Method for generating immunoregulatory cells in blood-derived sample

Abstract

To provide a method for generating immunoregulatory cells in a blood-derived sample.SOLUTION: The present invention relates to a method comprising the steps of providing a sample derived from a blood sample of a subject that has received checkpoint-inhibitor therapy and is suspected of developing, or has developed, symptoms of immune-related adverse events (irAE); adding a photosensitizing agent to the sample; and subjecting the sample to irradiation, preferably generating immunoregulatory NK cells in the sample. In embodiments, the photosensitizing agent 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 a subject, adding a photosensitizing agent to the sample, and subjecting the sample to irradiation.SELECTED DRAWING: None

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 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. [Background technology]

[0002] Immune checkpoints are regulatory molecules of the immune system that play a critical role in maintaining immune homeostasis and self-tolerance. The first immune checkpoints identified included 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 and binds to B7-1 (CD80) or B7-2 (CD86) molecules on antigen-presenting cells, functioning as a negative regulator of T cells. PD-1 also negatively influences T cell activity through interactions with its ligands, including programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2). Unlike CTLA-4, PD-1 is not only found on T cells but is 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 cancerous cells interacts with PD-1 on the surface of T cells to inhibit T cell effector function. Furthermore, several studies have shown that high tumor expression of PD-L1 significantly correlates with poor cancer prognosis. These studies suggest that blockade of the PD-1 signaling pathway in cancer may be therapeutically effective.

[0004] Recent clinical trials have demonstrated that several anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors (ICIs) are effective against a variety of cancers, including melanoma, non-small cell lung cancer, renal cell carcinoma, and head and neck cancer. Additional clinical trials to expand the efficacy of ICIs are currently underway. To date, the U.S. Food and Drug Administration (FDA) has approved three anti-PD-1 antibodies, nivolumab, pembrolizumab, and cemiplimab, and three anti-PD-L1 inhibitors, atezolizumab, avelumab, and durvalumab, for the treatment of different types of cancer.

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

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

[0007] In summary, checkpoint inhibitor therapy, particularly anti-CTLA4 and anti-PD-1 antibody therapy, is an effective treatment for multiple forms of cancer, especially melanoma. However, treatment can be associated with substantial side effects that may result from immune system activation caused by checkpoint inhibitor therapy. These side effects can lead to autoimmune reactions and manifest in different clinical manifestations, summarized as irAEs. A prominent irAE is autoimmune colitis. Side effects of treatment, such as autoimmune reactions, particularly autoimmune colitis, may persist even after checkpoint inhibitor therapy is stopped.

[0008] Thus, there is a need in the art for alternative or improved means for treating immune-related adverse events such as autoimmune reactions, particularly autoimmune colitis, in patients receiving checkpoint inhibitor therapy. Summary of the Invention [Means for solving the problem]

[0009] In light of the prior art, the technical problem underlying the present invention is to provide improved or alternative means for the treatment and / or prevention of immune-related adverse events, such as autoimmune reactions, in particular 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 provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - 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; - subjecting the sample to irradiation.

[0012] The present invention is based on the completely surprising finding that patients who have undergone checkpoint inhibitor therapy, e.g., for cancerous diseases, and who have developed irAEs, particularly autoimmune colitis, can be effectively treated by applying ECP. The ECP used in the examples essentially corresponds to a method in which a photosensitizer is added to a blood-derived sample from an irAE patient and the sample is subjected to irradiation. Surprisingly, it has been found that preforming this method on a blood sample, particularly a blood sample containing mononuclear cells (MNCs), leads to the generation of immunoregulatory NK cells in said sample. In this context, "generation" of immunoregulatory NK cells is understood as inducing or inducing the formation of such cells in the sample. In other words, cells contained in the sample differentiate into or adopt the phenotype of immunoregulatory NK cells.

[0013] It has been found that samples resulting from the methods of the present invention, particularly the induced immunoregulatory NK cells contained therein, are useful for treating patients with irAEs. Administration of such samples or cells contained in such samples to subjects undergoing checkpoint inhibitor therapy can surprisingly be shown to be effective in preventing the occurrence of irAEs and also effective in treating irAEs already established in the subject. Importantly, the methods of the present invention can be performed on samples from the same subjects undergoing checkpoint inhibitor therapy. Thus, cells or samples resulting from the methods of the present invention can be administered to the same subjects who served as blood donors, and the resulting samples can therefore represent autologous cell therapy.

[0014] As used herein, the term "subject receiving checkpoint inhibitor therapy" includes a subject who is currently receiving checkpoint inhibitor therapy or a subject who has received checkpoint inhibitor therapy that has been discontinued, e.g., after the onset of irAE symptoms.

[0015] It was entirely unexpected that such cell therapy using samples or cells resulting from the methods of the present invention would be effective even in irAE patients who are refractory to other immunosuppressive therapies, such as steroids or anti-TNF antibodies, and who continue to show symptoms of or suffer from irAEs after checkpoint inhibitor treatment has been discontinued.

[0016] As shown in the examples, the positive effects and efficacy of administering a sample subjected to the method of the present invention may be at least partially attributed to modulation of NK cell function of NK cells contained in the sample, said modulation being the result of irradiation in the presence of a photosensitizer. Thus, the present invention also encompasses immunoregulatory NK cells for use in treating and / or preventing immune-related adverse events (irAEs) in subjects receiving checkpoint inhibitor therapy. Preferably, the immunoregulatory NK cells are generated by subjecting blood or MNC or NK cells from an individual, preferably a subject receiving checkpoint inhibitor therapy and suspected of developing or having developed irAEs, to the method of the present invention, and then used to treat a subject, preferably a donor, receiving checkpoint inhibitor therapy and suspected of developing or having developed irAEs.

[0017] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: - providing a sample derived from an isolated blood sample of a subject; - adding a photosensitizer to the sample; - subjecting the sample to irradiation.

[0018] This aspect of the invention is based on the observation that NK cells are present in blood-derived samples irradiated after the addition of a photosensitizer, and adopt an immunomodulatory phenotype that is advantageous for the use of the resulting cells in the treatment and / or prevention of irAEs, a property that has not previously been observed for any other NK cells 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 derived from a subject receiving checkpoint inhibitor therapy and suspected of developing or having developed symptoms of an immune-related adverse event (irAE). This embodiment is particularly advantageous because the cells are autologous to the patient, eliminating the adverse events that can occur when transplanting xenogeneic 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 receiving checkpoint inhibitor therapy, including subjects currently receiving checkpoint inhibitor therapy or subjects who have received checkpoint inhibitor therapy that has been discontinued, e.g., after the onset of irAE symptoms.

[0022] Preferably, immunoregulatory NK cells for use in such treatment or prevention have been 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 immunoregulatory NK cells in said sample.

[0024] In embodiments, the photosensitizer is 8-methoxypsoralen. Further, in embodiments, the radiation is UVA radiation. In preferred embodiments, the photosensitizer is 8-methoxypsoralen and the radiation is UVA radiation.

[0025] In the context of the present invention, the irradiation is preferably preformed by an extracorporeal photochemotherapy (ECP) system. The irradiation of the blood sample can be carried out 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 carried out in vitro or ex vivo. As used herein, the terms in vivo and ex vivo are used interchangeably. In the context of the present invention, the terms refer to a method carried out on a sample derived from blood removed from a human body, while the method of the present invention is carried out on a sample derived from blood outside the human body. For this purpose, the blood of a donor subject is removed extracorporeally, which means removing blood from the physiological circulatory system. As used herein, an "isolated blood sample" is a sample of blood (meaning a certain amount of blood) removed from a donor's circulatory system to a location outside the human body.

[0027] Such an isolated blood sample can be provided or introduced into an extracorporeal photochemotherapy (ECP) system or apheresis system for carrying out at least certain steps of the method of the present invention. In one embodiment, such a system can be an online system in fluid connection with the subject's blood circulatory system. In an alternative embodiment, the method can be carried out offline, with the blood-derived sample to be irradiated being disconnected from the donor subject's circulatory system.

