Methods for generating immunomodulatory cells in blood-derived samples

Abstract

The present invention relates to a method comprising the steps of providing a sample derived from a blood sample of a subject who has received checkpoint inhibitor therapy and is suspected of developing or has developed symptoms of an immune-related adverse event (irAE); adding a photosensitizing agent to the sample; and subjecting the sample to irradiation, which preferably generates immunomodulatory NK cells in the sample. In embodiments, the photosensitizing agent is 8-methoxy psoralen and / or the irradiation is UVA irradiation. In another aspect, the present invention relates to immunomodulatory NK cells obtained from a method comprising the steps of providing a sample derived from an isolated blood sample of a subject; adding a photosensitizing agent to the sample; and subjecting the sample to irradiation. Furthermore, the present invention encompasses immunomodulatory NK cells for use in the treatment and / or prevention of irAEs in subjects who have received checkpoint inhibitor therapy.

Description

[0001] illustrate

[0002] This invention relates to a method comprising the steps of: providing a blood sample derived from a subject who has received checkpoint inhibitor therapy and is suspected of having or has already experienced symptoms of an immune-related adverse event (irAE); adding a photosensitizer to the sample; and irradiating the sample, which preferably generates immunomodulatory NK cells in the sample. In various embodiments, the photosensitizer is 8-methoxypsoralen and / or the irradiation is UVA irradiation. In another aspect, this invention relates to immunomodulatory NK cells obtained from a method comprising the steps of: providing a sample of isolated blood derived from a subject; adding a photosensitizer to the sample; and irradiating the sample. Furthermore, this invention covers immunomodulatory NK cells for the treatment and / or prevention of irAEs in subjects who have received checkpoint inhibitor therapy. Background Technology

[0003] Immune checkpoints are regulatory molecules of the immune system and play a crucial role in maintaining immune homeostasis and self-tolerance. The first immune checkpoints identified include cytotoxic T-lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1). CTLA-4 is expressed on the surface of T cells, binds to B7-1 (CD80) or B7-2 (CD86) molecules on antigen-presenting cells, and acts as a negative regulator of T cells. PD-1 also has a negative effect on 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 CTLA-4 and PD-1 is tightly and dynamically regulated.

[0004] During cancer development, malignant cells suppress the immune response by activating immune checkpoints. Previous studies have shown that PD-L1 is expressed in various cancers. In the tumor microenvironment, PD-L1 expressed by cancer cells interacts with PD-1 on the surface of T cells to suppress T cell effector function. Furthermore, numerous studies have demonstrated that high tumor expression of PD-L1 is significantly associated with poor cancer prognosis. These studies suggest that blocking the PD-1 signaling pathway may have a therapeutic effect on cancer.

[0005] Recent clinical trials have revealed the effectiveness of several anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors (ICIs) in various cancers, such as melanoma, non-small cell lung cancer, renal cell carcinoma, and head and neck cancer. Further clinical trials are currently underway to expand the indications for ICIs. To date, the U.S. Food and Drug Administration (FDA) has approved three anti-PD-1 antibodies—nivolumab, pembrolizumab, and cemiplimab—for the treatment of different types of cancer, and three anti-PD-L1 inhibitors—atezolizumab, avelumab, and durvalumab.

[0006] With the increasing use of ICIs, adverse events associated with this class of drugs have become a significant concern. ICIs have a different toxicity spectrum compared to conventional cytotoxic chemotherapy. Side effects associated with increased immune system activity caused by ICIs are called immune-related adverse events (irAEs), which can affect multiple organs in the body, including the skin, gastrointestinal tract, endocrine system, liver, lungs, nervous system, and skeletal muscle system.

[0007] For example, combination immune checkpoint inhibitor therapy using anti-CTLA4 and anti-PD-1 antibodies is an effective first-line treatment for malignant melanoma. However, approximately 50% of patients experience serious immune-related adverse events (irAEs). 1,2 Autoimmune colitis occurs in 20% of cases and can become steroid-resistant. 3 .

[0008] In summary, while checkpoint inhibitor therapy, specifically checkpoint inhibitor therapy utilizing anti-CTLA4 and anti-PD-1 antibodies, is an effective treatment for various forms of cancer, specifically malignant melanoma, this treatment is associated with significant side effects, potentially due to immune system activation induced by checkpoint inhibitor therapy. These side effects can lead to autoimmune responses and manifest as various clinical symptoms, which can be summarized as irAEs. A prominent irAE is autoimmune colitis. Side effects of this treatment, such as autoimmune responses, specifically autoimmune colitis, can persist even after checkpoint inhibitor therapy has been discontinued.

[0009] Therefore, there is a need in the art for alternative or ameliorative measures for treating patients who have already received checkpoint inhibitor therapy, such as autoimmune reactions and, specifically, immune-related adverse events like autoimmune colitis. Summary of the Invention

[0010] In view of the prior art, a potential technical problem of the present invention is to provide an improved or alternative means for treating and / or preventing immune-related adverse events such as autoimmune reactions and, specifically, autoimmune colitis, in patients who have received checkpoint inhibitor therapy.

[0011] This problem is solved by the features of the independent claim. Preferred embodiments of the invention are provided by the dependent claims.

[0012] In a first aspect, the present invention relates to a method comprising the following steps:

[0013] - Provide a blood sample from a subject who has received checkpoint inhibitor therapy and is suspected of having or has already experienced symptoms of an immune-related adverse event (irAE).

[0014] - Add photosensitizer to the sample, and

[0015] - Expose the sample to irradiation.

[0016] This invention is based on the completely unexpected discovery that patients with irAEs, such as those already receiving checkpoint inhibitor therapy due to cancerous diseases, can be effectively treated by applying ECP. The ECP used in this example essentially corresponds to a method of adding a photosensitizer to a blood sample from an irAE patient and subjecting the sample to irradiation. Surprisingly, it was found that performing this method on blood samples, such as blood samples specifically comprising monocytes (MNCs), resulted in the generation of immunomodulatory NK cells in the sample. In this context, "generation" of immunomodulatory NK cells is understood as inducing or triggering the formation of such cells in the sample. In other words, the cells included in the sample differentiate into immunomodulatory NK cells or adopt an immunomodulatory NK cell phenotype.

[0017] It has been found that samples produced by the method of the present invention, and specifically the induced immunomodulatory NK cells included in such samples, can be used to treat patients with irAEs. Surprisingly, it has been demonstrated that administration of such samples or cells included in such samples to subjects already receiving checkpoint inhibitor therapy is effective in preventing the occurrence of irAEs and even in treating irAEs already present in said subjects. Importantly, the method of the present invention can be performed on samples from the same subject already receiving checkpoint inhibitor therapy. Therefore, the cells or samples produced by the method of the present invention can be administered to the same subject acting as a blood donor, and thus the resulting samples can represent autologous cell therapy.

[0018] As used herein, the term "subject who has received checkpoint inhibitor therapy" includes subjects who are currently undergoing ongoing checkpoint inhibitor therapy, or subjects who have received checkpoint inhibitor therapy and discontinued it, for example, after the onset of irAE symptoms.

[0019] Completely unexpectedly, such cell therapies using samples or cells produced by the methods of the present invention are also effective even for patients with irAE who are refractory to other immunosuppressive therapies such as steroids or anti-TNF antibodies and who still show symptoms of irAE or are still suffering from irAE after discontinuation of checkpoint inhibitor therapy.

[0020] As illustrated in the examples, the positive effects and efficacy of administering a sample subjected to the methods of the present invention can be at least partially attributed to the NK cell function regulation of the NK cells included in the sample, wherein the regulation is a result of irradiation in the presence of a photosensitizer. Therefore, the present invention also covers immunomodulatory NK cells for the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy. Preferably, the immunomodulatory NK cells are generated by subjecting blood or MNCs or NK cells from a human subject, preferably a subject who has received checkpoint inhibitor therapy and is suspected of having or has already experienced an irAE, to the methods of the present invention, and subsequently used to treat the subject, preferably the donor, who has received checkpoint inhibitor therapy and is suspected of having or has already experienced an irAE.

[0021] In another aspect, the present invention relates to immunomodulatory NK cells obtained from a method comprising the following steps:

[0022] - Provide a sample derived from a separated blood sample taken from the subject.

[0023] - Add photosensitizer to the sample, and

[0024] - Expose the sample to irradiation.

[0025] This aspect of the invention is based on the observation that NK cells included in blood-derived samples irradiated after photosensitization exhibit an immunomodulatory phenotype, which is advantageous when the resulting cells are used to treat and / or prevent irAEs. In this context, such advantageous and beneficial properties have not yet been observed in any other NK cells.

[0026] Preferably, the subject of the present invention is a human being. Preferably, the blood sample and cells of the present invention are human.

[0027] In a preferred embodiment, the blood sample used to generate the immunomodulatory NK cells of the present invention is obtained from a subject who has received checkpoint inhibitor therapy and is suspected of having or has already experienced symptoms of immune-related adverse events (irAEs). This embodiment is particularly advantageous because the cells are autologous to the patient, and there will be no adverse events that would occur after xenogeneic cell transplantation.

[0028] Another aspect of the invention relates to immunomodulatory NK cells for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy. The subjects include those currently undergoing ongoing checkpoint inhibitor therapy, or those who have received checkpoint inhibitor therapy and discontinued it, for example, after the onset of irAE symptoms.

[0029] Preferably, the immunomodulatory NK cells used for such treatment or prevention have been generated by adding a photosensitizer to a blood sample derived from a human subject and subjecting the sample to irradiation.

[0030] A photosensitizer is added to the blood-derived sample and the sample is irradiated to produce or induce the formation of immunomodulatory NK cells in the sample.

[0031] In various embodiments, the photosensitizer is 8-methoxypsoralen. Furthermore, in various embodiments, the irradiation is UVA irradiation. In a preferred embodiment, the photosensitizer is 8-methoxypsoralen and the irradiation is UVA irradiation.

[0032] In the context of this invention, irradiation is preferably performed using an in vitro photodisplacement (ECP) system. The irradiation of the blood sample can be performed using any suitable system or irradiation apparatus known to those skilled in the art.

[0033] In various embodiments, the methods of the present invention are performed in vitro or ex vivo. As used herein, the terms in vitro and ex vivo are used synonymously. In the context of the present invention, the terminology relates to a method performed on a blood-derived sample that has been removed from the human body, wherein the method of the present invention is performed on said blood-derived sample outside the human body. For this purpose, blood from a donor subject is removed from the body, meaning removed from the physiological circulatory system. As used herein, "isolated blood sample" is a sample of blood (meaning a certain volume of blood) removed from the donor's circulatory system to a location outside the human body.

[0034] The separated blood sample can be subjected to or directed to an in vitro photocatalytic separation and replacement (ECP) system or apheresis system for performing at least some steps of the method of the present invention. Such a system may be an online system fluidly connected to the subject's circulatory system. In an alternative embodiment, the method can be performed offline, wherein the irradiated blood sample is disconnected from the donor subject's circulatory system.

[0035] Therefore, in various embodiments, the method of the present invention is an in vitro method. In embodiments of the method of the present invention, the blood sample is a separated blood sample. In various embodiments, the method is an in vitro method and the blood sample is a separated blood sample.

