, the support is a membrane. Membranes as components of affinity matrices have been used in protein purification due to their simplicity, ease of handling, reduced surface area, and lower diffusion limitations compared to gels, resins, and beads. The membrane can also take the physical form of hollow fibers or, alternatively, the physical form of flat membranes. According to one embodiment, the support includes a hemodialysis hollow fiber membrane dialyzer and the filter is a hemodialyzer.

[0117] Hollow fiber or flat sheet membranes for use as a support in a device according to the present invention can be composed of cellulose, cellulose esters (cellulose acetate and cellulose triacetate), poly(methyl methacrylate) (PMMA), polyamide (PA), other nitrogen-containing polymers (polybenzimidazole, polyacrylonitrile (PAN), polyglycidyl methacrylate (PGMA), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES) or polyarylethersulfone (PAES). The hollow fiber membranes that can be advantageously utilized to provide a device according to the present invention preferably have an inner diameter in the range of 100 to 500 μm. According to another embodiment of the present invention, specifically, when the membrane support is a hemodialysis membrane as described above, the hollow fiber membranes are additionally or alternatively functionalized by the enzymes according to the present invention on the luminal side of the fibers, and they can directly interact with the target metabolites in the blood or plasma perfusing the lumen of the hollow fiber membranes. The enzymes may also alternatively be immobilized on the outside of the membrane.

[0118] Method for extracorporeal blood treatment: The present invention comprises means configured to be located in an extracorporeal blood circuit through which a patient's blood passes, to transport blood from the patient's vascular system to a blood treatment device at a defined flow rate and then return the treated blood to the patient, and further comprises a device configured to reduce the levels of ADMA and / or MMA in the blood. Furthermore, the present invention also includes a device that can be used for extracorporeal blood or blood cell treatment, and the device can be separated or disconnected from the extracorporeal blood circuit or can be separable / removable.

[0119] According to the present invention, the expression "extracorporeal blood purification" preferably refers to a process of removing substances from body fluids through their clearances by flowing blood in a bypass circuit outside (extracorporeal) the patient's body. The substances may include endogenous toxins (i.e., uremic toxins), exogenous toxins (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. Treatment procedures include hemodialysis, hemoperfusion, plasma exchange and therapeutic apheresis, including intermittent hemodialysis (HD, HDF, HF) and continuous renal replacement therapy (CRRT). Such methods are known to those skilled in the art, and the devices of the present invention can be incorporated accordingly.

[0120] As used herein, the expression "blood" refers to whole blood containing all components of a living organism's blood, 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 that form part of whole blood but no longer contain red blood cells, white blood cells, and platelets. In the context of the present invention, the expression "blood plasma" or "plasma" refers to a specific fraction of plasma as defined above in its standard meaning, such as serum.

[0121] According to one aspect, the blood flow rate in the extracorporeal blood purification circuit is 20 ml to 700 ml / min. In addition to the blood treatment device according to the present invention, or in either case where the hemodialyzer is configured to metabolize ADMA and / or MMA, the typical dialysate flow rate in the extracorporeal circuit including a hemodialyzer for treating renal insufficiency ranges from 0.5 l / hour to 800 ml / min.

[0122] In therapeutic apheresis, whole blood can be treated, or the blood is fractionated into its component fractions, for example by centrifugation or by means of a plasma membrane or filter, and the fraction containing the solute to be removed is specifically treated before being returned to the patient.

[0123] The present invention provides an apheresis treatment in which whole blood or plasma (including the target protein) is removed from the patient's flowing blood, contacted with a device or matrix according to the present invention, and then returned to the patient. The typical blood or plasma flow rates in an extracorporeal circuit in which a blood treatment device is perfused with whole blood or plasma are in the ranges of 30 ml / min to 200 ml / min and 7 ml / min to 50 ml / min, respectively.

[0124] According to one aspect, an extracorporeal blood circuit according to the present invention is configured to perform hemodialysis. In this case, the device according to the present invention is, for example, a hemodialyzer additionally configured to immobilize the target protein according to the present invention. The circuit can be operated in different treatment modes according to medical needs, such as hemodialysis, hemodiafiltration, hemofiltration mode, etc.

[0125] Immune-related adverse events (irAE) As used herein, the term "immune-related adverse event" (irAE) relates to any side effect that specifically occurs in the context of immune checkpoint inhibitor therapy, for example in the context of cancer therapy. IrAEs are idiosyncratic and typically have a delayed onset and long duration, unlike adverse events that occur in the context of conventional cancer therapy. IrAEs can involve any organ or system. These effects are frequently low-grade, treatable, and reversible. However, some side effects can be severe and lead to permanent disabilities. Management is mainly based on corticosteroids and other immunosuppressive agents and should be prescribed carefully to reduce the potential for short-term and long-term complications.

[0126] Specifically, this term includes autoimmune diseases and symptoms of autoimmune diseases such as (autoimmune) colitis, (autoimmune) hepatitis, (autoimmune) thyroiditis, and (autoimmune) dermatitis.

[0127] The irAEs due to the administration of checkpoint inhibitor therapy in the present invention are not particularly limited. IrAEs should be understood as adverse events presumed to be immune-related: irAEs (see, for example, the drug interview form (version 9) of OPDIVO (registered trademark) intravenous injection of 20 mg - 100 mg revised in April 2016, the characteristics and handling of adverse events of anti-CTLA-4 antibody ipilimumab (YERVOY (registered trademark)) issued by the Safety Committee for New Drugs for Malignant Melanoma of the Japanese Dermatological Association on August 24, 2015).

[0128] In the present invention irAE Specific embodiments include lung disorders such as interstitial lung disease, myasthenia gravis, myositis, colitis, type 1 diabetes, liver dysfunction (liver impairment), hepatitis (e.g., autoimmune pneumonia), hypopituitarism or hypophysitis such as hypopituitarism, hypothyroidism, neuropathy, nephropathy, encephalitis, adrenal disorders such as adrenal insufficiency, severe skin disorders, venous thromboembolism, infusion reactions, psoriasis, psoriasiform eruptions, diarrhea (e.g., severe diarrhea), rheumatoid arthritis, uveitis, dermatitis, synovitis, exacerbation of radiation keratitis, chronic inflammatory demyelinating polyneuropathy (hereinafter also referred to as demyelinating polyneuropathy), bowel disorders, or nephritis, and hypopituitarism is preferred.

[0129] Immune-related adverse events (IrAEs) are known to occur, for example, 8 weeks or 8 - 12 weeks after administration. Immune-related adverse events can be evaluated by "grade" or "IrAE assessment". Here, "IrAE assessment" is an index representing the severity of the disease and is represented by 1 - 3. In the IrAE assessment, 1 represents a state where "no additional therapeutic intervention due to IrAE is required", 2 represents a state where "drug intervention etc. due to IrAE is required, but hospitalization or interruption of treatment is not required", and 3 represents a state where "hospitalization due to IrAE is accompanied and interruption of treatment is required". The correspondence between "IrAE assessment" and "grade" varies depending on each disease.