[0028] Thus, 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 type of blood-derived sample, including blood cell samples such as MNC samples generated from blood.

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

[0031] In embodiments of the present invention, when a subject undergoing checkpoint inhibitor therapy and suspected of developing or having developed symptoms of an irAE serves as a blood donor, blood sample donation and / or isolation can occur at any of the time points described in the above embodiments. For example, sample isolation can occur before or after the onset of irAE symptoms. Furthermore, sample isolation can occur during checkpoint inhibitor therapy or after cessation of said therapy.

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

[0033] In a preferred embodiment, sample isolation occurs during the course of checkpoint inhibitor therapy, either before or after the onset of irAE symptoms. Samples or cells resulting from the exemplary methods 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 present invention, a subject undergoing checkpoint inhibitor therapy can receive immunoregulatory NK cells, preferably autologous and generated by irradiating a blood-derived sample according to the methods described herein, while undergoing checkpoint inhibitor therapy. Such embodiments are particularly advantageous, as checkpoint inhibitor therapy, such as anti-cancer checkpoint inhibitor therapy, can be maintained while the patient receives the cells of the present invention and / or cells resulting from the methods of the present invention.

[0034] Thus, in such embodiments, the administration of irradiated cells either prevents the occurrence of irAEs or ameliorates irAEs, allowing checkpoint inhibitor therapy to be continued, allowing checkpoint inhibitor therapy to be administered to the subject for a longer period of time. Surprisingly, it can be shown that the methods of the present invention, particularly the administration of such cells derived from the immunoregulatory NK cells of the present invention, do not interfere with the subject's anti-cancer response to checkpoint inhibitor therapy. This represents a significant advantage compared to known treatment / prevention measures for irAEs, particularly the administration of immunosuppressants such as corticosteroids. Surprisingly, performing ECP did not significantly alter the anti-cancer effect of checkpoint inhibitor therapy, while concurrent administration of glucocorticoids (a certain class of corticosteroids), particularly prednisolone, resulted in worse outcomes, indicating reduced efficacy of checkpoint inhibitor therapy.

[0035] Furthermore, in embodiments in which a (human) blood-derived sample that has undergone a method of adding a photosensitizer to the sample and subjecting the sample to irradiation is used to treat and / or prevent irAEs in a subject undergoing checkpoint inhibitor therapy, regardless of the source of the blood-derived sample (autologous or xenogeneic), administration of the sample or cells resulting from such a method can occur, for example, before or after the onset of irAE symptoms and / or during checkpoint inhibitor therapy or after cessation of checkpoint inhibitor therapy.

[0036] In embodiments of the invention, the irAE (in the blood donor and / or cell recipient) comprises symptoms of an autoimmune disease and / or is caused by an autoimmune response. In preferred embodiments, the irAE comprises or is autoimmune colitis.

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

[0038] In embodiments, the irAE comprises at least one irAE selected from the group comprising immune checkpoint inhibitor-associated colitis, immune checkpoint inhibitor-associated hepatitis, immune checkpoint inhibitor-associated thyroiditis, and immune checkpoint inhibitor-associated dermatitis.

[0039] In embodiments, the subject (blood donor and / or cell recipient) is afflicted with cancer, such as malignant 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 administration of cells generated 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 their irAE symptoms. For such patients, there are no effective treatments available to ameliorate irAE symptoms, and therefore the present invention represents a totally unexpected possibility for the treatment of irAEs in these patients.

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

[0043] In embodiments, immunomodulatory NK cells for use in treating and / or preventing irAEs in a subject receiving checkpoint inhibitor therapy are autologous to the subject. In alternative embodiments, the NK cells may be xenogeneic to the subject.

[0044] In embodiments, the immunoregulatory NK cells for use according to the present invention are administered at the onset of irAE symptoms. In embodiments, 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 embodiments, the immunoregulatory NK cells are administered during the progression of checkpoint inhibitor therapy or after checkpoint inhibitor therapy has been discontinued.

[0045] In embodiments, the immunoregulatory NK cells are administered to a subject at least once, preferably at least twice, preferably daily. Thus, a subject can receive more than one dose of NK cells, but preferably not more than one dose per day. In further embodiments, a subject receives at least two, three, four, or five doses of the NK cells of the present invention. In embodiments, the immunoregulatory NK cells of the present invention are administered to a subject at least every 8 weeks, preferably every 2 to 4 weeks.

[0046] Each optional or preferred feature of the invention that is disclosed or described in the context of one aspect of the invention is also disclosed herein in the context of any other aspect of the invention described herein.

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

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

[0049] The present invention is based on the entirely surprising finding that patients who have undergone checkpoint inhibitor therapy, e.g., for cancer disease, and who have developed irAEs, particularly autoimmune colitis, can be effectively treated by applying ECP, even in patients who are refractory to other immunosuppressive therapies, such as steroids or anti-TNF antibodies, and who continue to show symptoms of or suffer from irAEs after checkpoint inhibitor treatment has been discontinued.

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

[0051] 2. An ECP system for use in accordance with the present invention, wherein the ECP system is an online ECP system.

[0052] 3. An ECP system for use in accordance with 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 is symptomatic of or suffering from an irAE.

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

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

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

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

[0059] 10. An ECP system for use according to any of the present invention, wherein ECP treatment is initiated while checkpoint inhibitor therapy is ongoing or after 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 one, preferably at least two, cycles of ECP, preferably on consecutive days.

[0061] 12. An ECP system for use in accordance with the present invention, wherein the ECP treatment is administered at least 2, 3, 4, or 5 times.

[0062] 13. An ECP system for use in accordance with the present invention, wherein ECP treatment is administered 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 response.

[0065] 16. An ECP system for use according to the present invention, wherein the irAE comprises at least one irAE selected from the group comprising 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 comprising immune checkpoint inhibitor-associated colitis, immune checkpoint inhibitor-associated hepatitis, immune checkpoint inhibitor-associated thyroiditis, and immune checkpoint inhibitor-associated 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 in accordance with the present invention, wherein the subject is a human.

[0070] 21. An ECP system for use in accordance with the present invention, wherein the subject is afflicted with cancer, such as malignant melanoma or another cancer treatable by checkpoint inhibitor therapy.

[0071] 22. An ECP system for use in accordance with the present invention, wherein the subject is receiving and / or is refractory to immunosuppressive drugs such as steroids, corticosteroids, cyclosporine, and / or anti-TNF antibodies (e.g., infliximab).

[0072] 23. An ECP system for use according to the present invention, wherein the subject is refractory to immunosuppressive drugs such as steroids, corticosteroids, cyclosporine and / or anti-TNF antibodies (e.g., infliximab).

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

[0074] 25. An ECP system for use in accordance with the present invention, wherein the ECP system is used to perform blood irradiation therapy.

[0075] 26. An ECP system for use in accordance with the present invention, wherein the ECP system comprises an immunomodulatory molecule, preferably an immunomodulatory molecule, bound to a membrane of the ECP system, and wherein the immunomodulatory molecule contacts immune cells of a subject.

[0076] 27. An in vitro method comprising: - Providing a sample derived from an isolated blood sample of a subject receiving checkpoint inhibitor therapy and suspected of developing or having developed symptoms of an immune-related adverse event (irAE); - subjecting said sample to extracorporeal photochemotherapy.

[0077] 28. A method of treating a subject receiving checkpoint inhibitor therapy and suspected of developing or having developed symptoms of an immune-related adverse event (irAE), comprising subjecting the subject to extracorporeal photochemotherapy (ECP) therapy (ECP), such as blood irradiation therapy with an ECP system.

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

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

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

[0081] 32. Immunoregulatory NK cells for use according to the present invention, wherein the NK cells are administered intravenously to said 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 subjection to extracorporeal photochemotherapy.