[0036] As used herein, the term "blood sample" includes all types of blood-derived samples, including blood cell samples such as MNC samples derived from blood.

[0037] In embodiments of the invention, the subject (the blood donor and / or the recipient of the cells) exhibits symptoms of or suffers from irAE. In another embodiment, the subject has received checkpoint inhibitor therapy, but the checkpoint inhibitor therapy is discontinued after symptoms and / or manifestations of irAE appear in the subject. In various embodiments, symptoms and / or manifestations of irAE appear in the subject after the discontinuation of checkpoint inhibitor therapy. In various embodiments, symptoms and / or manifestations of irAE persist after the discontinuation of checkpoint inhibitor therapy.

[0038] In embodiments of the invention, subjects who have received checkpoint inhibitor therapy and are suspected of having or have already developed symptoms of irAE serve as blood donors, and the provision and / or separation of blood samples can occur at any of the time points described in the above embodiments. For example, sample separation can occur before or after the onset of irAE symptoms. Furthermore, sample separation can occur during checkpoint inhibitor therapy or after the discontinuation of said therapy.

[0039] In a preferred embodiment of the invention, while checkpoint inhibitor therapy is ongoing, a sample generated by the method of the invention or NK cells available according to the invention are administered to the subject.

[0040] In a preferred embodiment, sample separation occurs during ongoing checkpoint inhibitor therapy, either before or after the onset of irAE symptoms. The sample or cells produced by the method of the described example can be used prophylactically or therapeutically by administering the sample / cells to the subject during ongoing checkpoint inhibitor therapy. Therefore, in a preferred embodiment of the invention, a subject already receiving checkpoint inhibitor therapy can receive immunomodulatory NK cells, preferably autologous and generated by irradiating a blood-derived sample according to the method described herein, while the checkpoint inhibitor therapy continues. Such embodiments are particularly advantageous because checkpoint inhibitor therapy, such as anticancer checkpoint inhibitor therapy, can be maintained while the patient receives the cells of the invention and / or cells generated by the method of the invention.

[0041] Therefore, in such embodiments, checkpoint inhibitor therapy can be administered to subjects for a longer period of time because the application of irradiated cells prevents or mitigates irAEs, allowing checkpoint inhibitor therapy to continue. Surprisingly, the administration of such cells produced by the method of the present invention, specifically the immunomodulatory NK cells of the present invention, does not interfere with the subject's anticancer response to checkpoint inhibitor therapy. This represents a significant advantage compared to known treatments / preventions of irAEs, specifically the administration of immunosuppressive drugs such as corticosteroids. The results surprisingly show that performing ECP does not significantly alter the anticancer efficacy of checkpoint inhibitor therapy, while the concomitant administration of glucocorticoids (a specific class of corticosteroids), specifically prednisolone, worsens the results, indicating a reduction in the effectiveness of checkpoint inhibitor therapy.

[0042] Furthermore, in various embodiments in which (human) blood-derived samples—which are subjected to a method of adding a photosensitizer to the sample and irradiating the sample—are used to treat and / or prevent irAEs in subjects who have received checkpoint inhibitor therapy, regardless of the source of the blood-derived sample (autologous or xenogeneic), the administration of the sample or the cells produced by such methods may occur, for example, before or after the onset of irAE symptoms and / or during or after checkpoint inhibitor therapy.

[0043] In embodiments of the invention, the irAE (of the blood donor and / or the cell recipient) includes symptoms of autoimmune diseases and / or is caused by an autoimmune response. In a preferred embodiment, the irAE includes autoimmune colitis or autoimmune colitis.

[0044] In various embodiments, the irAE includes at least one irAE selected from the group consisting of: autoimmune colitis, autoimmune hepatitis, autoimmune thyroiditis, and autoimmune dermatitis.

[0045] In various embodiments, the irAE includes at least one irAE selected from the group consisting of: immune checkpoint inhibitor-associated colitis, immune checkpoint inhibitor-associated hepatitis, immune checkpoint inhibitor-associated thyroiditis, and immune checkpoint inhibitor-associated dermatitis.

[0046] In each embodiment, the subject (blood donor and / or cell recipient) suffers from cancer, such as malignant melanoma or another cancer that can be treated with checkpoint inhibitor therapy.

[0047] In each embodiment, the subject is receiving immunosuppressive drugs such as steroids, corticosteroids, cyclosporine and / or anti-TNF antibodies (e.g., infliximab) and / or is refractory to immunosuppressive drugs.

[0048] Completely unexpectedly, the application of cells produced by the methods described herein, such as immunomodulatory NK cells, has been therapeutically effective in patients with irAE who are refractory to immunosuppressive drugs already administered for the treatment of irAE symptoms. For such patients, there are no effective treatments available to alleviate irAE symptoms, and therefore this invention represents a completely unexpected possibility for treating irAE in these patients.

[0049] In various embodiments, the checkpoint inhibitor therapy includes administration of at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody.

[0050] In various embodiments, the immunomodulatory NK cells used to treat and / or prevent irAEs in subjects who have received checkpoint inhibitor therapy are autologous to the subject. In alternative embodiments, the NK cells may be xenogeneic to the subject.

[0051] In each embodiment, the immunomodulatory NK cells according to the invention are administered after the onset of irAE symptoms. In each embodiment, the cells are administered 1, 2, 3, 4, 5, 6, or 7 days after the onset of irAE symptoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50 weeks after the onset of irAE symptoms. In each embodiment, the immunomodulatory NK cells are administered during or after checkpoint inhibitor therapy has been discontinued.

[0052] In various embodiments, the immunomodulatory NK cells are administered to the subject at least once, preferably at least twice, and preferably over several consecutive days. Therefore, the subject may receive more than one dose of NK cells, preferably no more than one dose per day. In other embodiments, the subject receives at least 2, 3, 4, or 5 doses of the NK cells of the present invention. In various embodiments, the immunomodulatory NK cells of the present invention are administered to the subject at least every 8 weeks, preferably every 2-4 weeks.

[0053] Each optional or preferred feature of the invention disclosed or described in the context of one aspect of the invention is also disclosed in the context of the other aspects of the invention described herein.

[0054] Furthermore, the present invention also relates to the following embodiments:

[0055] 1. An in vitro photodisplacement (ECP) system for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy, wherein the ECP is preferably performed with administration of 8-methoxypsoralen.

[0056] This invention is based on the completely unexpected discovery that patients who have received checkpoint inhibitor therapy for, for example, cancerous diseases, resulting in irAEs such as autoimmune colitis, can be effectively treated with ECP. This is even applicable to patients who are refractory to other immunosuppressive therapies such as steroids or anti-TNF antibodies and who still exhibit symptoms of or suffer from irAEs after discontinuing checkpoint inhibitor therapy.

[0057] Surprisingly, the positive effects and efficacy of ECP treatment have been demonstrated specifically due to the regulation of NK cell function following ECP. Therefore, the present invention also specifically relates to immunomodulatory NK cells for the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy, wherein the immunomodulatory NK cells are generated by subjecting blood or MNCs or NK cells derived from human, preferably from subjects of the present invention, to ECP treatment.

[0058] 2. The ECP system provided for use according to the present invention, wherein the ECP system is an online ECP system.

[0059] 3. The ECP system provided for use according to the present invention, wherein the ECP system is an offline ECP system.

[0060] 4. The ECP system provided for use according to the present invention, wherein the subject exhibits symptoms of or suffers from irAE.

[0061] 5. The ECP system provided according to the invention, wherein the checkpoint inhibitor therapy is discontinued after symptoms and / or manifestations of irAE occur in the subject.

[0062] 6. The ECP system provided according to the invention, wherein symptoms and / or manifestations of irAE occur after the discontinuation of the checkpoint inhibitor therapy.

[0063] 7. The ECP system provided according to the invention, wherein symptoms and / or manifestations of irAE persist after discontinuation of the checkpoint inhibitor therapy.

[0064] 8. An ECP system according to any of the present invention, wherein the ECP treatment is initiated after the onset of irAE symptoms.

[0065] 9. The ECP system for use according to the invention, wherein said ECP treatment is initiated 1, 2, 3, 4, 5, 6 or 7 days or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks or 50 weeks after the onset of irAE symptoms.

[0066] 10. An ECP system according to the invention, wherein ECP treatment is initiated during or after checkpoint inhibitor therapy is discontinued.

[0067] 11. The ECP system for use according to the invention, wherein ECP treatment comprises at least one, preferably at least two, ECP cycles, preferably for several consecutive days.

[0068] 12. The ECP system according to the invention, wherein ECP treatment is performed at least 2, 3, 4 or 5 times.

[0069] 13. The ECP system according to the invention, wherein ECP treatment is performed at least every 8 weeks, preferably every 2-4 weeks.

[0070] 14. The ECP system provided for use according to the invention, wherein the irAE includes symptoms of autoimmune diseases.

[0071] 15. The ECP system provided according to the invention, wherein the irAE is caused by an autoimmune response.

[0072] 16. The ECP system for use according to the invention, wherein the irAE includes at least one irAE selected from the group consisting of: autoimmune colitis, autoimmune hepatitis, autoimmune thyroiditis and autoimmune dermatitis.

[0073] 17. The ECP system for use according to the invention, wherein the irAE includes at least one irAE selected from the group consisting of: immune checkpoint inhibitor-associated colitis, immune checkpoint inhibitor-associated hepatitis, immune checkpoint inhibitor-associated thyroiditis, and immune checkpoint inhibitor-associated dermatitis.

[0074] 18. The ECP system provided according to the invention, wherein the irAE includes autoimmune colitis.

[0075] 19. The ECP system provided according to the invention, wherein the irAE is autoimmune colitis.

[0076] 20. The ECP system provided for use according to the invention, wherein the subject is a human.

[0077] 21. The ECP system for use according to the invention, wherein the subject suffers from cancer, such as malignant melanoma or another cancer that can be treated with checkpoint inhibitor therapy.

[0078] 22. The ECP system provided for use according to the invention, wherein the subject is receiving an immunosuppressive drug, such as a steroid, corticosteroid, cyclosporine and / or an anti-TNF antibody (e.g., infliximab).

[0079] 23. The ECP system provided for use according to the invention, wherein the subject is refractory to immunosuppressive drugs such as steroids, corticosteroids, cyclosporine and / or anti-TNF antibodies (e.g., infliximab).

[0080] 24. The ECP system for use according to the invention, wherein the checkpoint inhibitor therapy comprises administering at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody.

[0081] 25. An ECP system for use according to the invention, wherein the ECP system is used to perform blood irradiation therapy.

[0082] 26. An ECP system for use according to the present invention, wherein the ECP system comprises an immunomodulatory molecule, preferably an immunomodulatory molecule membrane-coupled to the ECP system, wherein the immunomodulatory molecule contacts the immune cells of the subject.

[0083] 27. An in vitro method comprising the following steps:

[0084] - Provide a sample of isolated blood from a subject who has received checkpoint inhibitor therapy and is suspected of having or has already experienced symptoms of immune-related adverse events (irAEs).

[0085] - The sample is subjected to receptor-based photodisplacement method.