[0130] Immune checkpoint molecules and checkpoint regulators In the context of the present invention, an immune checkpoint inhibitor is a drug that activates immune cells by regulating immune checkpoint molecules.

[0131] An immune checkpoint molecule is a molecule within the immune system that either raises or lowers the signal (co-stimulatory molecule) 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 that are lymphocyte surface receptors and can bring about the activation or stimulation of lymphocyte effector functions. Co-inhibitory checkpoint molecules include co-inhibitory lymphocyte receptors that are lymphocyte surface receptors and can bring about the inhibition of lymphocyte effector functions.

[0132] Co-stimulatory checkpoint molecules include, but are not limited to, HVEM, CD27, CD40, OX40, GITR, CD137, CD28, and ICOS.

[0133] In a preferred embodiment of the present invention, the term co-stimulatory lymphocyte receptor does not refer to CD122.

[0134] 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 can bind to BTLA.

[0135] CD27 supports the antigen-specific proliferation of naive T cells, is essential for the generation of T cell memory, and is also a memory marker for B cells. The activity of CD27 is governed by the transient availability of its ligand, CD70, on lymphocytes and dendritic cells. CD27 co-stimulation is known to suppress Th17 effector cell function. The agonistic monoclonal antibody CDX-1127 / Varlilumab against CD27 has been shown to be effective in the context of T cell receptor stimulation in animal models.

[0136] CD28 is constitutively expressed on almost all human CD4+ T cells and on approximately half of all CD8 T cells. For example, binding of one of its two ligands, CD80 and CD86, expressed on dendritic cells, promotes T cell proliferation.

[0137] CD40 is expressed on various immune system cells, including antigen-presenting cells. The ligand of CD40, called CD40L and also known as CD154, is transiently expressed on the surface of activated CD4+ T cells as its ligand. CD40 signaling is known to "license" dendritic cells to mature, thereby triggering T cell activation and differentiation.

[0138] 4-1BB (CD137) is bound by the CD137 ligand, resulting in T cell proliferation. CD137-mediated signaling is also known to protect T cells, particularly CD8+ T cells, from activation-induced cell death. The fully human IgG2 agonistic monoclonal antibody Utomilumab (PF-05082566) targets 4-1BB and stimulates a stronger immune system attack against cancer.

[0139] OX40 (CD134) has OX40L (CD252) as its ligand. OX40 is also known for its ability to promote the proliferation of effector T cells and memory T cells and to suppress the differentiation and activity of regulatory T cells. OX40 is transiently expressed after T cell receptor ligation, which is why it is upregulated only on recently antigen-activated T cells within inflammatory lesions, and this is the reason why OX40 is a valuable drug target. Agonist anti-OX40 monoclonal antibodies have been shown to have clinical utility in advanced cancers. Pharmaceutical company AstraZeneca is developing three drugs targeting OX40: MEDI0562, a humanized OX40 agonist; MEDI6469, a mouse OX40 agonist; and MEDI6383, an OX40 agonist.

[0140] GITR (glucocorticoid-induced TNFR family-related gene) promotes T cell proliferation. The ligand for GITR (GITRL) is mainly expressed on antigen-presenting cells. Antibodies against GITR have been shown to promote antitumor responses through loss of Treg lineage stability.

[0141] 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 functions.

[0142] 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.

[0143] A2AR (adenosine A2A receptor) is considered an important checkpoint in cancer therapy because adenosine in the immune microenvironment that leads to activation of the A2a receptor is a negative immune feedback loop, and the tumor microenvironment has a relatively high concentration of adenosine.

[0144] B7-H3, also known as CD276, was initially understood to be a costimulatory molecule but is now regarded as a coinhibitory molecule. MacroGenics is researching MGA271 (Enoblituzumab), an Fc-optimized monoclonal antibody targeting B7-H3.

[0145] B7-H4 (or VTCN1) is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor evasion.

[0146] BTLA (B and T lymphocyte attenuator, also known as CD272) is a coinhibitory receptor with HVEM (Herpesvirus Entry Mediator) as its ligand. The surface expression of BTLA is gradually downregulated during the differentiation of human CD8+ T cells from naive to effector cell phenotypes. However, tumor-specific human CD8+ T cells express high levels of BTLA.

[0147] CTLA-4 (cytotoxic T lymphocyte-associated protein 4, also known as CD152) is expressed on Treg cells and plays a role in controlling T cell proliferation. CTLA-4 (CD152) is a protein receptor that functions as an immune checkpoint. It is expressed by activated T cells and transmits an inhibitory signal to T cells. CTLA4 is homologous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 has a higher affinity and binding activity for CD80 and CD86 than CD28. CTLA4 transmits an inhibitory signal to T cells. Antagonist antibodies against CTLA4 include ipilimumab and tremelimumab.

[0148] IDO (Indoleamine 2,3-dioxygenase) is a tryptophan catabolic enzyme with immunosuppressive properties. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis.

[0149] KIR (killer cell immunoglobulin-like receptor) is a receptor for MHC class I molecules on natural killer cells. Lirilumab is a monoclonal antibody against KIR.

[0150] LAG-3 (lymphocyte activation gene-3) acts to suppress the immune response by its action on Tregs and direct effects on CD8+ T cells.

[0151] PD-1 (programmed death 1, or CD279) is a cell surface receptor that plays an important role in promoting self-tolerance by downregulating the immune system and suppressing the inflammatory activity of T cells. PD-1 has two ligands, PD-L1 and PD-L2. The advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. PD-L1, a ligand of PD1, is highly expressed in some cancers and may lead to inhibition of the anti-cancer immune response by T cells. Several cancer immunotherapy agents targeting the PD-1 receptor have been developed, including the antagonist antibodies nivolumab (Opdivo - Bristol Myers Squibb), pembrolizumab (Keytruda, MK-3475, Merck), pidilizumab (CT-011, Cure Tech), and BMS-936559 (Bristol Myers Squibb). Both atezolizumab (MPDL3280A, Roche) and avelumab (Merck KGaA, Darmstadt, Germany & Pfizer) are monoclonal antibodies directed against PD-L1, a ligand of PD-1.

[0152] 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 functions as a negative regulator of Th1 / Th17 function by inducing cell death upon interaction with its ligand, galectin-9.

[0153] VISTA (V-domain Ig suppressor of T cell activation) is a protein mainly expressed on hematopoietic cells such that consistent expression of VISTA on leukocytes within tumors may enable blockade of VISTA to be effective across a wide range of solid tumors.

[0154] 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 to CD155 on DCs and macrophages with high affinity and to CD112 with lower affinity.

[0155] The coinhibitory lymphocyte receptors of the present invention include PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, BTLA or VISTA. The costimulatory lymphocyte receptors of the present invention include OX40, 4-1BB, GITR, CD27, HVEM, CD28, or CD40.