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

[0084] All cited references, both patent and non-patent literature, are incorporated herein 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 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. Irradiation of the blood sample can be performed using any suitable system or irradiation device known to those skilled in the art. Preferably, irradiation is performed by an extracorporeal photochemotherapy (ECP) system.

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

[0087] Radiation and extracorporeal photochemotherapy (ECP) Photochemotherapy, or extracorporeal photochemotherapy (ECP), is a form of apheresis and photodynamic therapy in which blood is treated with a photosensitizer and then irradiated with specific wavelengths of light to achieve its effects. For example, buffy coat (WBCs + platelets) can be separated from whole blood, chemically treated with 8-methoxypsoralen (either dripped into a collection bag or administered several times in advance), exposed to ultraviolet (UVA) light, and returned to the patient. Activated 8-methoxypsoralen crosslinks DNA within exposed cells, ultimately leading to apoptosis of nucleated cells. 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 developed in 1987 by New England Journal of Medicine publication (Edelson, R, et al. (1987) "Treatment of This was first described in "Cutaneous T-cell lymphoma by extracorporeal photochemotherapy. Preliminary results," New England Journal of Medicine. 316(6):297-303. Photochemotherapy is currently the standard of care approved by the U.S. Food and Drug Administration (FDA) for cutaneous T-cell lymphoma. It has been suggested that this treatment may be effective in treating graft-versus-host disease. Photochemotherapy has also been successful in treating epidermolytic bullous disease when all other treatments have failed.

[0089] As used herein, ECP includes blood irradiation therapy. In embodiments, the ECP and ECP system 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 levels of red light, often laser light, for therapeutic reasons. Blood irradiation therapy can be administered in three ways: externally, by drawing blood and irradiating it in a special cuvette; this method is used for ultraviolet (UV) blood irradiation (UVBI) with a UV lamp; laser light is monochromatic, i.e., has a wavelength such that the light can be coupled into an optical fiber and used for intravenous irradiation via a catheter in a vein; this method is simpler and more effective; blood irradiation therapy can also be administered externally through the skin over the projections of large blood vessels.

[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 powered at 1–3 mW, into vascular channels, usually veins in the forearm, under the assumption that any therapeutic effect will circulate through the circulatory system. Most commonly, wavelengths of 365, 405, 525, or 635 nm and a power of 2.3 mW are used. This technique is currently widely used in Russia, but less commonly in Asia and not widely used in other parts of the world. ILBI has been shown to improve blood flow and its transport activity, and thus tissue trophism, favorably affecting the immune system and cellular metabolism. This issue is subject to skepticism. There has been some demand for increased research on this topic. Transdermal therapy involves applying laser light to unbroken skin in areas with numerous blood vessels (such as the forearm). Because the skin acts as a barrier to the blood and absorbs low levels of laser energy, the power of the laser is often increased to compensate. The problem can be solved by using a pulsed matrix laser source. External beam irradiation is used exclusively for ultraviolet blood irradiation, which involves drawing blood through veins and irradiating it outside the body. Although promoted as a cancer treatment, a 1952 review in the Journal of the American Medical Association and another in 1970 by the American Cancer Society concluded that the treatment was ineffective.

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

[0093] Two fundamentally different methods for performing the ECP procedure have been described and are encompassed by the present invention. These differ in the devices used for leukocyte collection and UVA irradiation: a "closed system" and a so-called "open system." The closed system, based on the original design by Edelson and colleagues, is the only FDA-approved system. The open system incorporates various separation instruments and is mostly used outside the United States. ECP has been an effective treatment for 30 years, with over 2 million treatments performed without any reported negative cytogenetic effects.

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

[0095] Extracorporeal photochemotherapy (ECP) is a process that involves (1) collection of mononuclear cells (MNCs) from a patient, (2) photoactivation of the collected MNCs, and (3) reinfusion of the treated cells (MNCs) back into the patient. More specifically, ECP involves the extracorporeal exposure of peripheral blood mononuclear cells to 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 believed to induce apoptosis, or programmed cell death, of T cells treated with ECP.

[0096] Although the exact mechanism of action of ECP treatment (in different disease states) is not fully known, early theories suggest that photoactivation irreversibly covalently binds 8-MOP to DNA strands contained in T cell nuclei. When photochemically damaged T cells are reinfused, a cytotoxic effect is triggered. For example, cytotoxic T cells, or "CD8+ cells," release cytotoxins when exposed to infected or damaged cells or otherwise attack cells carrying specific foreign or abnormal molecules on their surface. The cytotoxins target the membranes of damaged cells, enter the target cells, and ultimately lead to apoptosis, or programmed cell death, of the target cells. After the treated mononuclear cells are infused back into the body, the immune system recognizes the dying, abnormal cells and begins to generate healthy lymphocytes (T cells) to combat them.

[0097] In addition to the above, it is theorized that extracorporeal phototherapy also induces monocytes (a type of mononuclear cell) to differentiate into dendritic cells that can phagocytose and process apoptotic T cell antigens. Reinjection of these activated dendritic cells into the systemic circulation may trigger a systemic cytotoxic CD8+ T lymphocyte-mediated immune response against such processed apoptotic T cell antigens. It will be appreciated that other possible mechanisms of action may be involved in the observed benefits of ECP mononuclear cell treatment and subsequent benefit to patients undergoing ECP-based therapy.

[0098] Recently, it has been hypothesized that ECP may result in immune tolerance responses in patients. For example, in the case of graft-versus-host disease, infusion of apoptotic cells may stimulate the generation of regulatory T cells, inhibit inflammatory cytokine production, cause effective T cell deletion, and result in other responses. Peritt, “Potential Mechanisms of Photopheresis in Hematopoietic Stem Cells” See, Transplantation,” Biology of Blood and Marrow Transplantation 12:7-12 (2006). Currently, the theory of immune tolerance appears to be among the leading explanations, but other theories exist regarding the mechanism of action of ECP on graft-versus-host disease, as well as other disease states.

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

[0100] Currently, there are two commonly used methods for performing photochemotherapy—online and offline systems and methods.

[0101] In the online method, a dedicated photochemotherapy device, such as the Therakos device described above, is used to perform the entire treatment, including the reinfusion of the processed MNCs. Such devices are "dedicated" photochemotherapy devices designed only to perform photochemotherapy and are incapable of performing other collection protocols required in a hospital or blood processing setting, including, for example, multifunctional apheresis protocols for the collection of platelets, plasma, RBCs, node cells, and / or plasma / RBC exchange protocols.

[0102] In offline photochemotherapy methods, a multifunctional apheresis device may be used to collect mononuclear cells. Collected MNCs, typically contained in one or more collection containers, are detached or otherwise separated from the tubing used during collection, and then treated with a separate irradiation or UVA light device. The treated cells are then manually reinfused into the patient. However, during such offline methods, when the cells are transferred from the apheresis device to the irradiation device (which may be located in a separate room or laboratory), communication with the donor must be severed, and the cells must therefore 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] Immobilization of the device and enzymes within the device: In embodiments of the present invention, the ECP system may include a device containing a matrix with immobilized immunomodulatory molecules or other biomolecules, such as enzymes. Preferably, the matrix with the bound molecules is exposed to blood or MNCs in the context of the present invention.

[0104] Thus, as used herein, "matrix" refers to the material within a blood processing device that provides an interior material or surface through which blood or plasma passes. A matrix as used in the context of the present invention preferably comprises a support to which immobilized immunomodulatory molecules or other biomolecules are attached. Thus, the support functions as a carrier for the immobilized immunomodulatory molecules or other biomolecules, although it may serve other functions.

