Modified immune cells and calmodulin inhibitors and uses thereof

Abstract

Provided herein are methods of using modified immune cells (e.g., CAR-NK cells) and CNIs to treat a disease or disorder or reduce immune clearance.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 121132, filed on September 25, 2023, and Patent Application No. PCT / CN2023 / 141177, filed on December 22, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to methods for treating diseases or disorders using modified immune cells and calcineurin inhibitors (CNIs). Background Technology

[0004] Developing universal, off-the-shelf cell therapy products (e.g., CAR-T) is a primary goal of next-generation cell therapies. Universal cell therapies based on allogeneic cells often suffer from the problem of transplant rejection, where the transplanted cells induce a host-vG (HvG) response and are cleared by the patient's immune system, particularly by natural killer (NK) cells and T lymphocytes. This reduces the durability and efficacy of allogeneic cell therapy in patients.

[0005] Natural killer (NK) cells have emerged as a promising cell source for CAR-based therapies due to their availability and safety profile. One limitation of cell therapy products (e.g., CAR-NK therapy products) is their low tumor invasiveness.

[0006] Therefore, it is necessary to prevent or reduce HvG response while improving the efficacy and tumor infiltration capacity of allogeneic cell therapies (e.g., CAR-NK therapy). Summary of the Invention

[0007] This disclosure relates to the prevention or mitigation of host anti-graft (HvG) responses associated with cell therapies (e.g., CAR-NK therapies) and to enhancing the efficacy of cell therapies. Specifically, this disclosure relates to a combination therapy using a calcineurin inhibitor (CNI) and modified immune cells (e.g., CAR-NK cells) to prevent or mitigate HvG responses or immune clearance and to enhance the efficacy of cell therapies.

[0008] In one aspect, this disclosure relates to methods for treating a subject’s disease or disorder, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to a subject in need, wherein the subject is receiving or will receive treatment with modified immune cells.

[0009] In some embodiments, the disease or disorder is cancer, an autoimmune disease, or an infection.

[0010] This disclosure also relates to methods for reducing immune clearance of modified immune cells in subjects, including administering an effective amount of a calcineurin inhibitor (CNI) to the subject who is receiving or will receive treatment with modified immune cells.

[0011] In some embodiments, these modified immune cells are allogeneic cells isolated from a donor for administration to a subject.

[0012] In some embodiments, the immune clearance of these modified immune cells is achieved through host immune cell-mediated cytotoxicity.

[0013] In some embodiments, the immune clearance of these modified immune cells is achieved through host T cell-mediated cytotoxicity.

[0014] In some embodiments, the application of the CNI increases the amplification of these modified immune cells.

[0015] This disclosure also relates to methods for increasing the in vivo infiltration of modified immune cells into the tissues of a subject, methods including administering an effective amount of a calcineurin inhibitor (CNI) to the subject.

[0016] In some embodiments, the tissue is tumor tissue.

[0017] In some embodiments, the modified immune cells have increased expression of CX3CR1.

[0018] In some embodiments, the increased in vivo infiltration of the modified immune cells into the tissue is achieved via an interaction between CX3CR1 expressed on the modified immune cells and CX3CL1 expressed on cells in the tissue.

[0019] In some embodiments, the modified immune cell is a modified natural killer (NK) cell.

[0020] In some embodiments, the modified NK cell is a CAR-NK cell expressing a chimeric antigen receptor (CAR).

[0021] In some embodiments, the CNI is cyclosporine A (CsA).

[0022] In some embodiments, the CsA is administered at a dose of about 1 mg / kg / day to about 25 mg / kg / day. In some embodiments, the CsA is administered at a dose of about 0.5 to about 12.5 mg / kg / day twice daily. In some embodiments, the CsA is administered at a dose of about 5 mg / kg / day to about 15 mg / kg / day. In some embodiments, the CsA is administered at a dose of about 2.5 to about 7.5 mg / kg / day twice daily. In some embodiments, the trough blood concentration of CsA is about 50 ng / ml to about 300 ng / ml.

[0023] In some embodiments, the CNI is tacrolimus.