[0156] Inhibitors of the receptors prevent the generation of signals by the respective receptors. Thus, inhibitors of coinhibitory lymphocyte receptors are molecules that prevent the activation of the respective receptors and thereby prevent the generation of inhibitory signals. Conversely, activators of the receptors induce the generation of signals by the respective receptors, and activators of costimulatory lymphocyte receptors result in the generation of stimulatory signals.

[0157] Checkpoint regulators are molecules that interfere with the activity of immune checkpoint molecules by stimulating or inhibiting the activity of immune checkpoint molecules.

[0158] Soluble checkpoint regulators are molecules that can diffuse freely, for example, molecules that are not bound to the cell membrane or do not remain within the cell.

[0159] Checkpoint inhibitors in the context of the present invention include lymphocyte-stimulating checkpoint regulators, which are molecules that result in the activation of any lymphocyte, preferably effector T cells, either through the activation of co-stimulatory checkpoint molecules or through the inhibition of co-inhibitory checkpoint molecules. Furthermore, soluble lymphocyte-stimulating checkpoint regulators include molecules that interfere with the activation of membrane-bound immune checkpoint molecules, for example, the soluble forms of the respective immune checkpoint molecules.

[0160] Checkpoint regulators can be naturally occurring or engineered molecules that have the respective function of interfering with or regulating the activity of immune checkpoint molecules. Examples of checkpoint regulators include antibody or antibody fragment activity against immune checkpoint molecules having an agonist or antagonist, and ligands or modified ligands of immune checkpoint molecules.

[0161] Immune cells: The immune cells described herein relate to living cells involved in the immune response in a subject. The 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.

[0162] The term "immune cells" includes MNCs contained in the blood, which may also be referred to as peripheral blood mononuclear cells (PBMCs). PBMCs include any peripheral blood cells having a round nucleus and mainly consist of lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets do not have a nucleus and granulocytes (neutrophils, basophils, and eosinophils) have a multi-lobed nucleus. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, and the proportion of dendritic cells is small. These cells can be extracted from whole blood using density gradient centrifugation with ficoll, a hydrophilic polysaccharide that separates the layers of the blood, and the upper layer of plasma, followed by the layer of PBMCs, and the bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and red blood cells. Polymorphonuclear cells can be further isolated by lysing red blood cells. Basophils may be found in both the higher density fraction and the PBMC fraction.

[0163] T cells, also known as T lymphocytes, are a type of lymphocyte (a subtype of white blood cells) that play 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 their cell surface. Some subsets of T cells have different functions. Examples of T cell subtypes include, but are not limited to, T helper type 1 (Th1) cells, T helper type 2 (Th2) cells, T helper type 9 (Th9) cells, T helper type 17 (Th17) cells, T helper type 22 (Th22) cells, follicular helper T (Tfh) cells, regulatory T (Treg) cells, natural killer T (NKT) cells, gamma delta T cells, and CD8+ cytotoxic T lymphocytes (CTLs). Further 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 (CTLs, CD8+ T cells), CD4+CD8+ T cells, CD4CD8 T cells, or any other subset of T cells.

[0164] The present invention relates, in particular, to immunomodulatory NK cells (which may also be referred to as immunomodulatory NK cells) for use in the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy, preferably cell therapy.

[0165] Immune regulatory NK cells are a specific form of innate lymphoid cells (ILCs). ILCs belong to the lymphoid lineage (lymphocytes), but lack B cell or T cell receptors and thus are a group of innate immune cells that do not respond in an antigen-specific manner. This relatively newly described cell group has different physiological functions, some of which are similar to helper T cells, but also includes cytotoxic NK cells. According to this, they play important roles in protective immunity as well as in the regulation of homeostasis and inflammation. Natural killer (NK) cells are cytotoxic natural effector cells similar to the cytotoxic T cells of the adaptive immune system. They are distributed throughout the blood, organs, and lymphoid tissues and account for approximately 15% of peripheral blood lymphocytes. NK cells play a role in tumor surveillance and the rapid elimination of virus-infected cells. They do not require the "self" signal lacking MHC class I, can recognize stressed cells in the absence of antibodies, and are able to respond much more rapidly than the adaptive immune system. Natural killer (NK) cells play an important role in host immunity against cancer. In response to this, cancer develops mechanisms to escape NK cell attack or induce defective NK cells (Cheng M et al. Cell Mol Immunol. 2013 May;10(3):230-52.doi:10.1038 / cmi.2013.10.Epub 2013 Apr 22).

[0166] The immune regulatory NK cells of the present invention preferably have low expression (or downregulated expression) of CD16, particularly CD56 dim and are characterized on NK cells. CD16 is FcRγIII, an activating NK cell receptor capable of inducing strong cytokine production. The shedding / downregulation of CD16 may be an immune regulatory mechanism of NK cells to prevent autoimmunity. CD16 downregulation regulates NK cell responses and contributes to the maintenance of immune homeostasis in both antibody and T cell-dependent pathways. Furthermore, the immune regulatory NK cells of the present invention can show low expression of GM-CSF, IFN-γ, TNF, and / or IL-2.

[0167] In an embodiment, the generation or induction of immunomodulatory NK cells in a sample can be measured by, for example, flow cytometry, gene expression, and / or mass spectrometry of protein abundance, by comparing the phenotype and population distribution of NK cells in the sample before and after performing the method of the present invention. For example, the transition from CD16-high NK cells to CD16-low NK cells after irradiation indicates the induction of an immunomodulatory phenotype. Furthermore, a decrease in GM-CSF, IFN-γ, TNF and / or IL-2 expression in the NK cell population will also indicate the induction of immunomodulatory NK cells. Those skilled in the art are familiar with suitable methods and protocols for identifying different NK cell populations in a sample. For example, in a blood-derived sample, the total NK cell population can preferably be defined and identified as a CD45-positive, CD14-negative, CD3-negative, CD19-negative and CD56-positive cell population by flow cytometry. Within this population, further identification and quantification of immunomodulatory NK cells can be performed, for example, by determining CD16, GM-CSF, IFN-γ, TNF and / or IL-2 expression. Immunomodulatory NK cells are preferably positive for CD56, but exhibit a relatively low expression level of CD56 ("dim" expression).

[0168] Further features and definitions of immunomodulatory NK cells are well established in the art and are the subject of a number of studies and review articles known to or identifiable by those skilled in the art.

[0169] The term "immunomodulatory function" relates to the function or property of a molecule or cell that induces a change or regulation in the function, action or state of any component of the immune system.

[0170] Cell therapy typically involves the administration of immune cells isolated from a patient's blood. Cell types that can be used in this way 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 induces an anti-tumor response by presenting tumor antigens to dendritic cells. Dendritic cells present antigens to lymphocytes, which activate the antigens and prime them to kill other cells that present the antigens.