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

[0106] The support may be, for example, a resin, a membrane, or a nonwoven material. "Nonwoven" materials are broadly defined as sheets, fabrics, or web structures that are bonded together mechanically, thermally, or chemically by winding fibers or filaments (and by perforating films), but not by weaving or knitting. "Resin" refers to an insoluble material that can take the form of a gel, gel beads, microporous beads, or sponge. Such resins can be natural or biopolymers, synthetic polymers, and inorganic materials. Agarose, dextrose, and cellulose beads are commonly used natural supports. Synthetic polymeric or organic supports are primarily based on acrylamide, polystyrene, and polymethacrylate derivatives, while porous silica and glass are some frequently used inorganic supports.

[0107] According to one embodiment of the present invention, the resin is 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 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 Polypropylene (PTFE), polyacrylate (PAA), polymethyl methacrylate (PMMA), polyacrylamide, polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN), polyester, polycarbonate, polyethylene terephthalate (PET), polyamide, polyaramid, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES), polyarylethersulfone (PEAS), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), polyamide-imide, polyaryletherketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyhydroxyalkanoate, polyetheretherketone (PEEK), polyetherketoneketone (PEKK), polyetherimide (PEI), polyimide, polylactic acid (PLA), polymethylpentene (PMP), poly(p-phenylene ether) (PPE), polyurethane (PU), styrene acetate The polymer is composed of a synthetic polymer selected from the group consisting of acrylic nitrile (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 any of these polymers modified by the introduction of functional groups.

[0108] A variety of known methods can be used to immobilize immunomodulatory molecules or other biomolecules to supports and / or matrices in accordance with the present invention. Such immobilization is preferably specific or selective in that it immobilizes the enzyme, while other proteins and components present in blood or plasma or samples thereof (in vitro) are not immobilized to any significant extent.

[0109] "Immobilization" of an immobilized immunomodulatory molecule or other biomolecule to a support to provide a matrix that can be used in a device according to the invention refers to a non-covalent or covalent interaction that holds the two molecules together. According to one embodiment of the present invention, the immobilization refers to a 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 the two molecules to each other. The binding may otherwise be specific, selective, or non-specific.

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

[0111] According to another embodiment of the present invention, the immobilized immunomodulatory molecules or other biomolecules are covalently bound to a support, as described in more detail below and / or as described in the prior art. Covalent binding generally involves either covalent non-site-directed binding of proteins or site-directed binding of proteins. The support that forms the basis for the generation of the matrix must provide or facilitate chemical activation, thus allowing for chemical coupling of the immobilized immunomodulatory molecules or other biomolecules. Many coupling methods for immobilizing immobilized immunomodulatory molecules or other biomolecules are well known in the art.

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

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

[0114] Alternatively, the support may carry specific functional groups for linking the linker and / or enzyme thereto. For example, functionalized resins are commercially available and known to those skilled in the art. A wide range of coupling chemistries, including primary amines, sulfhydryls, aldehydes, hydroxyls, and carboxylic acids, are available on 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, with pore sizes ranging from 10 to 200 nm. According to another embodiment of the present invention, the support is in the form of beads. According to yet another embodiment, the support according to the present invention comprises magnetic beads. The magnetic beads are prepared by encapsulating magnetite in agarose or other polymeric material to 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 are used in protein purification due to their simplicity, ease of handling, reduced surface area, and low diffusion limitations compared to gels, resins, and beads. The membrane can take the physical form of a hollow fiber, or alternatively, a flat membrane. According to one embodiment, the support comprises a hemodialysis hollow fiber membrane dialyzer, and the filter is a hemodialyzer.

[0117] Hollow fiber or flat sheet membranes for use as supports in devices according to the invention may 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). Hollow fiber membranes that can be advantageously utilized to provide devices according to the invention preferably have an inner diameter in the range of 100-500 μm. According to another embodiment of the invention, specifically when the membrane support is a hemodialysis membrane as described above, the hollow fiber membrane is additionally or alternatively functionalized on the luminal side of the fiber with an enzyme according to the invention, which can directly interact with target metabolites in the blood or plasma perfusing the lumen of the hollow fiber membrane. The enzyme may also alternatively be immobilized on the outside of the membrane.

[0118] Methods for extracorporeal blood treatment: The present invention includes a device configured to be placed in an extracorporeal blood circuit through which a patient's blood passes, includes a means for transporting blood from the patient's vasculature to a blood treatment device at a prescribed flow rate, and then returning the treated blood to the patient, and further configured to reduce levels of ADMA and / or MMA in the blood. Additionally, the present invention also includes devices that can be used for extracorporeal blood or blood cell therapy, which devices can be separate or disconnected from the extracorporeal blood circuit, or can be separable / detachable.

[0119] According to the present invention, the term "extracorporeal blood purification" preferably refers to the process of removing substances from body fluids through their clearance from blood flowing in a bypass circuit outside the patient's body (extracorporeal). Such substances may include endogenous toxins (i.e., uremic toxins), exogenous toxins (i.e., ethylene glycol or fungal toxins), administered drugs, viruses, bacteria, antibodies, metabolites and proteins (i.e., IMHA, myasthenia gravis), abnormal cells (i.e., leukemia), and excess water. Therapeutic procedures include hemodialysis, including intermittent hemodialysis (HD, HDF, HF) and continuous renal replacement therapy (CRRT), hemoperfusion, plasma exchange, and therapeutic apheresis. Such methods are known to those skilled in the art, and the device of the present invention can be incorporated accordingly.

[0120] The term "blood" as used herein refers to whole blood, containing all components of an organism's blood, including red blood cells, white blood cells, and platelets suspended in plasma. The term "plasma" refers to a fluid consisting of approximately 92% water, 7% proteins, such as albumin, gamma globulin, fibrinogen, complement factors, and clotting factors, and 1% mineral salts, sugars, fats, electrolytes, hormones, and vitamins, which form part of whole blood but no longer contain red blood cells, white blood cells, or platelets. In the context of the present invention, the terms "blood plasma" or "plasma" refer to a specific fraction of plasma, as defined above, in its standard sense, such as serum.

[0121] According to one embodiment, the blood flow rate in the extracorporeal blood purification circuit is 20 ml to 700 ml / min. Typical dialysate flow rates in an extracorporeal circuit including a hemodialyzer for treating renal failure, either in addition to a blood treatment device according to the present invention or when the hemodialyzer is configured to metabolize ADMA and / or MMA, range from 0.5 l / h to 800 ml / min.

[0122] In therapeutic apheresis, whole blood can be treated, or the blood is divided into its component fractions, for example by centrifugation or by plasma membranes or filters, and the fractions containing the solutes to be removed are specifically treated before being returned to the patient.

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

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

[0125] Immune-related adverse events (irAEs) As used herein, the term "immune-related adverse events" (irAEs) refers to any side effect that occurs specifically in the context of immune checkpoint inhibitor therapy, e.g., cancer therapy. IrAEs are unique and, unlike adverse events occurring in the context of conventional cancer treatments, typically have delayed onset and long-term persistence. IrAEs may 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 disability. Management is primarily based on corticosteroids and other immunomodulatory agents, which should be prescribed judiciously to reduce the potential for short- and long-term complications.

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

[0127] The irAEs caused by the administration of checkpoint inhibitor therapy in the present invention are not particularly limited. IrAEs should be understood as adverse events that are presumed to be immune-related: irAEs (see, for example, the Drug Interview Form (Version 9) for OPDIVO® Intravenous Infusion 20 mg-100 mg, revised in April 2016, and the Characteristics and Treatment of Adverse Events of the Anti-CTLA-4 Antibody, Ipilimumab (YERVOY®), dated August 24, 2015, issued by the Committee on Safety of New Drugs for Malignant Melanoma of the Japanese Dermatological Association).