[0086] 28. A method of treating a subject who has received checkpoint inhibitor therapy and is suspected of having or has already had symptoms of an immune-related adverse event (irAE), the method comprising subjecting the subject to extrasensory photodisplacement (ECP) therapy (ECP), such as blood irradiation therapy using an ECP system.

[0087] 29. An immunomodulatory NK cell for the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy.

[0088] 30. The immunomodulatory NK cells for use according to the invention, wherein the immunomodulatory NK cells have been generated by subjecting a human blood sample or cells included in a human blood sample to a receptor-dependent photodisplacement method.

[0089] 31. The immunomodulatory NK cells for use according to the invention, wherein the NK cells are autologous or xenogeneic relative to the subject.

[0090] 32. The immunomodulatory NK cells for use according to the invention, wherein the NK cells are administered intravenously to the subject.

[0091] 33. The immunomodulatory NK cells for use according to the invention, wherein the NK cells are isolated from the human blood sample before or after receptor-mediated photodissociation.

[0092] All features disclosed in the context of an ECP system for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy of the present invention also relate to in vitro methods, therapeutic methods, and NK cells of the present invention for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy of the present invention, and therefore are also disclosed in the context of said in vitro methods, said therapeutic methods, and said NK cells, and vice versa. Detailed Implementation

[0093] All cited documents in patent and non-patent literature are hereby incorporated in their entirety by reference.

[0094] This document discloses a method comprising the following steps: providing a blood sample from a subject, preferably a subject who has received checkpoint inhibitor therapy and is suspected of having or has already experienced symptoms of an immune-related adverse event (irAE); adding a photosensitizer to the sample; and subjecting the sample to irradiation. The irradiation of the blood sample may be performed using any suitable system or irradiation device known to a person skilled in the art. Preferably, the irradiation is performed using an extracorporeal photodisplacement (ECP) system.

[0095] The present invention also relates to an in vitro photodisplacement (ECP) system for treating and / or preventing immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy, wherein the ECP is preferably performed in the presence of 8-methoxypsoralen.

[0096] Irradiation and in vitro photodisplacement (ECP) method

[0097] Photodissociation, or extracorporeal photodissociation or ECP, is a form of apheresis and photodynamic therapy in which blood is treated with a photosensitizer and then irradiated with light of a specified wavelength to achieve a particular effect. For example, erythrocyte sedimentation rate (ESR) amber (WBC+ platelets) can be separated from whole blood, chemically treated with 8-methoxypsoralen (infused into a collection bag or administered orally beforehand), exposed to ultraviolet (UVA), and returned to the patient. The activated 8-methoxypsoralen cross-links with the DNA in the exposed cells, ultimately leading to apoptosis of nucleated cells. The photochemically damaged T cells returned to the patient appear to induce cytotoxic effects on T cell formation.

[0098] The photodissociation method involving 8-methoxypsoralen was first described in the following: a 1987 New England Journal of Medicine publication (Edelson, R et al. (1987). "Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy. Preliminary results." New England Journal of Medicine. 316(6):297–303.). Photodissociation is currently the standard treatment approved by the U.S. Food and Drug Administration (FDA) for cutaneous T-cell lymphoma. There is evidence that this treatment is effective in treating graft-versus-host disease. Photodissociation has also been successfully used to treat acquired epidermolysis bullosa when all other treatments have failed.

[0099] As used herein, ECP includes blood irradiation therapy. In various 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 other than blood irradiation therapy.

[0100] Blood irradiation therapy is a procedure that exposes blood to low levels of red light (usually laser) for therapeutic purposes. Blood irradiation therapy can be administered in three ways. Externally, blood is drawn and irradiated into a special cuvette. This method is used for ultraviolet (UV) blood irradiation (UVBI) via a UV lamp. Lasers are monochromatic, meaning their wavelength allows light to enter an optical fiber and irradiate intravenously 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 a protruding portion of a large blood vessel.

[0101] Assuming any therapeutic effect circulates through the circulatory system, intravenous or intravascular laser blood irradiation (ILBI) involves irradiating blood within the body by feeding a low-level laser at a wavelength of 632.8 nm, generated by a 1-3 mW helium-neon laser, into a vascular channel, typically a vein in the forearm. Most commonly, wavelengths of 365, 405, 525, and 635 nm and a power of 2.3 mW are used. This technique is currently widely used in Russia, less so in Asia, and not widely used in other parts of the world. ILBI has been shown to improve blood flow and its transport activity, thus improving tissue nutrition and having a positive effect on the immune system and cellular metabolism. This argument is questionable. There have been calls for increased research on this subject. Percutaneous therapy applies lasers to unbroken skin in areas with abundant blood vessels, such as the forearm. Because the skin, acting as a barrier to blood, absorbs low-level laser energy, the laser power is often increased to compensate. This problem can be addressed by using a pulsed matrix laser source. External beam irradiation is used only for ultraviolet blood irradiation, which involves drawing blood through a vein and irradiating the blood outside the body. Although promoted as a treatment for cancer, a 1952 review published in the Journal of the American Medical Association and another review by the American Cancer Society in 1970 concluded that the treatment was ineffective.

[0102] Extracorporeal photolysis replacement (ECP), also known as extracorporeal photoimmunotherapy or photochemotherapy, is a leukocyte-removal-based therapy originally used for patients with cutaneous T-cell lymphoma (CTCL). Specifically, ECP was approved by the FDA (U.S. Food and Drug Administration) in 1988 to treat patients with treatment-resistant CTCL suffering from a leukemia variant, Sézary Syndrome. During ECP, whole blood is collected from the patient via the antecubital vein or a permanently implanted catheter to separate leukocytes from plasma and enucleated cells. Using a specially constructed device for this procedure, the collected leukocytes, known as the erythrocyte sedimentation rate (ESR) amber layer, are then exposed to ultraviolet-A (UVA) radiation in the presence of the photosensitizer 8-methoxypsoralen before being infused into the patient.

[0103] Two fundamentally different methods for performing ECP procedures have been described and are included within this invention. These two methods differ in the apparatus used for leukocyte collection and UVA irradiation: a “closed system” and a so-called “open system.” The closed system is based on the original design of Edelson and colleagues and is the only system approved by the FDA. The open system is a system incorporated with different separation instruments and is primarily used outside the United States. Although ECP has been an effective treatment method for 30 years and has been performed over 2 million times, no negative cytogenetic effects have been reported.

[0104] Since its introduction, the indications for ECP have been continuously expanding. ECP treatment is generally well tolerated by patients and has few or no significant undesirable side effects. In summary, ECP combines excellent safety profiles with efficacy.

[0105] In vitro photodissociation (sometimes also called in vitro photochemotherapy) involves the following procedures: (1) collecting monocytes (MNCs) from a patient; (2) photoactivating the collected MNCs; and (3) re-infusing the treated cells (MNCs) back into the patient. More specifically, ECP involves the in vitro exposure of peripheral blood monocytes to a photoactive compound such as 8-methoxypsoralen or “8-MOP”, followed by photoactivation with ultraviolet light, and then re-infusion of the treated monocytes. It is believed that the combination of 8-MOP and UV radiation induces apoptosis or programmed cell death in ECP-treated T cells.

[0106] While the exact mechanism of action of ECP therapy (in different disease states) is not fully understood, early theories suggest that photoactivation causes 8-MOP to irreversibly covalently bind to the DNA strand contained in the nucleus of T cells. When photochemically damaged T cells are re-infused, a cytotoxic effect is induced. For example, cytotoxic T cells, or "CD8+ cells," release cytotoxins or otherwise attack cells carrying certain foreign or abnormal molecules on their surface when exposed to infected or damaged cells. The cytotoxins target the membrane of the damaged cell and enter the target cell, ultimately leading to apoptosis or programmed cell death of the target cell. In other words, after the treated monocytes are returned to the body, the immune system recognizes the dying, abnormal cells and begins to produce healthy lymphocytes (T cells) to fight them.

[0107] In addition to the above, it is theoretically believed that in vitro photodissociation can also induce monocytes (a type of mononuclear cell) to differentiate into dendritic cells capable of phagocytizing and processing apoptotic T-cell antigens. When these activated dendritic cells are re-infused into the systemic circulation, they can elicit a systemic cytotoxic CD8+ T lymphocyte-mediated immune response to the processed apoptotic T-cell antigens, as described above. It should be understood that other possible mechanisms of action may involve achieving the benefits observed from ECP treatment of monocytes and subsequent benefits to patients receiving ECP-based therapies.

[0108] Recently, it has been hypothesized that ECP can induce an immune tolerance response in patients. For example, in graft-versus-host disease (GVHD), the infusion of apoptotic cells can stimulate the production of regulatory T cells, inhibit the production of inflammatory cytokines, induce the loss of effective T cells, and generate other responses. See Peritt, “Potential Mechanisms of Photopheresis in Hematopoietic Stem Cell Transplantation,” *Biology of Blood and Marrow Transplantation*, 12:7-12 (2006). While the theory of immune tolerance response currently appears to be one of the main explanations, other theories exist regarding the role of ECP in GVHD and other disease states.

[0109] For example, systems used to perform ECP include the UVAR XTS optical separation and replacement system and the CellEx optical separation and replacement system, which are available from Therakos, Inc. of Exton, Pa. Further details regarding performing ECP on a Therakos system can be found, for example, in U.S. Patent No. 5,984,887.

[0110] There are currently two commonly used methods for performing optical separation and replacement: online and offline systems and methods.

[0111] In online methods, dedicated photodissociation devices, such as the Therakos device mentioned above, are used to perform the entire therapy, including the reinfusion of processed MNCs. These devices are "dedicated" photodissociation devices, designed solely for performing photodissociation and incapable of performing other collection protocols required in hospital or blood processing settings, including, for example, multi-functional apheresis protocols for collecting platelets, plasma, RBCs, and granulocytes, and / or incapable of performing plasma / RBC exchange protocols.

[0112] In offline photodissociation methods, multifunctional apheresis devices can be used to collect mononuclear cells (MNCs). The collected MNCs, typically contained in one or more collection containers, are disconnected from or otherwise separated from the tubing used during collection. The collected MNCs are then processed later in a separate irradiation or UVA light device, and the processed cells are manually re-infused to the patient. However, during such offline methods, when transferring cells from the apheresis device to an irradiation device (which may be located in another room or laboratory), the connection to the donor must be severed, and the cells detached from the donor accordingly. Therefore, additional traceability procedures are required to ensure that the processed MNC product is ultimately re-infused to the correct donor.

[0113] The apparatus and the immobilization of enzymes within the apparatus:

[0114] In embodiments of the invention, the ECP system may include a device comprising a matrix having immobilized immunomodulatory molecules or other biomolecules, such as enzymes. Preferably, in the context of the invention, the matrix having conjugated molecules is exposed to blood or MNC.

[0115] Therefore, as used herein, "matrix" refers to the material inside a blood processing device that provides an internal material or surface through which blood or plasma passes or traverses. As used in the context of this invention, the matrix preferably comprises a support to which immobilized immunomodulatory molecules or other biomolecules are bound. Thus, the support acts as a carrier of immobilized immunomodulatory molecules or other biomolecules, but it may also perform other functions.