[0024] In some embodiments, tacrolimus is administered at a concentration of about 0.01 to 1 mg / kg / day. In some embodiments, tacrolimus is administered at a concentration of about 0.1 to 0.3 mg / kg / day. In some embodiments, the trough blood concentration of Tac is about 5 ng / ml to about 15 ng / ml.

[0025] In some embodiments, the CAR receptor specifically binds to the target antigen.

[0026] In some embodiments, the target antigen is selected from the group consisting of: BCMA, CLL1, CD4, GPC3, GPRC5D, GUCY2C, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α. 2. κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivability protein, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, duracin 18.2, duracin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1 and PD-L2.

[0027] In some embodiments, these modified immune cells are not modified by gene editing or knockout of the FKBP12 or PPIA genes, which would make the cells resistant to CNI.

[0028] In some embodiments, these modified immune cells are not modified by overexpression of at least one miRNA of the miR-17-92 cluster or its paralogs, or by inactivation of at least one miR-17-92 cluster target gene, which overexpression or inactivation makes the cells resistant to CNI.

[0029] In some embodiments, these modified immune cells do not contain any modifications that make the cells resistant to CNI.

[0030] In some embodiments, these modified immune cells are enriched in the subject's bone marrow.

[0031] In some embodiments, the subject has a bone marrow-related disease or disorder, or a blood cancer such as lymphoma or leukemia.

[0032] In some embodiments, the subject has acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenström macroglobulinemia, follicular lymphoma, B-cell lymphoma, cutaneous T-cell lymphoma, multiple myeloma, plasmacytoma, or amyloidosis.

[0033] In some embodiments, the subject has a bone marrow-related disease. In some embodiments, the subject has a bone marrow-related autoimmune disease, such as a B-cell-regulated autoimmune disease.

[0034] In some embodiments, the subject has systemic lupus erythematosus (SLE), rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, ulcerative colitis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), systemic sclerosis / scleroderma (SSc), idiopathic inflammatory myopathy (IIM), antiphospholipid syndrome (APS), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis (MG), ANCA (anti-neutrophil cytoplasmic antibody)-associated vasculitis, diabetes, Raynaud's syndrome, Sjögren's syndrome, neuromyelitis optica (NMO), glomerulonephritis, and myelin oligodendrocyte glycoprotein-IgG-associated disorder (MOGAD).

[0035] In some embodiments, the CNI is administered before, during, and / or after the administration of these modified immune cells.

[0036] In some embodiments, the CNI is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days prior to the administration of these modified immune cells.

[0037] In some embodiments, the CNI is administered throughout the administration of these modified immune cells.

[0038] In some embodiments, the CNI is administered for at least 15 days, at least 20 days, at least 1 month, 2 months, or 3 months after the administration of these modified immune cells.

[0039] In some embodiments, lymphocyte clearance is performed in the subject before or after administration of the CNI and before administration of these modified immune cells.

[0040] In some embodiments, the CNI is administered via intravenous injection and / or oral administration.

[0041] In some embodiments, the CNI is administered orally after the subject is discharged from the hospital.

[0042] In some embodiments, these modified immune cells have been expanded in vitro before being administered to the subject.

[0043] This disclosure also relates to a method for enhancing the degranulation of modified immune cells (e.g., CAR-NK cells) in a subject, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to the subject, who is receiving or will receive treatment with modified immune cells.

[0044] In some embodiments, degranulation of these modified immune cells (e.g., CAR-NK cells) is enhanced.

[0045] In some embodiments, CD107a expression is increased on these modified immune cells (e.g., CAR-NK cells).

[0046] Other features and advantages of the invention will be apparent from the following detailed description and accompanying drawings, as well as from the claims. Attached Figure Description

[0047] Figure 1A-1B A model of allogeneic cell therapy is shown. The window of therapeutic efficacy can be increased by enhancing the efficacy of allogeneic cell therapy products and reducing host resistance to graft (HvG) responses, as illustrated by the transition from Figure 1A to Figure 1B.

[0048] Figure 2The results showed that the calcineurin inhibitors tacrolimus (Tac) and cyclosporine A (CsA) inhibited the killing of target cells by CAR-T cells.