[0128] The present invention irAE Specific embodiments of the present invention include interstitial lung disease, myasthenia gravis, myositis, colitis, type 1 diabetes, liver dysfunction (hepatopathy), pulmonary disorders such as hepatitis (e.g., autoimmune pneumonia), hypopituitarism such as hypopituitarism or hypophysitis, hypothyroidism, neuropathy, nephropathy, encephalitis, adrenal disorders such as hypoadrenalism, severe skin disorders, venous thromboembolism, infusion reactions, psoriasis, psoriasiform rash, diarrhea (e.g., severe diarrhea), rheumatoid arthritis, uveitis, epitheliitis, bursitis, exacerbation of radiation keratitis, chronic inflammatory demyelinating multiple sclerosis (hereinafter also referred to as demyelinating multiple sclerosis), intestinal disorders, or nephritis, with hypopituitarism being preferred.

[0129] Immune-related adverse events (IrAEs) are known to occur, for example, 8 weeks or 8 to 12 weeks after administration. Immune-related adverse events can be evaluated by "grade" or "IrAE assessment." Here, "irAE assessment" is an index that indicates the severity of the disease and is expressed on a scale of 1 to 3. In the irAE assessment, 1 indicates a state in which "additional therapeutic intervention due to irAE is not required," 2 indicates a state in which "pharmacologic intervention or other treatment is required due to irAE, but hospitalization or treatment interruption is not required," and 3 indicates a state in which "hospitalization due to irAE is required and treatment interruption is required." The correspondence between "irAE assessment" and "grade" varies depending on the disease.

[0130] Immune checkpoint molecules and checkpoint modulators In the context of the present invention, immune checkpoint inhibitors are drugs that activate immune cells by modulating immune checkpoint molecules.

[0131] Immune checkpoint molecules are molecules in the immune system that either increase or decrease signals (costimulatory molecules) provided to immune effector cells. Therefore, immune checkpoint molecules can be subdivided into costimulatory checkpoint molecules and coinhibitory checkpoint molecules. Costimulatory checkpoint molecules include costimulatory lymphocyte receptors, which are lymphocyte surface receptors that can result in activation or stimulation of lymphocyte effector function. Coinhibitory checkpoint molecules include costimulatory lymphocyte receptors, which are lymphocyte surface receptors that can result in inhibition of lymphocyte effector function.

[0132] Costimulatory 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 costimulatory 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 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. CD27 activity is governed by the transient availability of its ligand, CD70, on lymphocytes and dendritic cells. CD27 costimulation 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 nearly all human CD4+ T cells and on approximately half of all CD8 T cells. Binding to one of its two ligands, CD80 and CD86, expressed, for example, on dendritic cells, promotes T cell proliferation.

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

[0138] 4-1BB (CD137) is bound by the CD137 ligand, leading to 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 agonist monoclonal antibody utomilumab (PF-05082566) targets 4-1BB to stimulate a more potent immune system attack against cancer.

[0139] OX40 (CD134) has OX40L (CD252) as its ligand. OX40 is known for its ability to promote the proliferation of effector and memory T cells and suppress the differentiation and activity of T regulatory cells. OX40 is transiently expressed after T cell receptor engagement, which is why it is only upregulated on recently antigen-activated T cells within inflammatory lesions, making it 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 murine OX40 agonist; and MEDI6383, a non-human OX40 agonist.

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

[0141] ICOS (inducible T cell costimulator, also known as CD278) is expressed on activated T cells. Its ligand is ICOSL, which is expressed primarily on B cells and dendritic cells. This molecule appears to be important in T cell effector function.

[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 leading to activation of A2a receptors is a negative immune feedback loop, and the tumor microenvironment has relatively high concentrations of adenosine.

[0144] B7-H3, also known as CD276, was originally understood to be a costimulatory molecule but is now considered a coinhibitory molecule. MacroGenics is investigating MGA271 (Enoblituzumab), an Fc-optimized monoclonal antibody that targets 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 co-inhibitory receptor whose ligand is HVEM (Herpesvirus Entry Mediator). Surface expression of BTLA is gradually downregulated during 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 regulating 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 inhibitory signals 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 higher affinity and avidity for CD80 and CD86 than CD28. CTLA4 transmits inhibitory signals 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 immune responses by its action on Tregs and by a direct effect on CD8+ T cells.

[0151] PD-1 (programmed death 1, or CD279) is a cell surface receptor that plays a key role in downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 has two ligands, PD-L1 and PD-L2. Targeting PD-1 has the advantage of restoring immune function in the tumor microenvironment. PD-L1, the ligand for PD1, is highly expressed in some cancers and may result in the inhibition of anti-cancer immune responses by T cells. Several cancer immunotherapies 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, CureTech), and BMS-936559 (Bristol Myers Squibb). Both atezolizumab (MPDL3280A, Roche) and avelumab (Merck KGaA, Darmstadt, Germany & Pfizer) are monoclonal antibodies directed against PD-L1, the ligand for 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 inhibitor of T-cell activation) is a protein expressed primarily on hematopoietic cells, such that consistent expression of VISTA on leukocytes within tumors may allow 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 with high affinity to CD155 on DCs and macrophages and with lower affinity to CD112.

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

[0156] Receptor inhibitors prevent the generation of signals by the respective receptors. Thus, inhibitors of co-inhibitory lymphocyte receptors are molecules that prevent the activation of the respective receptors, thereby preventing the generation of inhibitory signals. Conversely, receptor activators induce the generation of signals by the respective receptors, and co-stimulatory lymphocyte receptor activators result in the generation of stimulatory signals.

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

[0158] Soluble checkpoint regulators are molecules that are freely diffusible, eg, not bound to cell membranes or retained intracellularly.

[0159] Checkpoint inhibitors in the sense of the present invention include lymphocyte-stimulatory checkpoint modulators, which are molecules that result in the activation of lymphocytes, preferably effector T cells, either through the activation of costimulatory checkpoint molecules or through the inhibition of costimulatory checkpoint molecules. Furthermore, soluble lymphocyte-stimulatory checkpoint modulators include molecules that prevent the activation of membrane-bound immune checkpoint molecules, e.g., soluble forms of the respective immune checkpoint molecules.

[0160] Checkpoint modulators can be naturally occurring or engineered molecules that function to interfere with or modulate the activity of immune checkpoint molecules, respectively. Checkpoint modulators include, for example, antibodies or antibody fragments active against immune checkpoint molecules with agonists or antagonists, and ligands or modified ligands of immune checkpoint molecules.

[0161] Immune cells: The immune cells described herein relate to biological 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 (ILCs), megakaryocytes, monocytes / macrophages, natural killer (NK) cells, platelets, red blood cells (RBCs) and / or thymocytes.

[0162] The term "immune cells" includes MNCs contained in blood, which may also be referred to as peripheral blood mononuclear cells (PBMCs). PBMCs include any peripheral blood cell with a round nucleus, primarily consisting of lymphocytes (T cells, B cells, and NK cells) and monocytes, whereas red blood cells and platelets lack nuclei and granulocytes (neutrophils, basophils, and eosinophils) have multilobed nuclei. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, with a small proportion of dendritic cells. These cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates blood layers, and gradient centrifugation, which separates 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 can be found in both the denser fraction and the PBMC fraction.

[0163] T cells or T lymphocytes are a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of T cell receptors on the cell surface. Several subsets of T cells each have different functions. 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 examples 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-large 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 particularly relates to immunoregulatory NK cells (which may also be referred to as immunoregulatory NK cells) for use in the treatment and / or prevention of immune-related adverse events (irAEs) in subjects receiving checkpoint inhibitor therapy, preferably cell therapy.