[0116] As used herein, “support” refers to the portion of the matrix to which the immobilized immunomodulatory molecules or other biomolecules according to the invention bind as a “substrate” or “support material.” Such supports or support materials are sometimes also referred to as “adsorbent materials” or “adsorbers,” as used in “adsorption columns” or “columns” or “adsorption tubes.” Suitable supports according to the invention should be homogeneous, hydrophilic, physically and chemically stable within the relevant pH range and temperature, exhibit no or negligible enzyme leaching during use, possess good flow properties for whole blood and / or plasma, and provide a large surface area for enzyme attachment.

[0117] For example, the support can be resin, membrane, or nonwoven material. "Nonwoven" material is broadly defined as a sheet, fabric, or mesh structure bonded together by physical, thermal, or chemical entanglement of fibers or filaments (and by perforating the membrane) rather than by weaving or braiding. "Resin" refers to an insoluble material that can take the form of 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 polymers or organic supports are primarily based on acrylamide, polystyrene, and polymethacrylate derivatives, while porous silica and glass are some commonly used inorganic supports.

[0118] According to one embodiment of the present invention, the resin comprises: a polymer selected from the group consisting of: alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin, and agarose; an inorganic material selected from the group consisting of: zeolite, ceramics, diatomaceous earth, silica, glass, activated carbon, and charcoal; or a synthetic polymer selected from the group consisting of: polyethylene (PE), polyoxymethylene (POM), polypropylene (PP), polyvinyl chloride (PVC), polyvinyl acetate (PVA), polyvinylidene chloride (PVDC), polystyrene (PS), polytetrafluoroethylene (PTFE), polyacrylate (PAA), polymethyl methacrylate (PMMA), polyacrylamide, polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN), polyester, polycarbonate, polyethylene terephthalate (PET), polyamide, polyarylamide, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polysulfone (PS), and polyethersulfone (PES). Polyarylene ether sulfone (PEAS), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), polyamide imide, polyarylene ether ketone (PAEK), polybutadiene (PBD), polybutene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyhydroxyalkanoates, polyether ether ketone (PEEK), polyether ketone ketone (PEKK), polyetherimide (PEI), polyimide, polylactic acid (PLA), polymethylpentene (PMP), poly(p-phenylene ether) (PPE), poly Urea (PU), styrene-acrylonitrile (SAN), polybutenoic acid, poly(4-allylbenzoic acid), poly(glycidyl acrylate), poly(PGMA), acrylonitrile butadiene styrene (ABS), polydivinylbenzene (PDVB), poly(allyl glycidyl ether), poly(vinyl glycidyl ether), poly(vinyl glycidyl urethane), polyallylamine, polyethyleneamine, copolymers of said polymers, and any of these polymers modified by introducing functional groups.

[0119] Immobilized immunomodulatory molecules or other biomolecules can be immobilized onto the support and / or matrix according to the invention using various known methods. Such immobilization is preferably specific or selective because it immobilizes enzymes while other proteins and components present in blood or plasma or their samples (in vitro) are not significantly immobilized.

[0120] "Immobilizing" an immobilized immunoregulatory molecule or other biomolecule to a support for providing a matrix that can be used in a device according to the invention refers to a non-covalent or covalent interaction that holds two molecules together. According to one embodiment of the invention, the expression refers to a covalent interaction, i.e., a covalently bound immobilized immunoregulatory molecule or other biomolecule. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions between charged groups, van der Waals interactions, and hydrophobic interactions between nonpolar groups. One or more of these interactions can mediate the binding of two molecules to each other. The binding can be specific, selective, or non-specific in other ways.

[0121] According to one embodiment, the immobilized immunoregulatory molecules or other biomolecules include affinity tags for immobilizing them onto a support. The affinity tags can be used to purify proteins during production and / or to immobilize them onto a support of the matrix of the present invention. The affinity tags can be short polypeptide sequences or full proteins co-expressed with a fusion partner of the enzyme. Different types of affinity tags are well known in the art, among which multihistidine or His6 tags, C-myc tags, and FLAG tags are described in particular detail and are options for binding enzymes according to the present invention to the support material. Non-covalent linkage of biotin to streptavidin or avidin can also be used to immobilize the immobilized immunoregulatory molecules or other biomolecules onto the support.

[0122] According to another embodiment of the invention, immobilized immunoregulatory molecules or other biomolecules are covalently linked to a support, as further detailed below and / or as described in the prior art. Covalent coupling typically involves covalent non-site-directed linkage of proteins or site-directed linkage of proteins. The support constituting the basis for matrix generation must provide or facilitate chemical activation, thereby allowing the chemical coupling of immobilized immunoregulatory molecules or other biomolecules. Many coupling methods for immobilizing immobilized immunoregulatory molecules or other biomolecules are well known in the art.

[0123] For example, the activation chemistry should be stable over a wide range of pH, buffer conditions, and temperatures, resulting in negligible enzyme leaching. Coupling methods should avoid misalignment, multi-site linkages, or steric hindrance of immobilized immunoregulatory molecules or other biomolecules. Enzyme density per volume of matrix can be optimized to improve target accessibility and reaction.

[0124] Covalent coupling 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.

[0125] Alternatively, the support may carry specific functional groups for coupling the linker and / or enzyme to it. For example, functionalized resins are commercially available and known to those skilled in the art. These commercial supports may contain a variety of coupling chemicals, involving primary amines, thiol groups, aldehydes, hydroxyl groups, and carboxylic acids. Examples of commercially available activated resins are CarboLink coupling resin and Profinity. TM Epoxy resin, Affi-Gel 10 and 15, epoxy-activated Sepharose TM 6B. Agarose activated with trifluoroethanesulfonyl chloride and functionalized with epoxy groups Lifetech TM Methacrylate polymers.

[0126] According to one embodiment of the invention, the support material should be porous, wherein the pore size is in the range of 10 to 200 nm. According to another embodiment of the invention, the support is in the form of beads. According to yet another embodiment, the support according to the invention comprises magnetic beads. Magnetic beads are prepared by embedding magnetite within agarose or other polymer material immobilized with an enzyme according to the invention.

[0127] According to another embodiment of the invention, the support is a membrane. Membranes, as components of the affinity matrix, have been used for protein purification due to their simplicity, ease of handling, reduced surface area compared to gels, resins, and beads, and less diffusion restriction. The membrane can be in the physical form of a hollow fiber membrane, or alternatively, in the physical form of a sheet membrane. According to one embodiment, the support comprises a hemodialysis hollow fiber membrane dialyzer, wherein the filter is a hemodialysis unit.

[0128] Hollow fiber or sheet membranes used as supports in the devices according to the invention can be composed of cellulose, cellulose esters (cellulose acetate and cellulose triacetate), poly(methyl methacrylate) (PMMA), polyamide (PA), other nitrogen-containing polymers (polybenzimidazole, polyacrylonitrile (PAN), polyglycidyl methacrylate (PGMA), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES), or polyarylethersulfone (PAES). The inner diameter of the hollow fiber membrane used to provide the devices according to the invention is preferably in the range of 100 to 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, on the luminal side, where the enzyme can directly interact with target metabolites in the blood or plasma perfusing the lumen of the hollow fiber membrane. The enzyme can also or alternatively be immobilized to the exterior of the membrane.

[0129] Methods for extracorporeal blood processing:

[0130] The present invention includes an apparatus configured to be located in an extracorporeal blood circuit through which patient blood passes and including components for delivering blood from the patient's vascular system to a blood processing device at a defined flow rate and then returning the processed blood to the patient, wherein the apparatus is further configured to reduce the level of ADMA and / or MMA in the blood. Furthermore, the present invention also includes an apparatus that can be used for extracorporeal blood or blood cell processing, wherein the apparatus can be disconnected or detached from the extracorporeal blood circuit.

[0131] According to the present invention, the expression "extracorporeal blood purification" preferably refers to a process of removing substances from bodily fluids by removing substances from flowing blood in a transfer circuit outside the patient's body (extracorporeal). The 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. Treatment 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 can be accordingly incorporated into the apparatus of the present invention.

[0132] As used herein, the term "blood" refers to whole blood containing all the components of a living 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, forming a portion of whole blood but no longer containing red blood cells, white blood cells, or platelets. In the context of this invention, the term "blood plasma" (or plasma) refers to a specific fraction of plasma as defined above in the standard sense, such as serum.

[0133] According to one aspect, the blood flow rate in the extracorporeal blood purification circuit is between 20 ml / min and 700 ml / min. In addition to the blood treatment device according to the invention, it also includes a hemodialyzer for treating renal failure, or, in cases where the hemodialyzer is further configured to metabolize ADMA and / or MMA, a typical dialysate flow rate in the extracorporeal circuit is in the range of 0.5 liters / hour to 800 ml / min.

[0134] In therapeutic apheresis, whole blood can be processed or separated into fractions, for example by centrifugation or by using a plasma membrane or filter, and fractions containing solutes that should be removed are specially processed before being returned to the patient.

[0135] This invention provides apheresis treatment in which whole blood or plasma (containing target proteins) is removed from a patient's flowing blood, and the whole blood or plasma is returned to the patient after contact with a device or matrix according to the invention. Typical blood or plasma flow rates in the extracorporeal circuit perfused with whole blood or plasma by the blood processing device are in the range of 30 mL / min to 200 mL / min or 7 mL / min to 50 mL / min, respectively.

[0136] According to one aspect, an 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, which has been further configured to immobilize the target protein according to the invention. The circuit can operate in different treatment modes according to medical needs, including hemodialysis, hemodialysis filtration, and blood filtration mode.

[0137] Immune-related adverse events (irAEs)

[0138] As used herein, the term "immune-related adverse event" (irAE) refers to any specific side effect that occurs in the context of immune checkpoint inhibitor therapy, such as in the context of cancer therapy. irAEs are unique and distinct from adverse events occurring in the context of conventional cancer therapy, and typically have delayed onset and prolonged duration. irAEs can involve any organ or system. These effects are usually low-grade and treatable and reversible; however, some adverse reactions can be serious and lead to permanent condition. Treatment is primarily based on corticosteroids and other immunomodulatory agents, and should be prescribed with caution to minimize the possibility of short-term and long-term complications.

[0139] The terminology specifically includes symptoms of autoimmune diseases and autoimmune diseases such as (autoimmune) colitis, (autoimmune) hepatitis, (autoimmune) thyroiditis, and (autoimmune) dermatitis.

[0140] In this invention, there are no particular limitations on irAEs caused by the administration of checkpoint inhibitor therapy. irAEs are understood to be presumed to be immune-related adverse events: irAEs (see, for example, Drug interview form for intravenous infusion of 20mg-100mg, revised in April 2016 (9th edition); anti-CTLA-4 antibody ipilimumab. The nature and handling of the adverse event, dated August 24, 2015, was released by the Committee on Safety of New Drugs for Malignant Melanoma of the Japanese Dermatological Association.

[0141] The present invention irAE Specific embodiments preferably include interstitial lung disease, myasthenia gravis, myositis, colitis, type 1 diabetes, liver dysfunction (liver disease), lung disease such as hepatitis (e.g., autoimmune pneumonia), pituitary dysfunction such as hypopituitarism or hypophysitis, thyroid dysfunction such as hypothyroidism, neuropathy, nephropathy, encephalitis, adrenal disease such as adrenal insufficiency, severe skin disease, venous thromboembolism, infusion reaction, psoriasis, psoriasis-like rash, diarrhea (e.g., severe diarrhea), rheumatoid arthritis, uveitis, episcleritis, bursitis, exacerbation of radiation dermatitis, chronic inflammatory demyelinating polyneuropathy (hereinafter also referred to as demyelinating polyneuropathy), biliary tract disease or nephritis, and pituitary dysfunction.