[0171] Therapeutic applications of the present invention As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy. Subjects such as subjects or patients in need of treatment or prevention can be animals, vertebrates, mammals, rodents (e.g., guinea pigs, hamsters, rats, mice), mice (e.g., mice), dogs (e.g., dogs), cats (e.g., cats), horses (e.g., horses), primates, monkeys (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, orangutans, langurs), or humans. The meanings of terms such as "animal", "mammal", etc. are well known in the art and can be inferred, for example, from Wehner und Gehring (1995; Thieme Verlag). In the context of the present invention, in particular, animals are assumed to receive treatments that are economically, agriculturally, or scientifically important. Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human.

[0172] In embodiments of the present invention, a subject who has received checkpoint inhibitor therapy suffers from cancer such as melanoma or another cancer treatable by checkpoint inhibitor therapy.

[0173] In the context of the present invention, the term "cancer" relates to the treatment of all types of cancer, as in the case of certain leukemias, regardless of whether the cancer is associated with the formation of solid tumors or whether cancer cells do not form solid tumors.

[0174] Cancer can affect any part of the body and includes a group of diseases 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 to form metastases. Worldwide, there were 14 million new cancer patients and 8.2 million cancer-related deaths in 2012 (World Cancer Report 2014). Most cancers are caused by environmental signals such as tobacco use, obesity, and infections, but about 5 - 10% are genetic cases. Cancers can be classified into subcategories based on the origin cells. The most common subcategories are cancers derived from epithelial cells, sarcomas and lymphomas derived from connective tissues, and leukemias derived from hematopoietic cells. Cancers are associated with various local and systemic symptoms and often cannot be cured. Given the large number of new cancer patients and cancer-related deaths, new treatment strategies are needed.

[0175] The cancer according to the present invention refers to all types of cancers or neoplasms or malignant tumors found in mammals, including leukemia, sarcoma, melanoma and cancer. Either solid tumors and / or liquid tumors (such as leukemia or lymphoma) can be treated.

[0176] Examples of melanoma include, but are not limited to, acro-leiomyoma melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, vulvar melanoma, and superficially spreading melanoma.

[0177] 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, leukemic leukemia, leukemic leukemia, basement membrane leukemia, blast leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblast leukemia, hemocytoblast leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphocytic leukemia, lymphocytic leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryoblast leukemia, micromyeloblast leukemia, monocytic leukemia, myeloblast leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myeloid monocytic leukemia, Naegeli leukemia, plasmacytic leukemia, plasmacytic leukemia, promyelocytic leukemia, leader cell leukemia, Schilling's leukemia, stem cell leukemia, sub-leukemic leukemia, and undifferentiated cell leukemia.

[0178] Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Abt's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, sarcoma botryoides, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, interstitial sarcoma, Ewing's sarcoma, fascial sarcoma, fibrosarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's lymphoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, white matter sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulum sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0179] Examples of cancers include, but are not limited to, acinar carcinoma, acinous carcinoma, adenoid cystic carcinoma, adenoid cystic carcinoma, cancer adenoma, adrenal cortex cancer, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, villous carcinoma, colloidal carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, cuirus carcinoma, cutaneous carcinoma, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, cancer dura mater, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, cancer epithelial adenoid, exophytic tumor, ulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, adenocarcinoma, granular cell carcinoma, hair matrix cell carcinoma, hemocytoblastoma, hepatocellular carcinoma, Hurthle cell carcinoma, vitreous carcinoma, hypernephroma, infantile embryonal carcinoma, intraepithelial carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma, chondrosarcoma, myxosarcoma, myxosarcoma, mucinous cell carcinoma, mucoepidermoid carcinoma, cancer mucous, mucinous carcinoma, cancerous myxoma, nasopharyngeal carcinoma, oat cell carcinoma, osteocarcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spinous cell carcinoma, medullary carcinoma, renal cell carcinoma of the kidney, precursor cell carcinoma, carcinosarcoma, schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, globular cell carcinoma, spindle cell carcinoma, cancerous cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, filiform carcinoma, cancerous telangiectasia, cancerous telangiectasia, transitional cell carcinoma, nodular carcinoma, tuberous carcinoma, verrucous carcinoma, and villous carcinoma.

[0180] 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, Waldenström's macroglobulinemia, small cell lung tumors, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, pre-cancerous skin lesions, testicular tumors, lymphoma, thyroid cancer, esophageal cancer, genitourinary cancer, hypercalcemia of malignancy, cervical cancer, endometrial cancer, adrenocortical cancer, and prostate cancer.

[0181] In some embodiments, "tumor" includes, but is not limited to, prostate tumors, pancreatic tumors, squamous cell carcinomas, breast tumors, melanomas, basal cell carcinomas, hepatocellular carcinomas, cholangiocellular carcinomas, testicular tumors, neuroblastomas, gliomas or malignant astrocytic tumors such as glioblastoma multiforme, colorectal tumors, endometrial cancer, lung cancer, ovarian tumors, cervical tumors, osteosarcomas, rhabdomyosarcomas / smooth muscle sarcomas, synovial sarcomas, angiosarcomas, Ewing's sarcoma / PNET, and malignant lymphomas. These include primary tumors and metastatic tumors (both vascularized and non-vascularized).

[0182] As used herein, "treatment" or "therapy" generally means obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, for example, by reducing the risk of a subject having a particular disease or condition, by preventing the disease and / or condition completely or partially, or may be therapeutic, by considering curing the disease and / or its adverse effects partially or completely. In the present invention, "treatment" includes any treatment of a disease or condition in a mammal, particularly a human, such as the following treatments (a)-(c): (a) prevention of the onset of a disease, condition or symptom in a patient, (b) inhibition of the symptoms of a condition, i.e., prevention of the progression of the symptoms, (c) improvement of the symptoms of a condition, i.e., induction of regression of the disease or symptom.

[0183] As used herein, the term "administering" means providing to a subject a cell or liquid or composition containing the composition, and includes, but is not limited to, administration by a medical professional and self-administration. Administering a composition, substance, compound, or agent to a subject can be done using one of a variety of methods known to those of skill in the art. For example, a compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intravitreally, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracranially, and transdermally (by absorption, e.g., through a skin patch). A compound or agent can also be appropriately introduced by a rechargeable or biodegradable polymeric device, or other device that provides sustained, slow, or controlled release of the compound or agent, such as a patch or pump, or formulation. Administration can be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0184] The present invention encompasses the treatment of a patient by introducing a therapeutically effective number of cells, particularly the immunomodulatory NK cells of the present invention, into the bloodstream of the subject. As used herein, "introducing a 'cell' into the bloodstream of a subject" shall include, but is not limited to, introducing such cells into one of the subject's veins or arteries via injection. Such administration can be carried out, for example, once, multiple times, and / or over one or more extended periods of time. A single injection is preferred, although in some cases repeated injections (e.g., weekly, monthly, quarterly, semi-annually, or annually) over time may be required. Such administration is preferably carried out using a mixture of CD34-negative cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those of skill in the art and include, but are not limited to, 0.01 - 0.1 M, preferably 0.05 M, phosphate buffer or 0.8% saline, as well as commonly used proprietary cryopreservation media. Administration may also be carried out locally, for example, by injection into a region of the subject's body in proximity to a symptomatic organ or tissue.