[0165] Immunoregulatory NK cells are a specific form of innate lymphoid cells (ILCs). ILCs belong to the lymphoid lineage (lymphocytes) but lack B or T cell receptors, making them a group of innate immune cells that do not respond in an antigen-specific manner. This relatively newly described cell group has distinct physiological functions, some of which resemble helper T cells, but also includes cytotoxic NK cells. They play an important role in protective immunity and regulating homeostasis and inflammation. Natural killer (NK) cells are cytotoxic innate effector cells similar to cytotoxic T cells of the adaptive immune system. They are distributed throughout the blood, organs, and lymphoid tissues, accounting for approximately 15% of peripheral blood lymphocytes. NK cells play a role in tumor surveillance and rapid elimination of virus-infected cells. They lack the MHC class I "self" signal and can recognize stressed cells in the absence of antibodies, allowing them to respond much more rapidly than the adaptive immune system. Natural killer (NK) cells play a key role in host immunity against cancer. In response, cancers develop mechanisms to evade 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 immunoregulatory NK cells of the present invention preferably have low expression (or downregulated expression) or CD16, particularly CD56 dim Characterized on NK cells. CD16 binds to FcRγIII, It is an activating NK cell receptor that can induce potent cytokine production. CD16 shedding / downregulation may be an immunoregulatory mechanism of NK cells to prevent autoimmunity. CD16 downregulation regulates NK cell responses and contributes to the maintenance of immune homeostasis via both antibody and T cell-dependent pathways. Furthermore, the immunoregulatory NK cells of the present invention may exhibit low expression of GM-CSF, IFN-γ, TNF, and / or IL-2.

[0167] In embodiments, the generation or induction of immunoregulatory NK cells in a sample can be measured by, for example, comparing the phenotype and population distribution of NK cells in the sample before and after performing the methods of the present invention, using flow cytometry, gene expression, and / or mass spectrometry of protein abundance. For example, a transition from CD16-high NK cells to CD16-low NK cells after irradiation indicates the induction of an immunoregulatory phenotype. Furthermore, a decrease in GM-CSF, IFN-γ, TNF, and / or IL-2 expression in the NK cell population would also indicate the induction of immunoregulatory NK cells. Those skilled in the art will recognize 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 be defined and distinguished, preferably by flow cytometry, as a CD45-positive, CD14-negative, CD3-negative, CD19-negative, and CD56-positive cell population. Within this population, further identification and quantification of immunoregulatory NK cells can be performed, for example, by determining CD16, GM-CSF, IFN-γ, TNF, and / or IL-2 expression. Immunoregulatory NK cells are preferably positive for CD56, but exhibit relatively low expression levels of CD56 ("dim" expression).

[0168] Further characteristics and definitions of immunoregulatory NK cells are well established in the art and are the subject of multiple studies and review articles that are known to or can be identified by those skilled in the art.

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

[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 using dendritic cells to present tumor antigens. Dendritic cells present antigens to lymphocytes, which then activate and prime them to kill other cells that present the antigen.

[0171] Therapeutic applications of the present invention As used herein, the term "subject" refers to a human or non-human animal selected for treatment or therapy. A subject, such as a subject or patient in need of treatment or prevention, may be an animal, vertebrate, mammal, rodent (e.g., guinea pig, hamster, rat, mouse), mouse (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human. The meaning of the terms "animal," "mammal," and the like is well known in the art and can be inferred, for example, from Wehner and Gehring (1995; Thieme Verlag). In the context of the present invention, it is particularly envisioned that animals will receive economically, agriculturally, or scientifically important treatments. Preferably, the subject / patient is a mammal. More preferably, the subject / patient is human.

[0172] In embodiments of the invention, the subject receiving checkpoint inhibitor therapy has a cancer, such as malignant 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, whether or not the cancer is associated with the formation of a solid tumor or whether or not the cancer cells form a solid tumor, as is the case with certain leukemias.

[0174] Cancer encompasses a group of diseases that can affect any part of the body and are 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 forming metastases. Globally, there were 14 million new cancer cases and 8.2 million cancer-related deaths in 2012 (World Cancer Report 2014). The majority of cancers are caused by environmental signals, such as tobacco use, obesity, and infections, while approximately 5–10% are genetic. Cancers can be classified into subcategories based on their cell of origin. The most common subcategories are epithelial cell-derived cancers, connective tissue-derived sarcomas and lymphomas, and hematopoietic cell-derived leukemias. Cancer is associated with a variety of local and systemic symptoms and is often incurable. Given the high number of new cancer cases and cancer-related deaths, new treatment strategies are needed.

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

[0176] Melanomas include, but are not limited to, for example, cusp melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo melanoma, malignant melanoma, nodular melanoma, vulvar melanoma, and superficially spreading melanoma.

[0177] Leukemias include acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukemic leukemia, leukemic leukemia, basement membrane leukemia, blastic leukemia, bovine leukemia, and chronic leukemia. myelocytic leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hematoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, phosphorus These include, but are not limited to, lymphocytic leukemia, lymphocytic leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryoblastic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, promyelocytic leukemia, leader cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and anaplastic cell leukemia.

[0178] Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibrosarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's lymphoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulosarcoma, Rous sarcoma, serum cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0179] Cancers include acinar carcinoma, acinous carcinoma, and glandular carcinoma. cystic carcinoma, adenoid cystic carcinoma, carcinoma adenoma, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebrocellular carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, cuirass carcinoma, skin cancer, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, cerebrocellular carcinoma, epidermoid carcinoma, carcinoma epithelial adenoid, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix cell Cancer, hematopoietic cell carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, vitreous carcinoma, high renal carcinoma, infantile embryonal carcinoma, carcinoma in situ, carcinoma in situ, carcinoma in situ, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular Carcinoma (carcinoma lenticulare), lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma , melanotic carcinoma, soft carcinoma, mucinous carcinoma, mucinous carcinoma, mucous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucinous, mucinous carcinoma, carcinoma myxoma, nasopharyngeal carcinoma, oat cell carcinoma, bone cancer, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spinous cell carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, carcinosarcoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, carcinomatous cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, carcinomatous telangiectasia, carcinomatous telangiectasia, transitional cell carcinoma, nodular carcinoma, tuber carcinoma, verrucous carcinoma, and choriocarcinoma.

[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, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular tumor, lymphoma, thyroid cancer, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, and prostate cancer.

[0181] In some embodiments, "tumor" includes, but is not limited to, prostate tumors, pancreatic tumors, squamous cell carcinoma, breast tumors, melanoma, basal cell carcinoma, hepatocellular carcinoma, cholangiohemangiocarcinoma, testicular tumors, neuroblastoma, malignant astrocytic tumors such as glioma or glioblastoma multiforme, colorectal tumors, endometrial cancer, lung cancer, ovarian tumors, cervical tumors, osteosarcoma, rod body / leiomyosarcoma, synovial sarcoma, angiosarcoma, Ewing's sarcoma / PNET, and malignant lymphomas. These include primary 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 preventative, considering that the disease and / or condition is completely or partially prevented, for example, by reducing the subject's risk of having a particular disease or condition, or may be therapeutic, considering that the disease and / or its adverse effects are partially or completely cured. 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) to (c): (a) preventing the onset of a disease, condition, or condition in a patient; (b) inhibiting the symptoms of a condition, i.e., preventing the progression of the symptoms; and (c) ameliorating the symptoms of a condition, i.e., inducing regression of the disease or condition.

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

[0184] The present invention encompasses the treatment of patients by introducing a therapeutically effective number of cells, particularly immunoregulatory NK cells of the present invention, into the subject's bloodstream. As used herein, "introducing cells into a subject's bloodstream" includes, but is not limited to, introducing such cells into one of the subject's veins or arteries via injection. Such administration may be performed, for example, once, multiple times, and / or over one or more extended periods. A single injection is preferred, but in some cases repeated injections may be necessary over time (e.g., weekly, monthly, quarterly, semi-annually, or annually). Such administration is preferably performed 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 performed locally, for example, by injection into an area of ​​the subject's body adjacent to a symptomatic organ or tissue.

[0185] Furthermore, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions, most preferably aqueous solutions. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, those based on Ringer's dextrose, and the like. 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 S-Wilkins (2000). Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like, may also be present.