[0142] Immune-related adverse events (IrAEs) are known to occur, for example, 8 weeks or 8 to 12 weeks after administration. IrAEs can be assessed using a "grading" or "IrAE assessment." Here, the "irAE assessment" is an index representing the severity of the disease and is represented by a number from 1 to 3. In the irAE assessment, 1 indicates a condition "where no further treatment intervention is required due to the irAE," 2 indicates a condition "where pharmacological intervention is required due to the irAE, but hospitalization or treatment interruption is not required," and 3 indicates a condition "where pharmacological intervention is required due to the irAE, and hospitalization and treatment interruption are required." The correspondence between the "irAE assessment" and the "grading" varies depending on the disease.

[0143] Immune checkpoint molecules and checkpoint modulators

[0144] In the context of this invention, immune checkpoint inhibitors are drugs that activate immune cells by modulating immune checkpoint molecules.

[0145] Immune checkpoint molecules are molecules in the immune system that enhance (co-stimulatory) signals (co-stimulatory molecules) or weaken signals provided to immune effector cells. Therefore, immune checkpoint molecules can be further subdivided into co-stimulatory checkpoint molecules and co-inhibitory checkpoint molecules. Co-stimulatory checkpoint molecules contain co-stimulatory lymphocyte receptors, which are lymphocyte surface receptors that can induce activation or stimulation of lymphocyte effector function. Co-inhibitory checkpoint molecules contain co-inhibitory lymphocyte receptors, which are lymphocyte surface receptors that can induce inhibition of lymphocyte effector function.

[0146] Co-stimulatory checkpoint molecules include, but are not limited to, HVEM, CD27, CD40, OX40, GITR, CD137, CD28, and ICOS.

[0147] In a preferred embodiment of the invention, the term co-stimulatory lymphocyte receptor does not refer to CD122.

[0148] HVEM (herpesvirus entry mediator, CD270) is also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14) and is a receptor of the TNF receptor superfamily that can bind to BTLA.

[0149] CD27 supports antigen-specific amplification of primordial T cells and is crucial for the development of T cell memory, as well as serving as a memory marker for B cells. CD27 activity is controlled by the transient availability of its ligand CD70 on lymphocytes and dendritic cells. CD27 co-stimulation is known to inhibit Th17 effector cell function. The agonistic monoclonal antibody against CD27, CDX-1127 / varlilumab, has been shown to be effective in animal models in the context of T cell receptor stimulation.

[0150] CD28 is constitutively expressed on almost all human CD4+ T cells and about half of all CD8 T cells. For example, binding to one of its two ligands, CD80 and CD86, expressed on dendritic cells, promotes T cell proliferation.

[0151] CD40 is expressed on a variety of immune system cells, including antigen-presenting cells. The ligand for CD40 is called CD40L, also known as CD154, and is transiently expressed on the surface of activated CD4+ T cells. CD40 signaling is known to “permit” dendritic cell maturation, thereby triggering T cell activation and differentiation.

[0152] 4-1BB (CD137) binds to its ligand, thereby inducing T cell proliferation. CD137-mediated signaling is also known to protect T cells, specifically protecting 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 aggressive attack by the immune system against cancer.

[0153] OX40 (CD134) uses OX40L (CD252) as its ligand. OX40 promotes the expansion of effector and memory T cells and is also known for its ability to inhibit the differentiation and activity of regulatory T cells. OX40 is transiently expressed after T cell receptor engagement, which is why it is upregulated only on T cells activated by the most recent antigen within inflammatory lesions, making OX40 a valuable drug target. Agonisttic anti-OX40 monoclonal antibodies have shown clinical utility in advanced cancers. Pharmaceutical company AstraZeneca is developing three drugs targeting OX40: MEDI0562, a humanized OX40 agonist; MEDI6469, a mouse OX40 agonist; and MEDI6383, an OX40 agonist.

[0154] GITR (a glucocorticoid-induced TNFR family-associated gene) promotes T cell proliferation. The GITR ligand (GITRL) is primarily expressed on antigen-presenting cells. Antibodies against GITR have been shown to enhance antitumor responses by destabilizing the Treg lineage.

[0155] ICOS (inducible T cell co-stimulatory factor, also known as CD278) is expressed on activated T cells. Its ligand is ICOSL, which is primarily expressed on B cells and dendritic cells. This molecule appears to play an important role in T cell effector function.

[0156] 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.

[0157] A2AR (adenosine A2A receptor) is considered an important checkpoint in cancer therapy because adenosine in the immune microenvironment that causes A2a receptor activation forms a negative immune feedback loop, and the concentration of adenosine in the tumor microenvironment is relatively high.

[0158] B7-H3, also known as CD276, was initially considered a co-stimulatory molecule but is now considered a co-inhibitory molecule. MacroGenics is investigating MGA271 (enoblituzumab), an Fc-optimized monoclonal antibody that targets B7-H3.

[0159] B7-H4 (or VTCN1) is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape.

[0160] BTLA (B and T lymphocyte attenuator, also known as CD272) is a co-inhibitory receptor whose ligand is HVEM (herpesvirus entry mediator). During the differentiation of human CD8+ T cells from the primordial to the effector cell phenotype, the surface expression of BTLA is gradually downregulated; however, tumor-specific human CD8+ T cells express high levels of BTLA.

[0161] CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152) is expressed on Treg cells and is used to control T cell proliferation. CTLA-4 (CD152) is a protein receptor that acts as an immune checkpoint and is expressed by activated T cells, transmitting inhibitory signals to them. CTLA4 is homologous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 has a greater affinity for CD80 and CD86 than for CD28. CTLA4 transmits inhibitory signals to T cells. Antagonistic antibodies against CTLA4 include ipilimumab and tremelimumab.

[0162] IDO (indoleamine 2,3-dioxygenase) is an immunosuppressive tryptophan-catabolizing enzyme. Another important molecule is TDO (tryptophan 2,3-dioxygenase). IDO is known to inhibit T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis.

[0163] KIR (Killer Cell Immunoglobulin-like Receptor) is a receptor for MHC class I molecules on natural killer cells. Lirilumab is a monoclonal antibody targeting KIR.

[0164] LAG-3 (lymphocyte activation gene-3) suppresses the immune response by acting on Tregs and directly on CD8+ T cells.

[0165] PD-1 (programmed death 1 or CD279) is a cell surface receptor that plays a crucial role in downregulating the immune system and promoting self-tolerance by inhibiting T-cell inflammatory activity. PD-1 has two ligands, PD-L1 and PD-L2. The advantage of targeting PD-1 is its ability to restore immune function in the tumor microenvironment. PD-L1, the ligand of PD-1, is highly expressed in several cancers and can lead to the suppression of T-cell anti-cancer immune responses. Numerous cancer immunotherapies targeting the PD-1 receptor have been developed, including the antagonistic antibodies nivolumab (Opdivo, Bristol Myers Squibb), pembrolizumab (Keytruda, MK-3475, Merck), pitilizumab (CT-011, Cure Tech), and BMS-936559 (Bristol Myers Squibb). Atezolizumab (MPDL3280A, Roche, Switzerland) and avirumab (Merck KGaA, Darmstadt, Germany and Pfizer, Germany) are both monoclonal antibodies targeting PD-L1, the ligand of PD-1.

[0166] TIM-3 (T cell immunoglobulin domain and mucin domain 3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1 / Th17 function by triggering cell death upon interaction with its ligand galactoglobulin-9.

[0167] VISTA (a T-cell activation V-domain Ig inhibitor) is a protein primarily expressed on hematopoietic cells, and consistent expression of VISTA on leukocytes within tumors allows for effective VISTA blockade in a wide range of solid tumors.

[0168] TIGIT (a T-cell immune receptor with Ig and ITIM domains, also known as WUCAM and Vstm3) is an immune receptor present on certain T cells and natural killer cells and regulates T-cell-mediated immunity. TIGIT can bind with high affinity to CD155 on dendritic cells (DCs) and macrophages, and with lower affinity to CD112.

[0169] The co-inhibitory lymphocyte receptors of the present invention include PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, BTLA, or VISTA. The co-stimulatory lymphocyte receptors of the present invention include OX40, 4-1BB, GITR, CD27, HVEM, CD28, or CD40.

[0170] Receptor inhibitors prevent the corresponding receptor from generating signals. Therefore, inhibitors of co-inhibitory lymphocyte receptors are molecules that prevent the activation of the corresponding receptor and thus prevent the generation of inhibitory signals. Conversely, receptor activators induce the generation of signals by the corresponding receptor, and co-stimulatory lymphocyte receptor activators induce the generation of stimulatory signals.

[0171] Checkpoint modulators are molecules that interfere with the activity of immune checkpoint molecules by stimulating or inhibiting their activity.

[0172] Soluble checkpoint regulators are molecules that can diffuse freely and, for example, do not bind to the cell membrane or remain inside the cell.

[0173] The checkpoint inhibitors in this invention include lymphocyte-stimulating checkpoint modulators, which induce activation of lymphocytes, preferably effector T cells, through the activation of co-stimulatory checkpoint molecules or the inhibition of co-inhibitory checkpoint molecules. Furthermore, soluble lymphocyte-stimulating checkpoint modulators contain molecules that interfere with the activation of membrane-bound immune checkpoint molecules, such as the corresponding immune checkpoint molecules in soluble form.

[0174] Checkpoint modulators can be naturally occurring or engineered molecules that have the function of interfering with or modulating the activity of immune checkpoint molecules. Checkpoint modulators contain, for example, antibody or antibody fragment activity against immune checkpoint molecules that are agonistic or antagonistic, as well as ligands or modified ligands of the immune checkpoint molecules.

[0175] Immune cells:

[0176] The immune cells described herein refer to biological cells that participate in the immune response of a subject. The immune cells are preferably selected from T cells, B cells, dendritic cells, granulocytes, innate lymphoid cells (ILCs), megakaryocytes, monocytes / macrophages, natural killer (NK) cells, platelets, erythrocytes (RBCs), and / or thymocytes.

[0177] The term "immune cells" includes MNCs in the blood, which can also be referred to as peripheral blood mononuclear cells (PBMCs). PBMCs comprise any peripheral blood cell with a round nucleus and are primarily composed of lymphocytes (T cells, B cells, NK cells) and monocytes, while erythrocytes and platelets lack nuclei, and granulocytes (neutrophils, basophils, and eosinophils) have multilobed nuclei. In humans, lymphocytes constitute the majority of the PBMC population, followed by monocytes, with dendritic cells comprising only a small fraction. These cells can be extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates the layers of blood, and gradient centrifugation, which separates the blood into a top plasma layer, followed by a layer of PBMCs and a bottom polymorphonuclear cell fraction (such as neutrophils and eosinophils) and erythrocyte fraction. Polymorphonuclear cells can be further separated by lysing erythrocytes. Basophils are sometimes found in both dense fractions and PBMC fractions.