[0185] Furthermore, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions, and may most preferably be aqueous solutions. Aqueous carriers include water, alcohol / aqueous solutions, emulsions and suspensions, including physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluids and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, and those based on Ringer's dextrose. Fluids commonly used for intravenous administration can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams & Wilkins (2000). Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, inert gases, etc., may also be present.

[0186] In one embodiment, a therapeutically effective number of cells is administered. This can relate to either the NK cells or the therapeutic immune cells of the present invention as a combined immunotherapy. As used herein, "therapeutically effective number of cells" includes, but is not limited to, the following amounts and ranges of amounts: (i) about 1×10 2 ~ about 1×10 8 cells / kg body weight, (ii) about 1×10 3 ~ about 1×10 7 cells / kg body weight, (iii) about 1×10 4 ~ about 1×10 6 cells / kg body weight, (iv) about 1×10 4 ~ about 1×10 5 cells / kg body weight, (v) about 1×10 5 ~ about 1×10 6 cells / kg body weight, (vi) about 5×10 4 ~ about 0.5×10 5 cells / kg body weight, (vii) about 1×10 3 cells / kg body weight, (viii) about 1×10 4 cells / kg body weight, (ix) about 5×10 4cells / kg body weight, (x) approximately 1×10 5 cells / kg body weight, (xi) approximately 5×10 5 cells / kg body weight, (xii) approximately 1×10 6 cells / kg body weight, and (xiii) approximately 1×10 7 cells / kg body weight. The assumed human body weights include, but are not limited to, approximately 5 kg, 10 kg, 15 kg, 30 kg, 50 kg, approximately 60 kg, approximately 70 kg, approximately 80 kg, approximately 90 kg, approximately 100 kg, approximately 120 kg, and approximately 150 kg. These numerical values are based on preclinical animal experiments and human tests from the transplantation of CD34+ hematopoietic stem cells as well as standard protocols. Mononuclear cells (including CD34+ cells) usually contain CD34-negative cells at a ratio of 1:23000 to 1:300000. These cell numbers can be applied to the NK cells of the present invention.

[0187] The present disclosure also includes kits, packages, and multi-container units of the ECP systems and their components described herein, as well as materials for practicing the in vitro and therapeutic methods of the present invention, and materials for preparing and isolating the NK cells of the present invention, and other materials.

[0188] Figure The present invention is further illustrated by the following figures. These are provided for a larger exemplification of the present invention described herein and do not intend to limit the scope of the present invention, but represent preferred embodiments of the aspects of the present invention.

Brief Description of the Drawings

[0189] [Figure 1-1]Refractory autoimmune colitis responds to ECP and leads to the proliferation of immunomodulatory NK cells. (a) Severity of diarrhea in patients according to the Common Terminology Criteria for Adverse Events (y-axis) over time (x-axis) with different treatments. (b) Colorectal endoscopic images (left panel) and H&E-stained biopsy sections (right panel) from the initial diagnosis of immune checkpoint inhibitor-related colitis showing mucosal edema and ulcers. (c) Colorectal endoscopic images (left panel) and H&E-stained biopsy sections (right panel) after successful ECP treatment showing no signs of autoimmune colitis. Colonic crypts show a regular morphology without granulocyte infiltration, apoptosis, or crypt loss. [Figure 1-2] Refractory autoimmune colitis responds to ECP and leads to the proliferation of immunomodulatory NK cells. (d) tSNE plot visualizing the peripheral lymphocyte compartments of a patient before and 8 weeks after the start of ECP. (e) Relative NK cell numbers before and after ECP treatment. [Figure 1-3] Refractory autoimmune colitis responds to ECP and leads to the proliferation of immunomodulatory NK cells. (f) Expression intensities of CD16, CD56, and CD57 in NK cells (defined as single / live / CD45pos / CD14neg / CD3neg / CD19neg / CD56pos) of healthy age-matched donors (HD, n = 5) and patients at week 53 and 75 after the start of ECP, visualized by UMAP. FlowSOM-clustered NK cell subsets are overlaid on the two plots on the right. [Figure 1-4] Refractory autoimmune colitis responds to ECP and leads to the proliferation of immunomodulatory NK cells. (g) MFI of CD16 on CD56dim mature NK cells (median marker expression, value range: 0 - 1). HD (n = 5), whiskers of the box plot represent the minimum and maximum values of the HD dataset. (h) Survival of mice injected with T cells (Tc) with or without NK cells (4×104 or 4×105 NK doses per mouse) in the model of anti-PD1 antibody-induced irAE described in the Supplementary Appendix. **p = 0.003, ****p < 0.0001. [Figure 2-1](a) tSNE plots showing 1000 randomly selected CD45+ lymphocytes (lymphocytes defined by FSC / SSC) at each time point, along with the expression of the indicated markers. (b) Heatmap showing the median marker expression (value range: 0 - 1) for each annotated population. [Figure 2-2] (c) Absolute cell numbers of NK cells before and after ECP treatment. (d) Relative cell numbers of B cells, CD4+ T cells, CD8+ T cells, and CD4 - CD8 - T cells before and after ECP treatment. (e) Absolute cell numbers of B cells, CD4+ T cells, CD8+ T cells, and CD4 - CD8 - T cells before and after ECP treatment. (f) Expression intensity of the indicated markers in NK cells (defined as single / live / CD45pos / CD14neg / CD3neg / CD19neg / CD56pos) in HD (n = 5) and patients at week 53 and week 75, visualized by UMAP. [Figure 2-3] (g) Heatmap showing the median marker expression (value range: 0 - 1) for the annotated NK cell subsets defined by FlowSOM clustering. (h - j) MFI (median marker expression, value range: 0 - 1) of the indicated markers on CD56brightCD16dim mature NK cells, CD56dim mature NK cells, and CD57dim terminally differentiated NK cells in healthy age - matched donors (HD) and patients (at week 53 and week 75 after ECP initiation). HD (n = 5), whiskers of the box plot represent the minimum and maximum values of the HD dataset. [Figure 3-1] (a) - (d) Percentages of cytokine - expressing cells in CD56bright NK cells, CD56dim NK cells, CD4+ memory T cells, CD4+ naive cells, and CD8+ T cells in healthy age - matched donors (HD) and patients (at week 53 and week 75 after ECP initiation). HD (n = 5), whiskers of the box plot represent the minimum and maximum values of the HD dataset. [Figure 3-2](a)–(d) Percentage of cytokine-expressing cells in CD56bright NK cells, CD56dim NK cells, CD4+ memory T cells, CD4+ naive cells, and CD8+ T cells of healthy age-matched donors (HD) and patients (53rd and 75th weeks after ECP initiation). HD (n = 5), whiskers of box plots represent the minimum and maximum values of the HD dataset. [Figure 4] (a) Experimental model to induce immune checkpoint inhibitor-related autoimmunity by adoptively transferring patient T cells alone or together with NK cells into Rag2− / −Il2rg− / − mice and treating with anti-PD-1 antibody. (b) Histopathological neutrophil and lymphocyte infiltration scores in the liver, skin, lung, and colon isolated from Rag2− / −Il2rg− / − mice on day 15 after injection of patient T and NK cells as shown in (a). [Figure 5] (a) Treatment scheme of Rag2− / −Il2rg− / − mice treated with anti-PD-1 antibody without injection of patient-derived cells (negative control). (b) Neutrophil infiltration scores in the liver, lung, skin, and colon isolated from Rag2− / −Il2rg− / − mice on day 15 after the start of treatment as shown in (a). (c) Survival of Rag2− / −Il2rg− / − mice treated as described in (a). [Figure 6-1] (a) Treatment schedule: As shown, mice were treated with DSS (3%), anti-PD1, and ECP. (b) Weight curves of mice untreated or treated with DSS (3%) alone or anti-PD1 in combination with ECP as shown. [Figure 6-2] (c) Colonic length quantified in 5 mice per group. Groups as shown in panel B. (d) Representative colon of 1 mouse per group. Groups as shown in panel B. [Figure 6-3] (e) Representative HE-stained sections of the colon of the groups shown in panel B. (f) Histopathological scores of the colon of the groups shown in panel B. [Figure 7-1] (a) Treatment schedule: Mice were intravenously injected with B16 melanoma cells and then treated with anti-PD1, prednisolone, or ECP as shown. [Figure 7-2](b) Survival of mice injected intravenously with B16 melanoma cells and then treated with anti-PD1, prednisolone, or ECP.