[0049] Figures 3A-3C The results showed that the calcineurin inhibitors tacrolimus (Tac) and cyclosporine A (CsA) enhanced the killing of target cells by CAR-NK cells. Figure 3A The percentage of live H929 cells is shown. Figure 3B The percentage of CD107a+ CAR-NK cells after CsA treatment is shown. Figure 3C The expression of CD107a on CAR-NK cells after CsA treatment was shown.

[0050] Figures 4A-4B The results showed that CsA enhanced the killing of target cells by CAR-NK cells in the NCG tumor mouse model. Figure 4A The results showed that 50 mg / kg CsA enhanced the targeted killing of tumor cells by CAR-NK cells. Figure 4B The results showed that CsA alone did not have a direct inhibitory effect on tumor growth.

[0051] Figures 5A-5B The expression of CX3CR1 in CAR-NK cells after CsA treatment was shown. Figure 5A The results of flow cytometry analysis of CX3CR1 expression in CAR-NK cells treated with CsA or Tac at clinically relevant drug concentrations are shown. Figure 5B The MFI results for CX3CR1 expression in CAR-NK cells treated with CsA or Tac at clinically relevant drug concentrations are shown. These results indicate that treatment with CsA or Tac upregulated CX3CR1 expression.

[0052] Figures 6A-6C The bone marrow after treatment with CsA was shown. Figure 6A ), peripheral blood ( Figure 6B Distribution of CAR-NK cells in bone marrow-separated CAR-NK cells and CX3CR1 in CAR-NK cells + CAR-NK percentage ( Figure 6C CsA was administered at a dose of 50 mg / kg, and Ctrl shows the results of the control group without CsA treatment.

[0053] Figures 7A-7BThe results showed that co-administration of CsA suppressed PBMC-mediated immune clearance of CAR-NK cells in the mixed lymphocyte response (MLR). A mixed lymphocyte system was established by co-culturing allogeneic PBMCs, CAR-NK cells, and H929 cells at a ratio of 50:1:4. CAR-NK cell counts were monitored every two days. CAR-NK cell counts were normalized to the initial CAR-NK cell count, and fold changes were recorded. Figure 7A The results showed that co-administration of CsA inhibited PBMC-mediated immune clearance of CAR-NK cells in the MLR. CsA was administered at a concentration of 200 ng / ml, and Ctrl shows the results of the control group without CsA treatment. Figure 7B The results showed that, in MLR, co-administration of Tac inhibited PBMC-mediated immune clearance of CAR-NK cells. Tac was administered at a concentration of 10 ng / ml, and Ctrl shows the results of the control group without Tac treatment.

[0054] Figures 8A-8C The in vivo efficacy, amplification, and gene enrichment analysis of CAR-NK cells treated with CsA or Tac are shown. Figure 8A The results showed that, compared with treatment with the medium, treatment with CsA or Tac did not inhibit the tumor-killing effect of CAR-NK, but rather increased it. Figure 8B The results showed that the CAR-NK count in peripheral blood was comparable among the groups treated with CsA, Tac, or the mediator. Figure 8C The enrichment pathways upregulated by CsA or Tac treatment compared to CAR-NK treated with the vector are listed. Specifically, CsA, Tac, or vector-treated CAR-NK were isolated from mouse spleens at the experimental endpoint. RNA sequencing was performed, and differentially expressed genes were counted (comparing CsA or Tac to CAR-NK treated with the vector), and KEGG enrichment was analyzed. CsA was administered at a dose of 50 mg / kg / day, and Tac at a dose of 5 mg / kg / day.

[0055] Figures 9A-9E The results showed that, in the HvG model, CsA suppressed CAR-NK rejection of allogeneic PBMCs and promoted CAR-NK persistence in vivo. Figure 9A A schematic diagram of the HvG model design is shown. Figure 9B The dynamic changes in CAR-NK counts in peripheral blood are shown in the HvG model. Figure 9C The dynamic changes in T cell counts derived from PBMCs were shown. Figure 9D The expression of CD38 in PBMC-derived T cells was shown. Figure 9E The expression of CD71 in PBMC-derived T cells was shown.