[0178] T cells, or T lymphocytes, are a type of lymphocyte (a subtype of leukocyte) that plays a central role in cell-mediated immunity. T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of T cell receptors on their cell surface. Several subsets of T cells each have distinct functions. T cell subsets include, but are not limited to, type 1 helper T cells (Th1), type 2 helper T cells (Th2), type 9 helper T cells (Th9), type 17 helper T cells (Th17), type 22 helper T cells (Th22), follicular helper T cells (Tfh), regulatory T cells (Treg), natural killer T cells (NKT), γδ T cells, and CD8+ cytotoxic T lymphocytes (CTLs). Other non-limiting embodiments of T cells include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3 and CD56-positive, non-major histocompatibility complex (MHC)-restricted, natural killer (NK)-like T lymphocytes. T cells can be CD4+ T cells, cytotoxic T cells (CTLs, CD8+ T cells), CD4+CD8+ T cells, CD4CD8 T cells, or any other T cell subset.

[0179] The present invention specifically relates to immunomodulatory NK cells (also referred to as immunomodulatory NK cells) preferably used as cell therapy for the treatment and / or prevention of immune-related adverse events (irAEs) in subjects who have received checkpoint inhibitor therapy.

[0180] Immunomodulatory NK cells are a specific form of innate lymphoid cells (ILCs). ILCs are a group of innate immune cells that belong to the lymphocyte lineage (lymphocytes) but do not respond in an antigen-specific manner because they lack B or T cell receptors. This relatively recently described group of cells has different physiological functions, some of which resemble helper T cells while also including cytotoxic NK cells. Therefore, they play an important role in protective immunity and the regulation of homeostasis and inflammation. Natural killer (NK) cells are cytotoxic innate effector cells that resemble cytotoxic T cells in the adaptive immune system. They are distributed in blood, organs, and lymphoid tissues and account for approximately 15% of peripheral blood lymphocytes. NK cells play a role in tumor surveillance and the rapid elimination of virally infected cells. They do not require the missing “self” signal of MHC class I and can recognize stress cells in the absence of antibodies, allowing them to respond faster than the adaptive immune system. Natural killer (NK) cells play a crucial role in the host's anti-cancer immunity. In response, cancer has developed mechanisms to evade NK cell attacks or induce defective NK cells (Cheng M et al. Cell Mol Immunol. May 2013; 10(3):230-52. doi:10.1038 / cmi.2013.10.Epub April 22, 2013).

[0181] The immunomodulatory NK cells of the present invention are preferably characterized by CD56. 弱 Low expression (or downregulated expression) of CD16 on NK cells. CD16 is FcRγIII, an activated NK cell receptor that can induce potent cytokine production. CD16 shedding / downregulation can be an immunomodulatory mechanism by which NK cells defend against autoimmunity. CD16 downregulation regulates NK cell responses and helps maintain immune homeostasis in antibody- and T cell-dependent pathways. Furthermore, the immunomodulatory NK cells of this invention can exhibit low expression of GM-CSF, IFN-γ, TNF, and / or IL-2.

[0182] In various embodiments, the generation or induction of immunoregulatory NK cells in a sample can be measured by comparing the NK cell phenotype and population distribution of NK cells in samples before and after performing the methods of the present invention, for example by flow cytometry, mass spectrometry analysis of gene expression and / or protein abundance. For example, a shift from CD16-high to CD16-low NK cells after irradiation indicates the induction of an immunoregulatory phenotype. Furthermore, a decrease in the expression of GM-CSF, IFN-γ, TNF, and / or IL-2 in the NK cell population also indicates the induction of immunoregulatory NK cells. Those skilled in the art know suitable methods and protocols for identifying different NK cell populations in a sample. For example, in blood-derived samples, the total NK cell population can preferably be defined and identified by flow cytometry as CD45-positive, CD14-negative, CD3-negative, CD19-negative, and CD56-positive cell populations. Within this population, further identification and quantification of immunoregulatory NK cells can be performed, for example, by determining the expression of CD16, GM-CSF, IFN-γ, TNF, and / or IL-2. Immunomodulatory NK cells are preferably positive for CD56, but show rather low ("weak") levels of CD56 expression.

[0183] Other characteristics and definitions of immunomodulatory NK cells are well known in the field and are the subject of numerous studies and review articles or can be identified by a technician.

[0184] The term “immunomodulatory function” refers to the function or property of a molecule or cell that induces changes or regulation in the function, action, or state of any component of the immune system.

[0185] Cell therapy typically involves the administration of immune cells isolated from a patient's blood. Cell types that can be used in this manner include, but are not limited to, natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, monocytes, macrophages, granulocytes, and dendritic cells. Dendritic cell therapy induces an anti-tumor response by causing dendritic cells to present tumor antigens. Dendritic cells present antigens to lymphocytes, which activates the lymphocytes, sensitizing them to kill other antigen-presenting cells.

[0186] Therapeutic applications of the present invention

[0187] As used herein, the term "subject" refers to a human or non-human animal selected for treatment or therapy. Subjects or patients, such as those requiring treatment or prevention, can be animals, vertebrates, mammals, rodents (e.g., guinea pigs, hamsters, rats, mice), rats (e.g., mice), dogs (e.g., dogs), felines (e.g., cats), equines (e.g., horses), primates, apes (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, brown apes, gibbons), or humans. The meanings of the terms "animal," "mammal," etc., are well known in the art and can be inferred, for example, from Wehner und Gehring (1995; Thieme Verlag). In the context of this invention, treatment of animals of economic, agronomical, or scientific importance is particularly contemplated. Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human.

[0188] In embodiments of the present invention, the subject who has received checkpoint inhibitor therapy has cancer, such as malignant melanoma or another cancer that can be treated with checkpoint inhibitor therapy.

[0189] In the context of this invention, the term "cancer" refers to the treatment of all kinds of cancer, regardless of whether the cancer is related to the formation of a solid tumor or whether cancer cells do not form a solid tumor, as is the case with some types of leukemia.

[0190] 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, where they form metastases. In 2012, there were 14 million new cases of cancer and 8.2 million cancer-related deaths worldwide (World Cancer Report 2014). Most cancers are caused by environmental factors such as tobacco use, obesity, and infection, while approximately 5-10% are genetic cases. Cancers can be classified into subcategories based on the cells of origin. The most common subcategories are carcinomas originating from epithelial cells, sarcomas originating from connective tissue, and lymphomas and leukemias originating from hematopoietic cells. Cancer is associated with a wide variety of local and systemic symptoms and is incurable in many cases. Given the large number of new cancer cases and cancer-related deaths, new treatment strategies are needed.

[0191] The cancer according to the present invention refers to all types of cancer or growths or malignant tumors found in mammals, including leukemia, sarcoma, melanoma, and carcinoma. Solid tumors and / or liquid tumors (such as leukemia or lymphoma) can be treated.

[0192] Melanoma includes, but is not limited to, acral lentigines melanoma, amelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, malignant lentigines, malignant melanoma, nodular melanoma, subungual melanoma, or superficial diffuse melanoma.

[0193] Leukemia includes, but is not limited to, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, non-leukemic leukemia, basophylic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, and hemoblastic leukemia. Leukemia, hemocytoid leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia. Leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0194] Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, liposarcoma, liposarcoma, alveolar soft partial sarcoma, ameloblastic sarcoma, botryoid sarcoma, green carcinosarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, and Kupffer cell sarcoma. Sarcoma, angiosarcoma, leukosarcoma, malignant mesothelioma sarcoma, extraperiosteal sarcoma, reticulum cell sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0195] Cancers include, but are not limited to, acinar carcinoma, acinar carcinoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basoid cell tumor, basal cell-like carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchial carcinoma, brain carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, uterine endometrial cancer, cribriform carcinoma, armored carcinoma, carcinoma cutaneum, columnar carcinoma, columnar cell carcinoma, tubular carcinoma, sclerosing carcinoma, embryonal carcinoma, medullary carcinoma, epidermoid carcinoma, adenoid epithelial cell carcinoma, explant carcinoma, ulcerative carcinoma, fibrocarcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granular cell carcinoma, and hair matrix carcinoma. Carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, adrenal carcinoma, juvenile embryonal carcinoma, carcinoma in situ, intraepithelial carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanoma, molluscum carcinoma, mucinous carcinoma, mucinous cell carcinoma, mucinous epidermoid carcinoma, mucinous carcinoma Mucosum, myxoid carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossificans carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, acanthosis cervix, pultaceous carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scleroderma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, potato-shaped carcinoma, spherical cell carcinoma, spindle cell carcinoma, medullary carcinoma(spongiosum), squamous cell carcinoma, ligamentous carcinoma, angiomyocarcinoma, telangiectodes carcinoma, transitional cell carcinoma, tuberosum carcinoma, tuberous carcinoma, verrucous carcinoma, and villous carcinoma.

[0196] Other cancers include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocytosis, essential macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid tumor, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, and prostate cancer.

[0197] In some embodiments, "tumor" may include, but is not limited to, prostate tumors, pancreatic tumors, squamous cell carcinoma, breast tumors, melanoma, basal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, testicular cancer, neuroblastoma, glioma or malignant astrocytomas such as glioblastoma multiforme, colorectal tumors, endometrial cancer, lung cancer, ovarian tumors, cervical tumors, osteosarcoma, rhabdomyosarcoma / leiomyosarcoma, synovial sarcoma, angiosarcoma, Ewing's sarcoma / PNET, and malignant lymphomas. These include primary tumors as well as metastatic tumors (vascularized and non-vascularized).

[0198] As used herein, “treatment” or “therapeutic method” generally refers to achieving a desired pharmacological and / or physiological effect. Such effect may be preventative, for example, by completely or partially preventing the disease and / or symptoms by reducing the risk of a subject having a particular disease or symptom, or therapeutic, by partially or completely curing the disease and / or the adverse effects of said disease. In this invention, “therapeutic method” includes any treatment of a disease or symptom in mammals, specifically humans, such as treatments of (a) through (c) the following: (a) preventing the onset of a disease, symptom, or condition in a patient; (b) suppressing the symptoms of the symptom, i.e., preventing the progression of the symptoms; (c) alleviating the symptoms of the symptom, i.e., inducing the resolution of the disease or symptom.

[0199] As used herein, the term "administration" means providing a subject with cells or containing a liquid or composition, and includes, but is not limited to, administration by a medical professional and self-administration. A composition, substance, compound, or agent may be administered to a subject using one of a variety of methods known to those skilled in the art. For example, a compound or agent may be administered intravenously, arterially, subcutaneously, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), nasally (by inhalation), spinally, intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). A compound or agent may also be suitably introduced via a rechargeable or biodegradable polymer device or other device, such as a patch and pump, or a formulation that provides an extended, slow, or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0200] This invention covers treating a patient by introducing a therapeutically effective amount of cells, specifically the immunomodulatory NK cells of this invention, into the bloodstream of a subject. As used herein, introducing cells “into the bloodstream of a subject” includes, but is not limited to, introducing such cells into one of the subject’s veins or arteries by injection. Such administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods. A single injection is preferred, but in some cases repeated injections over time may be necessary (e.g., weekly, monthly, quarterly, semi-annually, or annually). Such administration is also preferably performed using a mixture of CD34-negative cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and comprise, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate-buffered saline or 0.8% saline, and commonly used proprietary cryopreservation media. Administration can also be performed locally, for example, by injection into an area of ​​the subject’s body near a symptomatic organ or tissue.