Example

[0190] The present invention is further illustrated by the following examples. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of aspects of the invention provided for greater exemplification of the invention described herein.

[0191] Here, we report a patient with ipilimumab / nivolumab-induced colitis who was refractory to multiple immunosuppressive drugs and achieved complete remission after extracorporeal photochemotherapy (ECP) concomitant with the expansion of immunomodulatory natural killer (NK) cells.

[0192] Results of the Example A 29-year-old male patient was treated with ipilimumab and nivolumab for metastatic melanoma. After two doses, the patient developed dermatitis, thyroiditis, hepatitis, and colitis. Colitis was diagnosed based on macroscopic mucosal ulcers identified by biopsy and intraepithelial apoptosis and crypt loss (Figure 1a, b). Dermatitis, thyroiditis, and hepatitis resolved after discontinuation of ipilimumab / nivolumab and corticosteroid treatment, but the patient experienced three episodes of colitis (CTC-AE II-III°) within the next 20 weeks. These were treated with corticosteroids (23 weeks before the total of ECP), infliximab (two single doses 18 weeks and 15 weeks before ECP), and cyclosporine (14 weeks before ECP, Figure 1a and Table 1).

[0193] Due to the lack of a sustained response, the patient received ECP. Over the next eight months, the patient received two cycles of ECP every two to four weeks for consecutive days. ECP was well tolerated and resulted in complete remission (Figure 1a). Immunosuppression tapered off without symptom rebound. Continued remission of colitis was confirmed by colonoscopy (Figure 1c). Immunotherapy with immune checkpoint inhibitors was discontinued at the first signs of irAE and was not restarted.

[0194] The peripheral blood leukocyte compartment was analyzed at multiple time points before and during ECP treatment. We observed a four-fold increase in NK cells (Figs. 1d–e, 2a–j) using an immunomodulatory phenotype (Figs. 1f, g). Also, multiple pro-inflammatory cytokines were lower in patients compared to age-matched healthy donors (Figs. 3a–e). The idea that NK cells regulate autoimmunity was supported, and adoptive transfer of patient NK cells dose-dependently prevented irAE in a mouse irAE model induced by human T cells and anti-PD-1 antibody treatment (Figs. 1h and 4a, b). Control experiments confirmed that the morbidity was mediated by patient-derived T cells (Fig. 5).

[0195] ECP is an established therapy for the treatment of graft-versus-host disease (GVHD) 4 that leads to an increase in NK cells in GVHD patients 5 . Data on the safety and efficacy of ECP in IrAE treatment are currently lacking. This case report suggests that ECP may be an effective therapy for refractory checkpoint inhibitor-related colitis through the expansion of the protective NK cell population.

[0196] ECP reduces immune-mediated adverse events without blocking the anti-melanoma effect Based on the above results (Figs. 1-5), ECP aimed to reduce immune-mediated adverse events without blocking the anti-melanoma effect and then test this in an in vivo model of irAE. To induce colitis, mice were treated with 3% DSS and anti-PD1 according to a previous report (19) (Fig. 6A). Treatment decreased the body weight of the mice, which coincided with the onset of colitis, and the weight loss was reduced by ECP treatment (Fig. 6B). ECP treatment also increased the colon length compared to the group treated with anti-PD1 alone (Figs. 6C, D). The colon length has been reported to be a surrogate parameter for the severity of immunotherapy-induced colitis (19). Consistent with the decreased colitis severity, we observed a reduction in neutrophil infiltration in the colon wall of mice treated with ECP compared to the group treated with anti-PD1 alone (Figs. 6E, F). These findings indicate that ECP reduces anti-PD1-induced colitis in mice.

[0197] To understand whether the immunomodulatory effect of ECP is associated with the loss of anti-tumor activity, next, mice with melanoma were treated with anti-PD1 alone or in combination with the glucocorticoid prednisolone or ECP (Fig. 7A). We observed that prednisolone decreased the survival of mice with melanoma compared to the group treated with anti-PD1 alone (Fig. 7B). In contrast, the group treated with anti-PD and ECP had an outcome equivalent to that of the group treated with anti-PD1 alone (Fig. 7B). These findings indicate that ECP does not interfere with the anti-melanoma response induced by anti-PD1 treatment.

[0198] Discussion of the Examples Discussion of the mechanism: NK cells exert various heterogeneous immunological functions, including anti-inflammatory activity. In a mouse model of GVHD, the transcription of NK cells improved the survival rate that was dependent on intact TGF-β signaling. 6 In another preclinical GVHD study, NK cells induced a decrease in perforin- and Fas ligand-mediated alloreactive T cell proliferation and an increase in T cell apoptosis. 7 c-Kit - CD27 - CD11b+ identified a specific subset of effector NK cells that can control GVHD without interfering with the graft-versus-leukemia (GVL) effect 8 In humans, mismatch of killer cell immunoglobulin-like receptor (KIR) ligands in allogeneic HCT in the GVH direction reduced the risk of NK cell-mediated GVHD 9 .