[0186] In one embodiment, a therapeutically effective number of cells is administered. This can refer to either NK cells or therapeutic immune cells of the present invention as a combined immunotherapy. As used herein, a "therapeutically effective number of cells" includes, but is not limited to, the following amounts and ranges of amounts: (i) about 1 x 10 2 ~Approx. 1×10 8 cells / kg body weight, (ii) approximately 1 × 10 3 ~Approx. 1×10 7 cells / kg body weight, (iii) approximately 1 × 10 4 ~Approx. 1×10 6 cells / kg body weight, (iv) approximately 1 × 10 4 ~Approx. 1×10 5 cells / kg body weight, (v) approximately 1 x 10 5 ~Approx. 1×10 6 cells / kg body weight, (vi) approximately 5 × 10 4 ~about 0.5×10 5 cells / kg body weight, (vii) approximately 1 × 10 3 cells / kg body weight, (viii) approximately 1 × 10 4 cells / kg body weight, (ix) approximately 5 × 10 4cells / kg body weight, (x) approximately 1 x 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. Estimated 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 values ​​are based on preclinical animal experiments and human trials from transplantation of CD34+ hematopoietic stem cells and standard protocols. Mononuclear cells (including CD34+ cells) typically contain 1:23,000 to 1:300,000 CD34-negative cells. 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 components thereof described herein, as well as materials for practicing the in vitro and therapeutic methods of the invention, and materials for preparing and isolating the NK cells of the invention, and other materials.

[0188] figure The present invention is further illustrated by the following figures, which are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein. [Brief explanation of the drawings]

[0189] [Figure 1-1]Refractory autoimmune colitis responds to ECP, leading to the proliferation of immunoregulatory NK cells. (a) Severity of diarrhea in patients according to common toxicity criteria (y-axis) over time (x-axis) with different treatments. (b) Colonoscopy images (left panel) and H&E-stained biopsy sections (right panel) from the initial diagnosis of immune checkpoint inhibitor-associated colitis showing mucosal edema and ulcers. (c) Colonoscopy images (left panel) and H&E-stained biopsy sections (right panel) after successful ECP treatment showing no signs of autoimmune colitis. Colonic crypts show regular morphology without granulocytic infiltration, apoptosis, or crypt loss. [Figure 1-2] Refractory autoimmune colitis responds to ECP and leads to an expansion of immunoregulatory NK cells. (d) tSNE plot visualizing the peripheral lymphocyte compartment 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 expansion of immunoregulatory NK cells. (f) Expression intensity of CD16, CD56, and CD57 on NK cells (defined as single / live / CD45pos / CD14neg / CD3neg / CD19neg / CD56pos) from healthy age-matched donors (HD, n=5) and patients at 53 and 75 weeks after starting 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 expansion of immunoregulatory NK cells. (g) MFI (median marker expression, value range: 0-1) of CD16 on CD56dim mature NK cells. HD (n=5). Box plot whiskers represent the minimum and maximum values ​​of the HD dataset. (h) Survival of mice injected with T cells (Tc) with or without NK cells (4x104 or 4x105 NK doses per mouse) in a model of anti-PD1 antibody-induced irAE described in the Supplementary Appendix. **p=0.003, ****p<0.0001. [Figure 2-1](a) tSNE plot displaying 1000 stochastically selected CD45+ lymphocytes (lymphocytes defined by FSC / SSC) for 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 numbers of NK cells before and after ECP treatment. (d) Relative numbers of B cells, CD4+ T cells, CD8+ T cells, and CD4-CD8- T cells before and after ECP treatment. (e) Absolute 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 on NK cells (defined as single / live / CD45pos / CD14neg / CD3neg / CD19neg / CD56pos) in patients with HD (n=5) and at weeks 53 and 75 visualized by UMAP. [Figure 2-3] (g) Heatmap showing median marker expression (value range: 0-1) of 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 from healthy age-matched donors (HDs) and patients (53 and 75 weeks after ECP initiation). HDs (n=5). Boxplot whiskers represent the minimum and maximum values ​​for the HD dataset. [Figure 3-1] (a)-(d) Percentage of cytokine-expressing cells within CD56bright NK cells, CD56dim NK cells, CD4+ memory T cells, CD4+ naive cells, and CD8+ T cells in healthy age-matched donors (HDs) and patients (53 and 75 weeks after the start of ECP). HDs (n=5). Boxplot whiskers represent the minimum and maximum values ​​for the HD dataset. [Figure 3-2](a)-(d) Percentage of cytokine-expressing cells within CD56bright NK cells, CD56dim NK cells, CD4+ memory T cells, CD4+ naive cells, and CD8+ T cells in healthy age-matched donors (HDs) and patients (53 and 75 weeks after the start of ECP). HDs (n=5). Boxplot whiskers represent the minimum and maximum values ​​for the HD dataset. [Figure 4] (a) Experimental model in which patient T cells were adoptively transferred into Rag2- / -Il2rg- / - mice alone or together with NK cells, followed by treatment with anti-PD-1 antibody to induce immune checkpoint inhibitor-associated autoimmunity. (b) Histopathological neutrophil and lymphocyte infiltration scores of liver, skin, lung, and colon isolated from Rag2- / -Il2rg- / - mice 15 days 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 15 days 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: Mice were treated with DSS (3%), anti-PD1, and ECP as indicated. (b) Weight curves of mice untreated or treated with DSS (3%), anti-PD1 alone or in combination with ECP as indicated. [Figure 6-2] (c) Colon length quantified in five mice per group, groups as shown in panel B. (d) Representative colon of one mouse per group, groups as shown in panel B. [Figure 6-3] (e) Representative HE-stained sections of the colon of the group shown in panel B. (f) Histopathological scores of the colon of the group 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 indicated. [Figure 7-2](b) Survival of mice injected intravenously with B16 melanoma cells and subsequently treated with anti-PD1, prednisolone, or ECP. [Example]

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

[0191] Here, we report a patient with ipilimumab / nivolumab-induced colitis refractory to multiple immunosuppressive drugs who achieved a complete response 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 with metastatic melanoma was treated with ipilimumab and nivolumab. After two doses, the patient developed dermatitis, thyroiditis, hepatitis, and colitis. Colitis was diagnosed based on gross mucosal ulcers and intraepithelial apoptosis and crypt loss identified on biopsy (Figure 1a, b). Although the dermatitis, thyroiditis, and hepatitis resolved after discontinuing ipilimumab / nivolumab and corticosteroid treatment, the patient experienced three colitis episodes within the next 20 weeks (CTC-AEII-III°). These were treated with corticosteroids (a total of 23 weeks before ECP), infliximab (two single doses 18 and 15 weeks before ECP), and cyclosporine (14 weeks before ECP; Figure 1a and Table 1).

[0193] Due to the lack of a durable response, the patient underwent ECP. Over the next 8 months, she received two cycles of daily ECP every 2–4 weeks. ECP was well tolerated and resulted in a complete response (Figure 1a). Immunosuppression was tapered without symptom rebound. Colonoscopy confirmed continued remission of colitis (Figure 1c). Immune checkpoint inhibitor treatment was discontinued at the first sign of irAEs and was not restarted.

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

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

[0196] ECP reduces immune-mediated adverse events without blocking anti-melanoma effects Based on the above results (Figures 1-5), we hypothesized that ECP reduces immune-mediated adverse events without blocking anti-melanoma effects. We then aimed to test this in an in vivo model of irAEs. To induce colitis, mice were treated with 3% DSS and anti-PD1, as previously reported (19) (Figure 6A). Treatment reduced mouse body weight, consistent with the onset of colitis, and weight loss was reduced by ECP treatment (Figure 6B). ECP treatment also increased colon length compared with the group treated with anti-PD1 alone (Figure 6C, D). Colon length has been reported to be a surrogate parameter for the severity of immunotherapy-induced colitis (19). Consistent with the reduced colitis severity, we observed reduced neutrophil infiltration in the colon wall of ECP-treated mice compared with the group treated with anti-PD1 alone (Figure 6E, F). These findings indicate that ECP reduces anti-PD1-induced colitis in mice.