[0201] Additionally, pharmaceutically acceptable carriers of this type can be aqueous or non-aqueous solutions, suspensions, and emulsions, with aqueous solutions being the most preferred. Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions, and suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's solution, and fixed oils. Intravenous carriers include fluids and nutritional supplements, such as Ringer's dextrose, Ringer's dextrose-based supplements, and electrolyte supplements. Fluids commonly used for intravenous administration can be found, for example, in *Remington: The Science and Practice of Pharmacy*, 20th edition, p. 808, Lippincott Williams S.-Wilkins (2000). It may also contain preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, inert gases, etc.

[0202] In one embodiment, a therapeutically effective amount of cells is administered. This may involve NK cells of the present invention or therapeutic immune cells as part of a combination immunotherapy. As used herein, “therapeutically effective amount of cells” includes, but is not limited to, the following amounts and ranges: (i) about 1 × 10⁻⁶ 2 Up to approximately 1×10 8 (ii) Approximately 1 × 10⁻⁶ cells / kg body weight; 3 Up to approximately 1×10 7 (iii) Approximately 1 × 10⁻⁶ cells / kg body weight; 4 Up to approximately 1×10 6 (iv) Approximately 1 × 10⁻⁶ cells / kg body weight; 4 Up to approximately 1×10 5 Cells / kg body weight; (v) approximately 1×10 5 Up to approximately 1×10 6 Cells / kg body weight; (vi) Approximately 5 × 10 4 To approximately 0.5 × 10 5 Cells / kg body weight; (vii) Approximately 1×10 3 (viii) Approximately 1 × 10⁻⁶ cells / kg body weight; 4 Cells / kg body weight; (ix) approximately 5 × 10⁻⁶ 4 Cells / kg body weight; (x) approximately 1×10 5 Cells / kg body weight; (xi) approximately 5 × 10 5 Cells / kg body weight; (xii) approximately 1×10 6 Cells / kg body weight; and (xiii) approximately 1×107 Cells / kg body weight. Envisioned 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 figures are based on preclinical animal and human trials and standard protocols for CD34+ hematopoietic stem cell transplantation. Monocytes (containing CD34+ cells) typically contain 1:23,000 to 1:300,000 CD34-negative cells. These cell counts can be applied to the NK cells of this invention.

[0203] This disclosure also includes reagent kits, data packages, and multi-container units, as well as other materials for the ECP system and its components described herein, and materials for performing in vitro methods and treatments of the invention, and for preparing and isolating NK cells of the invention.

[0204] Attached Figure

[0205] The invention is further described with reference to the following accompanying drawings. These drawings are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention provided to better illustrate the invention described herein. Attached image description:

[0206] Figure 1 : The response of refractory autoimmune colitis to ECP, which induces the expansion of immunomodulatory NK cells.

[0207] (a) shows the change in diarrhea severity (y-axis) over time (x-axis) in patients under different treatments according to common toxicity criteria.

[0208] (b) Colonoscopy images (left inset) and H&E-stained biopsy sections (right inset) from the initial diagnosis of immune checkpoint inhibitor-associated colitis show mucosal edema and ulceration.

[0209] (c) Colonoscopy images (left inset) and H&E-stained biopsy sections (right inset) following successful ECP treatment showed no signs of autoimmune colitis. Colonic crypts showed a normal morphology without granulocyte infiltration, apoptosis, or crypt loss.

[0210] (d) tSNE plots of peripheral lymphocyte compartments in patients before and 8 weeks after ECP initiation.

[0211] (e) Number of relevant NK cells before and after ECP treatment.

[0212] (f) NK cells (defined as single / live / CD45) from healthy, age-matched donors (HD, n=5) and patients at weeks 53 and 75 after ECP initiation, visualized by UMAP. 阳性 / CD14 阴性 / CD3 阴性 / CD19 阴性 / CD56 阳性 The expression intensity of CD16, CD56, and CD57 on the cells. NK cell subsets from FlowSOM clustering are overlaid on the two plots on the right.

[0213] (g)CD56 弱 MFI (median marker expression, range: 0-1) of CD16 on mature NK cells. HD (n=5), the bins of the box plot represent the minimum and maximum values ​​of the HD dataset.

[0214] (h) In anti-PD-1 antibody-induced irAE models as described in the Supplementary Appendix, with or without NK cells (4 × 10⁶ cells per mouse). 4 Or 4×10 5 Survival rate of mice injected with T cells (Tc) at the NK dose. **p=0.003, ****p<0.0001.

[0215] Figure 2 :

[0216] (a) Displaying 1000 randomly selected CD45 samples at each time point using indicator markers. + tSNE plot of lymphocytes (lymphocytes as defined by FSC / SSC).

[0217] (b) shows a heatmap of median marker expression (value range: 0-1) for each annotated group.

[0218] (c) Absolute counts of NK cells before and after ECP treatment.

[0219] (d) B cells and CD4 counts before and after ECP treatment + T cells, CD8 + T cells and CD4 - CD8 - The relative number of T cells.

[0220] (e) B cells and CD4 counts before and after ECP treatment + T cells, CD8 + T cells and CD4 - CD8 - Absolute count of T cells.

[0221] (f) NK cells (defined as single / live / CD45) from HD (n=5) and patients at weeks 53 and 75, visualized by UMAP 阳性 / CD14 阴性 / CD3 阴性 / CD19 阴性 / CD56 阳性 The expressive strength of the indicator on the screen.

[0222] (g) shows a heatmap of median marker expression (value range: 0-1) of annotated NK cell subsets defined by FlowSOM clustering.

[0223] (hj) Healthy, age-matched donors (HD) and patients' CD56 强 CD16 弱 Immature NK cells, CD56 弱 Mature NK cells and CD57 弱 Median marker expression (MFI, range: 0-1) on terminally differentiated NK cells (at weeks 53 and 75 after ECP initiation). HD (n=5), the bins of the box plot represent the minimum and maximum values ​​of the HD dataset.

[0224] Figure 3 :

[0225] (a)-(d) CD56 of healthy, age-matched donors (HD) and patients 强 NK cells, CD56 弱 NK cells, CD4 + Memory T cells, CD4 + Primary cells and CD8 + The percentage of cytokine-expressing cells within the T cell group (at weeks 53 and 75 after ECP initiation). HD (n=5), the bins of the box plot represent the minimum and maximum values ​​of the HD dataset.

[0226] Figure 4 :

[0227] (a) Adopting patient T cells, alone or together with NK cells, to Rag2 - / - Il2rg - / - An experimental model of immune checkpoint inhibitor-related autoimmunity was induced in mice by treatment with anti-PD-1 antibodies.

[0228] (b) Day 15 following the injection of patient T cells and NK cells as shown in (a), from Rag2 - / - Il2rg - / -Histopathological scores of neutrophil and lymphocyte infiltration in isolated liver, skin, lung and colon samples from mice.

[0229] Figure 5 :

[0230] (a) Rag2 cells treated with anti-PD-1 antibody without injection of patient-derived cells - ' - I! 2rg - ' - Treatment protocol for mice (negative control).

[0231] (b) On day 15 after treatment begins as shown in (a), from Rag2 - / - Il2rg - / - Neutrophil infiltration scores of isolated liver, lung, skin, and colon from mice.

[0232] (c) Rag2 processed as described in (a) - / - Il2rg - / - Survival rate of mice.

[0233] Figure 6 :

[0234] (a) Treatment protocol: Mice were treated with DSS (3%), anti-PD1 and ECP as indicated.

[0235] (b) Weight curves of mice, as indicated, either untreated or treated with DSS (3%), anti-PD1 alone or in combination with ECP.

[0236] (c) Quantitative colon length was measured in 5 mice in each group. Groups are indicated in inset B.

[0237] (d) Representative colon of one mouse in each group. Groups are indicated in inset B.

[0238] (e) A representative HE-stained section of the colon of the group indicated in small figure B.

[0239] (f) Histopathological score of the colon in the group indicated in small figure B.

[0240] Figure 7 :

[0241] (a) Treatment protocol: Mice were intravenously injected with B16 melanoma cells and subsequently treated with anti-PD1, prednisolone or ECP, as indicated.

[0242] (b) Survival rate of mice that were intravenously injected with B16 melanoma cells and subsequently treated with anti-PD1, prednisolone or ECP.

[0243] Example

[0244] The invention is further described in the following examples. These figures are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention provided to better illustrate the invention described herein.

[0245] This report describes a patient with ipilimumab / nivolumab-induced colitis who was refractory to multiple immunosuppressive drugs achieving a complete response after in vitro photodissociation replacement (ECP) consistent with the expansion of immunoregulatory natural killer (NK) cells.

[0246] Instance results

[0247] A 29-year-old male patient was treated with ipilimumab and nivolumab for metastatic melanoma. After two doses, the patient developed dermatitis, thyroiditis, hepatitis, and colitis. The colitis was based on a biopsy ( Figure 1 The diagnosis was made based on the visible mucosal ulcers, intraepithelial cell apoptosis, and crypt loss identified in a) and b). Although the dermatitis, thyroiditis, and hepatitis resolved after discontinuation of ipilimumab / nivolumab and corticosteroid treatment, the patient experienced three flare-ups of colitis (CTC-AE II-III°) over the following 20 weeks. These colitis phases were treated with corticosteroids (23 weeks prior to ECP), infliximab (two single doses at 18 and 15 weeks prior to ECP), and cyclosporine (14 weeks prior to ECP). Figure 1 Treatment was administered according to a and Table 1).

[0248] Because there was no sustained response, the patient received ECP. Over the next 8 months, the patient underwent two ECP cycles every 2-4 weeks for several consecutive days. ECP was well tolerated and elicited a complete response. Figure 1 a) Immunosuppression gradually weakened without a rebound of symptoms. Colonoscopy confirmed the continued remission of colitis. Figure 1 c). Immune checkpoint inhibitor therapy was discontinued after the first presentation of irAE and was never restarted.

[0249] Peripheral blood leukocyte compartments were analyzed at multiple time points before and during ECP treatment. An immunomodulatory phenotype was observed. Figure 1 NK cells (f, g) Figure 1 de、 Figure 2 (aj) increased 4-fold. Furthermore, compared to age-matched healthy donors, patients had lower levels of multiple pro-inflammatory cytokines ( Figure 3This supports the view that NK cells regulate autoimmunity, and that adoptive transfer of NK cells from patients prevents dose-dependent irAEs (irAEs) triggered by human T cell and anti-PD-1 antibody treatment in a mouse irAE model. Figure 1 h and Figure 4 a, b). Controlled experiments confirmed that the incidence rate is mediated by patient-derived T cells. Figure 5 ).

[0250] ECP is a well-established therapy for treating graft-versus-host disease (GVHD) and induces an increase in NK cells in GVHD patients. 5 To date, data on the safety and efficacy of ECP for the treatment of irAEs have been lacking. This case report suggests that ECP, by expanding the protective NK cell population, may be an effective therapy for refractory checkpoint inhibitor-associated colitis.