[0199] ECP is an effective treatment for GVHD. An increase in NK cells during ECP for extensive chronic GVHD has been previously observed 10 Patients with acute GVHD have a higher frequency of CD56 bri NK subsets with stronger NKG2D and CD62L expression 11 . In the same study, in patients with chronic cGVHD, CD56 with higher expression of CD57 and CD11b - CD16 + NK cells increased. ECP shifted the NK cell population to a more immunomodulatory phenotype with protection of specialized antiviral and anti-leukemia CD57 + NKG2C + CD56 dim subsets 11 . We hypothesize that a similar mechanism may be involved in the protective effect of NK cells against irAE. When comparing the NK cell compartment in patients with that of age- and sex-matched healthy controls, we observed downregulation of CD16 expression, particularly in CD56 dim NK cells. CD16 is the FcRγIII, an activating NK cell receptor that can induce strong cytokine production. Previous studies have shown that shedding of CD16 may be an immunoregulatory mechanism to prevent autoimmunity 12 . CD16 downregulation regulates NK cell responses and contributes to the maintenance of immune homeostasis in both antibody- and T cell-dependent pathways 13 . Supporting this hypothesis, the expression of GM-CSF, IFN-γ, TNF, and IL-2 was lower in the NK cells of patients compared to the control group

[0200] Method used in the examples ECP procedure: Extracorporeal photochemotherapy was performed on the Therakos CellEx photochemotherapy system after administration of methoxysalen (Uvadex (copyright)). Two procedures were performed on consecutive days, and 1500 ml of blood was processed during each procedure. We collected all human samples after obtaining approval from the Ethics Committee of the Albert Ludwigs University in Freiburg, Germany (protocol number 300 / 16) and written informed consent in accordance with the Declaration of Helsinki.

[0201] Mouse model of IrAE T cells were isolated from the peripheral blood of patients using negative selection with a Pan T cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. NK cells were isolated from the peripheral blood of patients using an NK cell isolation kit, human (Miltenyi Biotec) according to the manufacturer's instructions. Rag2 - / - Il2rg - / - Mice were injected intravenously with 3 × 10 4 or 3 × 10 5 T cells with or without 4 × 10 5 NK cells. From day 1 to day 22 after injection, the mice were treated twice a week with 8 mg / kg body weight of anti-PD-1 antibody (clone J43) and once a week with 1 mg / kg body weight of LPS, both applied by intraperitoneal injection (Figure 4a). Sections of skin, liver, colon, and lung collected on day 15 after human T cell injection were stained with hematoxylin-eosin and scored based on histopathological characterization of human irAE including lymphocyte and neutrophil infiltration, crypt abscesses, and apoptotic cells 14,15 All animal experiments were approved by the University's Institutional Review Board for the Use and Care of Laboratory Animals at the Albert-Ludwigs University in Freiburg, Germany (protocol approval numbers: G17-049, X13-07J, X15-10A).

[0202] Flow cytometry For monitoring lymphocyte subsets during and after ECP therapy, peripheral blood lymphocytes from patients were isolated and stained with a standard panel of antibodies against CD45, CD19, CD3, CD4, CD8, CD16, CD56, and HLA-DR as part of routine diagnosis. Data were compensated with FlowJo (V10), lymphocytes were exported, and the R environment was used 17 . tSNE and FlowSOM clustering were performed as described above 16 .

[0203] For multiparameter NK cell phenotype and cytokine analysis, peripheral blood lymphocytes were isolated using density gradient medium (Lymphoprep, STEMCELL Technologies) according to the manufacturer's instructions. Thawed peripheral blood lymphocytes were stained with the antibodies listed in Table S2. The Zombie Aqua Fixable Viability kit (Biolegend) was used for live / dead discrimination. For cytokine production, cells were stimulated with 50 ng / ml PMA (Axon Lab) and 500 mg / ml ionomycin (Sigma) for 4 hours in the presence of GolgiPlug (BD Biosciences). Intracellular staining was performed using the BD Cytofix / Cytoperm kit (BD Biosciences) according to the manufacturer's protocol. Data were acquired on an Aurora flow cytometer (Cytek) and compensated using FlowJo (Flowjo V10.6.1, LLC) software. The cell populations designated in the figures were exported and analyzed using the R environment 17 . Data were processed for FlowSOM clustering as described 16 . The UMAP package was used for dimensionality reduction 18 .

[0204] Statistics Statistical analysis was performed using GraphPad Prism Lab Software V7.0. Comparison between two groups was carried out by two-sided unpaired Student's t-test. The difference in survival time (Kaplan–Meier survival curves) was evaluated using the Mantel–Cox (log-rank) test. Unless otherwise indicated, data are presented as mean ± SEM. A P value < 0.05 was considered significant.

[0205] Table of Examples

Table 1

[0206] The patient had the first episode of colitis 20 weeks before the start of ECP. At that time, the patient had been treated with steroids for 3 weeks due to previous immune checkpoint inhibitor-related hepatitis, thyroiditis, and dermatitis. Colitis developed during the tapering of steroids. Therefore, the steroid dose was increased, and infliximab 5 mg / kg BW was administered once because the response was insufficient. The symptoms subsided. During the tapering of steroids, the patient experienced a second episode of colitis 16 weeks before the start of ECP. The patient was treated with increased doses of methylprednisolone and a second dose of infliximab. Diarrhea was refractory to this therapy, and as a result, cyclosporine A was added. The symptoms subsided again. As the cyclosporine A dose decreased, the patient had a third episode of colitis. Here, ECP treatment was initiated. Two weeks after the start of ECP, the patient had a normal bowel frequency. Cyclosporine A treatment was discontinued 8 weeks after the start of ECP, and corticosteroid treatment was discontinued 12 weeks after the start of ECP, and there was no rebound of any symptoms. ECP was performed for a total of 32 weeks. At the 11-month follow-up from the last ECP, the patient maintained complete remission regarding both signs of irAE and melanoma.