[0197] To understand whether the immunomodulatory effects of ECP are associated with the loss of antitumor activity, we next treated melanoma-bearing mice with anti-PD1 alone or in combination with the glucocorticoid prednisolone or ECP (Figure 7A). We observed that prednisolone reduced the survival of melanoma-bearing mice compared with the group treated with anti-PD1 alone (Figure 7B). In contrast, the group treated with anti-PD1 and ECP had a comparable outcome to the group treated with anti-PD1 alone (Figure 7B). These findings indicate that ECP does not interfere with the anti-melanoma response induced by anti-PD1 treatment.

[0198] Discussion of Examples Mechanistic considerations: NK cells exert a variety of heterologous immunological functions, including anti-inflammatory activity. In a murine model of GVHD, NK cell upregulation improved survival, which 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 effector NK cell subset that can control GVHD without interfering with the graft-versus-leukemia (GVL) effect. 8 In humans, killer cell immunoglobulin-like receptor (KIR) ligand mismatch in GVH-directed allogeneic HCT reduced the risk of NK cell-mediated GVHD. 9 .

[0199] ECP is an effective treatment for GVHD. Increased NK cell activity during ECP for extensive chronic GVHD has been previously observed. 10 Patients with acute GVHD have stronger NKG2D and CD62L expression than CD56 bri have a higher frequency of NK subsets 11 The same study also found that patients with chronic cGVHD had higher expression of CD56 with higher expression of CD57 and CD11b. - CD16 + NK cells were increased. ECP stimulated the NK cell population with specialized antiviral and anti-leukemic CD57 + NKG2C + CD56 dim Shifted toward a more immunomodulatory phenotype with subset protection 11 We hypothesize that similar mechanisms may be involved in the protective effect of NK cells against irAEs. When comparing the NK cell compartment in patients with that of age- and sex-matched healthy controls, we found that CD56 dim We observed downregulation of CD16 expression on NK cells. CD16 is an activating NK cell receptor that can induce FcRγIII, a potent cytokine production. Previous studies have shown that CD16 shedding may be an immunoregulatory mechanism to prevent autoimmunity. 12 CD16 downregulation modulates NK cell responses and contributes to the maintenance of immune homeostasis through both antibody- and T cell-dependent pathways. 13 Supporting this hypothesis, expression of GM-CSF, IFN-γ, TNF, and IL-2 was lower in NK cells from patients compared with controls.

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

[0201] Mouse model of IrAE T cells were isolated from the patient's peripheral blood using negative selection with a Pan T cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. NK cells were isolated from the patient's peripheral blood using an NK cell isolation kit, human (Miltenyi Biotec) according to the manufacturer's instructions. Rag2 - / - Il2rg - / - Mouse, 4 x 10 4 or 4×10 5 3 x 10 with or without NK cells 5 T cells were injected intravenously. From day 1 to day 22 after injection, mice were treated twice weekly with 8 mg / kg body weight anti-PD-1 antibody (clone J43) and once weekly with 1 mg / kg body weight LPS, both administered intraperitoneally (Fig. 4a). Skin, liver, colon, and lung sections collected 15 days after human T cell injection were stained with hematoxylin-eosin and examined by an experienced pathologist for lymphocytic and neutrophilic infiltrates, crypt abscesses, and apoptotic cells. 14,15 Human irAEs were scored based on histopathological characterization, including All animal studies were approved by the Institutional Review Board for the Use and Care of Laboratory Animals at Albert-Ludwigs University, Freiburg, Germany (protocol approval numbers: G17-049, X13-07J, X15-10A).

[0202] Flow cytometry To monitor lymphocyte lineage populations during and after ECP therapy, patients' peripheral blood lymphocytes 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 diagnostics. Data were analyzed in FlowJo (V10), exported to the R environment, and analyzed using the R tool. 17 tSNE and FlowSOM clustering were performed as previously described. 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. Cell populations designated in the figures were exported and analyzed using the R environment. 17 Data were processed for FlowSOM clustering as described. 16 For dimensionality reduction, we used the UMAP package. 18 .

[0204] statistics Statistical analysis was performed using GraphPad Prism Lab Software V7.0. Comparisons between two groups were performed using a two-tailed, unpaired Student's t-test. Differences in survival time (Kaplan-Meier survival curves) were evaluated using the Mantel-Cox (log-rank) test. Data are presented as mean ± SEM unless otherwise indicated. A P value of <0.05 was considered significant.

[0205] Table of Examples [Table 1]

[0206] The patient had his first episode of colitis 20 weeks before initiating ECP. At that time, he had been treated with steroids for three weeks for previous immune checkpoint inhibitor-associated hepatitis, thyroiditis, and dermatitis. Colitis developed during steroid tapering. Therefore, the steroid dose was increased, and due to an inadequate response, infliximab 5 mg / kg BW was administered once. Symptoms subsided. During steroid tapering, the patient experienced a second episode of colitis 16 weeks before initiating ECP. The patient was treated with an increased dose of methylprednisolone and a second dose of infliximab. Diarrhea was refractory to this therapy, and as a result, cyclosporine A was added. Symptoms again subsided. As the cyclosporine A dose was tapered, the patient experienced a third episode of colitis. ECP treatment was then initiated. Two weeks after initiating 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 without any symptomatic rebound. ECP was administered for a total of 32 weeks. At 11 months of follow-up after the last ECP, the patient remained in complete remission with respect to both irAEs and melanoma symptoms. [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 2:1946-69. 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 6,03426v:03422(2018). 19. Perez-Ruiz E, Minute L, Otano I, Alvarez[[ID=I3]] 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) - 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; - subjecting said sample to irradiation. (Item 2) 2. The method according to item 1, wherein immunoregulatory NK cells are generated or induced in the sample by adding a photosensitizer to the sample and subjecting the sample to irradiation. (Item 3) Item 11. The method of any one of the preceding items, wherein the photosensitizer is 8-methoxypsoralen and / or the radiation is UVA radiation. (Item 4) The method of any one of the preceding items, wherein the subject exhibits symptoms of or is suffering from an irAE. (Item 5) The method of any one of the preceding items, wherein the subject developed symptoms and / or signs of an irAE before the checkpoint inhibitor therapy was discontinued, or the symptoms and / or signs of an irAE developed after the checkpoint inhibitor therapy was discontinued, and / or the symptoms and / or signs of an irAE persisted after the checkpoint inhibitor therapy was discontinued. (Item 6) The method of any one of the preceding items, wherein the irAE comprises symptoms of an autoimmune disease and / or is caused by an autoimmune response. (Item 7) The method of any one of the preceding items, wherein the irAE comprises autoimmune colitis. (Item 8) The method of any one of the preceding items, wherein the subject is afflicted with cancer, such as malignant melanoma or another cancer treatable by checkpoint inhibitor therapy. (Item 9) The method of any one of the preceding items, wherein the subject is receiving and / or is refractory to immunosuppressive drugs, such as steroids, corticosteroids, cyclosporine, and / or anti-TNF antibodies (e.g., infliximab). (Item 10) The method of 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) - providing a sample derived from an isolated blood sample of a subject; - adding a photosensitizer to the sample; - subjecting the sample to irradiation. (Item 12) The immunomodulatory NK cell of any one of the preceding items, wherein the blood sample is derived from a subject receiving checkpoint inhibitor therapy and suspected of developing or having developed symptoms of an immune-related adverse event (irAE). (Item 13) Immunomodulatory NK cells for use in the treatment and / or prevention of immune-related adverse events (irAEs) in subjects receiving checkpoint inhibitor therapy. (Item 14) 10. The immunoregulatory NK cells for use according to any one of the preceding items, wherein the immunoregulatory NK cells are 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) 15. The immunomodulatory NK cells for use according to item 13 or 14, wherein the NK cells are autologous to the subject and / or the NK cells are administered to the subject while checkpoint inhibitor therapy is ongoing.

Claims

[Claim 1] The invention described in the present specification.

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