[0251] ECP reduced immune-mediated adverse events without blocking its anti-melanoma effects.

[0252] Based on the above presentation, it is evident that extracorporeal photolysis replacement (ECP) reduces the incidence of immune-related colitis in patients with metastatic melanoma who have already been treated with combination immunotherapy (nivolumab, ipilimumab). Figure 1-5 The next step is to test these results in an in vivo model for irAE. To induce colitis, 3% DSS and anti-PD1 were used, as previously reported. Figure 6 A) The mice were treated (19). The treatment reduced the weight of the mice, which was consistent with the development of colitis, and the weight loss was reduced by ECP treatment ( Figure 6 B). Compared with the group treated with anti-PD1 alone, ECP treatment also increased colon length ( Figure 6 C, D). Colon length has been reported as a surrogate parameter for the severity of immunotherapy-induced colitis (19). Consistent with the reduced severity of colitis, decreased neutrophil infiltration was observed in the colonic wall of mice treated with ECP compared to the group treated with anti-PD1 alone ( Figure 6 E, F). These findings suggest that ECP alleviates anti-PD1-induced colitis in mice.

[0253] To understand whether the immunomodulatory effects of ECP are related to the loss of antitumor activity, mice with melanoma were treated with anti-PD1 alone or in combination with glucocorticoids such as prednisolone or ECP. Figure 7 A). Prednisolone was observed to reduce the survival rate of mice carrying melanoma compared to the group treated with anti-PD1 alone. Figure 7 B). In contrast, the group treated with anti-PD and ECP had comparable results to the group treated with anti-PD1 alone. Figure 7 B). These findings suggest that ECP does not interfere with the anti-melanoma response induced by anti-PD1 treatment.

[0254] Discussion of examples

[0255] Mechanism discussion: NK cells perform various heterologous immunological functions, including anti-inflammatory activity. In a mouse model of GVHD, NK cell transfer improved survival, which depended on intact TGF-β signaling. 6 In another preclinical GVHD study, NK cells induced perforin and Fas ligand-mediated allogeneic T cell proliferation reduction and increased T cell apoptosis. 7 c-Kit - CD27 - CD11b + The population was identified as a specific effector NK cell subset capable of controlling GVHD without interfering with graft-versus-leukemia (GVL) efficacy. 8 In humans, haplotype mismatch of killer cell immunoglobulin-like receptors (KIRs) in haplotype-congenial allogeneic hematocrits (HCTs) in the direction of GVH reduces the risk of NK cell-mediated GVHD. 9 .

[0256] ECP is an effective treatment for GVHD. Increased NK cells during ECP have been previously observed in patients with widespread chronic GVHD. 10 Patients with acute GVHD have higher frequencies of NKG2D and CD62L expression and stronger CD56 expression. 明 NK Subgroup 11 In the same study, CD56 showed higher expression of both CD57 and CD11b. - CD16 + NK cells are increased in patients with chronic cGVHD. ECP protects against specific antiviral and antileukemic CD57. + NKG2C + CD56 弱 In the case of subsets, the NK cell population shifts towards a more immunomodulatory phenotype. 11 It is hypothesized that a similar mechanism may explain the protective effect of NK cells against irAE. When NK cell compartments from patients were compared with those from age- and sex-matched healthy controls, CD16 expression, specifically CD56, was observed. 弱 The aforementioned expression is downregulated on NK cells. CD16 is FcRγIII, an activated NK cell receptor that can induce potent cytokine production. Previous studies have shown that CD16 shedding may be an immunomodulatory mechanism for preventing autoimmunity. 12CD16 downregulation regulates NK cell responses and helps maintain immune homeostasis in antibody- and T cell-dependent pathways. 13 Supporting this hypothesis is the lower expression of GM-CSF, IFN-γ, TNF, and IL-2 in the patient's NK cells compared to the control group.

[0257] The method used in the example

[0258] ECP procedure:

[0259] When applying methoxsalen In vitro photodisplacement was performed on the Therakos CellEx photodisplacement system. The procedure was repeated twice over several consecutive days, with 1500 ml of blood processed during each procedure. All human samples were collected after approval by the Ethics Committee of the Albert Ludwig University, Freiburg, Germany (Agreement No. 300 / 16) and after obtaining written informed consent in accordance with the Declaration of Helsinki.

[0260] irAE's mouse model

[0261] According to the manufacturer's instructions, T cells were isolated from the patient's peripheral blood using the Pan T Cell Isolation Kit (Miltenyi Biotec) with negative selection. NK cells were also isolated from the patient's peripheral blood using the Human NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. With or without using 4 × 10⁻⁶ cells... 4 Or 4×10 5 In the case of one NK cell, Rag2 - / - Il2rg - / - Mice were injected intravenously with 3×10 5 T cells. From day 1 to day 22 post-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 via intraperitoneal injection. Figure 4 a) Sections of skin, liver, colon, and lung collected on day 15 after human T-cell injection were stained with hematoxylin and eosin and scored by an experienced pathologist based on the histopathological features of human irAE, including lymphocytic and neutrophilic infiltration, crypt abscesses, and apoptotic cells. 14,15All animal studies have been approved by the Institutional Review Committee on the Use and Care of Laboratory Animals at the University of Freiburg, Germany (Agreement Approval Nos.: G17-049, X13-07J, X15-10A).

[0262] Flow cytometry

[0263] To monitor lymphocyte lineages during and after ECP therapy, peripheral blood lymphocytes were isolated from patients and stained with a standardized antibody panel against CD45, CD19, CD3, CD4, CD8, CD16, CD56, and HLA-DR as part of routine diagnostics. Data were compensated in FlowJo (V10) using the R environment. 17 Export lymphocytes. Perform tSNE and FlowSOM clustering as previously described. 16 .

[0264] 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 cell identification. To generate cytokines, cells were stimulated for 4 hours in the presence of GolgiPlug (BD Biosciences) with 50 ng / ml PMA (Axon Lab) and 500 mg / ml iomycin (Sigma). Intracellular staining was performed using the BDCytofix / 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. Using the R environment 17 Export and analyze the cell populations specified in the figure. Process the data for FlowSOM clustering as described. 16 To reduce dimensionality, UMAP data packets were used. 18 .

[0265] statistics

[0266] Statistical analyses were performed using GraphPad Prism Lab software V7.0. Comparisons between the two groups were performed using a two-tailed unpaired Student's t-test. Differences in survival rates (Kaplan-Meier survival curves) were assessed using the Mantel Cox (log-rank) test. Unless otherwise specified, data are presented as mean ± SEM. A p-value < 0.05 was considered significant.

[0267] Instance table

[0268] Table 1: Diseases and Treatment Courses

[0269]

[0270] The patient first experienced a colitis flare-up 20 weeks prior to ECP initiation. At that time, the patient had already been treated with steroids for 3 weeks for prior immune checkpoint inhibitor-associated hepatitis, thyroiditis, and dermatitis. The colitis occurred during steroid tapering. Therefore, the steroid dose was increased, and due to inadequate response, a single dose of infliximab 5 mg / kg BW was administered. Symptoms resolved. Following steroid tapering, the patient experienced a second colitis flare-up 16 weeks prior to ECP initiation. Treatment included an increased dose of methylprednisolone and a second dose of infliximab. Diarrhea was refractory to this therapy, and cyclosporine A was added. Symptoms again resolved. With reduction in the cyclosporine A dose, the patient experienced a third colitis flare-up. Here, ECP treatment was initiated. Two weeks after ECP initiation, the patient's bowel movement frequency was normal. Cyclosporine A treatment was discontinued 8 weeks after ECP initiation, and corticosteroid treatment was discontinued 12 weeks after ECP initiation, with no symptom rebound. ECP was administered for a total of 32 weeks. At the 11-month follow-up following the last ECP, the patient maintained complete remission in both irAE and melanoma presentation.

[0271] Table 2: Antibodies used in flow cytometry using human cells.

[0272] antigen Fluorescent dyes clone Manufacturer CD16 BUV495 3G BD CD14 BUV563 M5E2 BD CD45RO BUV615 UCHL1 BD CD3 BUV661 UCHT1 BD CD45 BUV805 HI-30 BD NKp46 BV421 9E2 Baijin Biotechnology Co., Ltd. CD56 BV480 NCAM16.2 BD CD8 BV570 RPA-T8 Baijin Biotechnology Co., Ltd. CD4 BV711 OKT4 Baijin Biotechnology Co., Ltd. CD4 APC-Cy7 RPA-T4 BD CD94 BV786 HP-3D9 BD CD57 PerCP-Cy5.5 HNK-1 Baijin Biotechnology Co., Ltd. TIGIT PE MBSA43 eBioscience CD62L PE-Cy5 DREG-56 BD KLRG1 PE-Cy7 13F12F2 Thermo Scientific NKG2D APC 1D11 Baijin Biotechnology Co., Ltd. NKG2C AF488 134591 R&D CD19 BUV737 SJ25C1 BD CD19 APC-Vio770 REA675 Miltenyi IL-2 BV711 MQ1-17H12 Baijin Biotechnology Co., Ltd. TNF BV785 Mab11 Baijin Biotechnology Co., Ltd. IFNγ PE-Cy7 4S.B3 eBioscience GM-CSF PE BVD2-21C11 BD

[0273] References

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[0277] 4. Zeiser R, Blazar BR. Acute Graft-versus-Host Disease-Biologic Process, Prevention, and Therapy. The New England Journal of Medicine; 2017; 377:2167-79.

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[0281] 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.

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Claims

1. A method for obtaining immunomodulatory NK cells, comprising the following steps: - Provide a blood sample from a subject who has received checkpoint inhibitor therapy and is exhibiting or has experienced an immune-related adverse event (irAE) including autoimmune colitis. - Add 8-methoxypsoralen to the sample, and - Expose the sample to UVA irradiation. The sample is added with 8-methoxypsoralen and subjected to UVA irradiation to induce the formation of immunomodulatory NK cells.

2. The method of claim 1, wherein the checkpoint inhibitor therapy is discontinued after symptoms and / or manifestations of irAE appear in the subject.

3. The method of claim 1, wherein the symptoms and / or manifestations of irAE occur after the checkpoint inhibitor therapy is discontinued.

4. The method of claim 1, wherein the symptoms and / or manifestations of irAE persist after discontinuation of the checkpoint inhibitor therapy.

5. The method according to any one of claims 1-4, wherein the subject has cancer.

6. The method according to any one of claims 1-5, wherein the subject suffers from malignant melanoma or another cancer that can be treated with checkpoint inhibitor therapy.

7. The method according to any one of claims 1-6, wherein the subject is receiving an immunosuppressive drug.

8. The method according to claim 7, wherein the immunosuppressive drug is a steroid, a corticosteroid, cyclosporine, an anti-TNF antibody, or any combination thereof.

9. The method of claim 8, wherein the anti-TNF antibody is infliximab.

10. The method according to any one of claims 1-6, wherein the subject is refractory to immunosuppressive drugs.

11. The method according to any one of claims 1-10, wherein the checkpoint inhibitor therapy comprises administering at least one of an anti-CTLA4 antibody and an anti-PD-1 antibody.

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