Table 2-1

Table 2-2

[0207] References 1. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med 2015;373:23 - 34. 2. Wolchok JD, Chiarion-Sileni V, Gonzalez R, et al. Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med 2017;377:1345 - 56. 3. Postow MA, Chesney J, Pavlick AC, et al. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N Engl J Med 2015;372:2006 - 17. 4. Zeiser R, Blazar BR. Acute Graft-versus-Host Disease - Biologic Process, Prevention, and Therapy. N Engl J Med;2017;377:2167 - 79. 5. Ni M, Wang L, Yang M, et al. Shaping of CD56(bri) Natural Killer Cells in Patients With Steroid-Refractory / Resistant Acute Graft-vs.-Host Disease via Extracorporeal Photopheresis. Front Immunol 2019;10:547. 6.Asai O,Longo DL,Tian ZG,et al.Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation.J Clin Invest 1998;101:1835-42. 7.Olson JA,Leveson-Gower DB,Gill S,Baker J,Beilhack A,Negrin RS.NK cells mediate reduction of GVHD by inhibiting activated,alloreactive T cells while retaining GVT effects.Blood 2010;115:4293-301. 8.Meinhardt K,Kroeger I,Bauer R,et al.Identification and characterization of the specific murine NK cell subset supporting graft-versus-leukemia-and reducing graft-versus-host-effects.Oncoimmunology 2015;4:e981483. 9.Ruggeri L,Capanni M,Urbani E,et al.Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants.Science 2002;295:2097-100. 10. Alcindor T, Gorgun G, Miller KB, et al. Immunomodulatory effects of extracorporeal photochemotherapy in patients with extensive chronic graft-versus-host disease. Blood 2001;98:1622-5. 11. Ni M, Wang L, Yang M, et al. Shaping of CD56(bri) Natural Killer Cells in Patients With Steroid-Refractory / Resistant Acute Graft-vs.-Host Disease via Extracorporeal Photopheresis. Front Immunol 2019;10: 547. 12. Romee R, Foley B, Lenvik T, et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17(ADAM17). Blood 2013;121:3599-608. 13. Goodier MR, Lusa C, Sherratt S, Rodriguez-Galan A, Behrens R, Riley EM. Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK Cell Function. Front Immunol 2016;7:384. 14. Beck KE, Blansfield JA, Tran KQ, et al. Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4. J Clin Oncol 2006;24:2283-9. 15. Johncilla M, Misdraji J, Pratt DS, et al. Ipilimumab-associated Hepatitis: Clinicopathologic Characterization in a Series of 11 Cases. Am J Surg Pathol 2015;39:1075-84. 16. Brummelman J, Haftmann C, Nunez NG, et al. Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry. Nat Protoc 2019;14:1946-69. 17. Team RDC. A language and Environment for Statistical Computing. R Foundation for Statistical Computing 2010. 18. Mcinnes L, Healy J, Melville J. UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. arXiv 6,03426v03422(2018). 19. Perez-Ruiz E, Minute L, Otano I, Alvarez M, et al. Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy. Nature. 2019 May;569(7756):428-432. The present invention provides, for example, the following items. (Item 1) - A step of providing a sample derived from a blood sample of a subject who has received checkpoint inhibitor therapy and is suspected of having or has developed symptoms of immune-related adverse events (irAE); - A step of adding a photosensitizer to the sample; - A step of subjecting the sample to irradiation, a method comprising the steps. (Item 2) The method according to item 1, wherein by adding a photosensitizer to the sample and subjecting the sample to irradiation, immunomodulatory NK cells in the sample are generated or induced (the formation thereof). (Item 3) The method according to any one of the preceding items, wherein the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation. (Item 4) The method according to any one of the preceding items, wherein the subject exhibits symptoms of irAE or suffers from irAE. (Item 5) After the symptoms and / or signs of irAE have occurred in the subject, the checkpoint inhibitor therapy has been discontinued, or after the checkpoint inhibitor therapy has been discontinued, the symptoms and / or signs of irAE have occurred, and / or after the checkpoint inhibitor therapy has been discontinued, the symptoms and / or signs of irAE have persisted, the method according to any one of the preceding items. (Item 6) The method according to any one of the preceding items, wherein the irAE includes symptoms of an autoimmune disease and / or is caused by an autoimmune reaction. (Item 7) The method according to any one of the preceding items, wherein the irAE includes autoimmune colitis. (Item 8) The method according to any one of the preceding items, wherein the subject suffers from cancer such as melanoma or another cancer treatable by checkpoint inhibitor therapy. (Item 9) The method according to any one of the preceding items, wherein the subject is receiving an immunosuppressive drug such as a steroid, corticosteroid, cyclosporine, and / or an anti-TNF antibody (e.g., infliximab) and / or is refractory to the immunosuppressive drug. (Item 10) The method according to any one of the preceding items, wherein the checkpoint inhibitor therapy comprises administration of at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody. (Item 11) - A step of providing a sample derived from an isolated blood sample of a subject; - A step of adding a photosensitizer to the sample; - A step of subjecting the sample to irradiation, an immunomodulatory NK cell obtained from a method comprising the steps. (Item 12) The immunomodulatory NK cell according to any one of the preceding items, wherein the blood sample is derived from a subject who has received a checkpoint inhibitor therapy and is suspected of having developed or has developed symptoms of an immune-related adverse event (irAE). (Item 13) An immunomodulatory NK cell for use in the treatment and / or prevention of an immune-related adverse event (irAE) in a subject who has received a checkpoint inhibitor therapy. (Item 14) The immunomodulatory NK cell for use according to any one of the preceding items, wherein the immunomodulatory NK cell is generated by adding a photosensitizer to a sample derived from a blood sample of a human subject and subjecting the sample to irradiation. (Item 15) The immunomodulatory NK cell for use according to item 13 or 14, wherein the NK cell is autologous to the subject and / or the NK cell is administered to the subject while the checkpoint inhibitor therapy is in progress.

Claims

**Claim 1** A method for producing immunomodulatory NK cells, the method comprising: - providing a sample derived from a blood sample of a subject who has received checkpoint inhibitor therapy and exhibits or suffers from symptoms of immune-related adverse events (irAE) including autoimmune colitis; - adding 8-methoxypsoralen to the sample; - subjecting the sample to UVA irradiation, wherein adding 8-methoxypsoralen to the sample and subjecting the sample to UVA irradiation generates immunomodulatory NK cells in the sample or induces the formation of immunomodulatory NK cells in the sample. **Claim 2** The method according to claim 1, wherein the checkpoint inhibitor therapy is discontinued after the occurrence of symptoms and / or signs of irAE in the subject. **Claim 3** The method according to claim 1, wherein the symptoms and / or signs of irAE occur after the discontinuation of the immune checkpoint inhibitor therapy. **Claim 4** The method according to claim 1, wherein the symptoms and / or signs of irAE are maintained after the discontinuation of the immune checkpoint inhibitor therapy. **Claim 5** The method according to any one of claims 1 to 4, wherein the subject is suffering from cancer. **Claim 6** The method according to any one of claims 1 to 5, wherein the subject is suffering from melanoma or another cancer treatable by checkpoint inhibitor therapy. **Claim 7** The method according to any one of claims 1 to 6, wherein the subject is receiving an immunosuppressive drug. **Claim 8** The method according to claim 7, wherein the immunosuppressive drug is a steroid, corticosteroid, cyclosporine, anti-TNF antibody, or any combination thereof. **Claim 9** The method according to claim 8, wherein the anti-TNF antibody is infliximab. **Claim 10** The method according to any one of claims 1 to 6, wherein the subject is refractory to the immunosuppressive drug. **Claim 11** The method according to any one of claims 1 to 10, wherein the checkpoint inhibitor therapy comprises administration of at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody.

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