[0056] Figure 10 The results showed that CsA promoted CAR-NK persistence (a type of CAR-NK cell inhibitor) in vivo in a dose-dependent manner. Figure 9A (Similar to the HvG model mentioned above). Higher CsA doses are associated with increased Cmax and prolonged CAR-NK amplification.

[0057] Figure 11A-11B The display shows that, in relation to Figure 9A In the HvG model, which is similar to the model mentioned above, the duration of CsA treatment determines the persistence of CAR-NK in vivo. Figure 11A The study showed that longer CsA treatment duration was associated with increased Cmax and prolonged CAR-NK amplification. Figure 11B The results showed that discontinuing CsA treatment led to the recovery of peripheral T cells.

[0058] Figure 12A-12B The results showed that CsA promoted the tumor-killing effect of CAR-NK in the tumor-bearing HvG model. This model, in which tumor cells were additionally inoculated, was based on a previously described... Figure 9A The HvG model in [the context of the text]. Figure 12A The results showed that CsA treatment inhibited tumor progression compared to the mediator treatment group. Figure 12B The results showed that CsA treatment promoted CAR-NK expansion. CsA was administered at a dose of 50 mg / kg / day. Detailed Implementation

[0059] Cell therapies based on allogeneic cells often have the problem of transplant rejection. Typically, transplanted cells can induce a host-vG (HvG) response, and these cells are killed by the recipient's immune system, for example, by T lymphocytes. This can reduce the durability and efficacy of allogeneic cell products.

[0060] In tissue transplant patients, immunosuppressants are used to prevent or reduce HvG responses. CNIs are also used to maintain immunosuppression long-term to control HvG responses.

[0061] This disclosure is based in part on the fact that CNIs (such as CsA or Tac) can inhibit the target-killing activity of certain immune cells (e.g., T cells or CAR-T cells) but enhance the target-killing activity of other immune cells (e.g., CAR-NK cells). Therefore, co-administration of CNIs and engineered immune cells (such as CAR-NK cells) can prevent or mitigate host immune clearance mediated by, for example, T cells, and enhance the efficacy of cell therapies using engineered immune cells (e.g., CAR-NK cells). Furthermore, CNIs can increase tumor infiltration of modified immune cells (e.g., CAR-NK cells) and promote the proliferation of modified immune cells (e.g., CAR-NK cells).

[0062] Therefore, in one respect, this disclosure provides a method for treating a subject’s disease or disorder, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to the subject in need, wherein the subject is receiving or will receive treatment with modified immune cells.

[0063] In one aspect, this disclosure provides a method for treating a subject’s disease or disorder, the method comprising administering to the subject in need an effective amount of modified immune cells and calcineurin inhibitors (CNIs).

[0064] In one aspect, this disclosure provides methods for reducing the immune clearance of modified immune cells in a subject, including administering an effective amount of a calcineurin inhibitor (CNI) to the subject who is receiving or will receive treatment with modified immune cells.

[0065] In one aspect, this disclosure provides methods for reducing the immune clearance of modified immune cells in a subject, methods including administering an effective amount of these modified immune cells and a calcineurin inhibitor (CNI) to the subject.

[0066] In one aspect, this disclosure provides methods for increasing the in vivo infiltration of modified immune cells into the tissues of a subject, methods including administering an effective amount of a calcineurin inhibitor (CNI) to the subject.

[0067] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells, such as NK cells. Some CARs are also referred to as "chimeric immune receptors." CARs may contain extracellular ligand-binding domains or extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains that are specific to one or more ligands or antigens, such as tumor antigens, autoimmune disease antigens. "CAR-NK cells" refers to NK cells that express a CAR.

[0068] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapid proliferative cell growth. The term is intended to include cancerous growth, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematopoietic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. “Naturally occurring” cancer includes any cancer not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by patient exposure to one or more carcinogens, cancer caused by insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infection (e.g., viral infection). The term “cancer” is generally accepted in the field as referring to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of both carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to cancer derived from glandular tissue or in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted in the field as referring to a malignant tumor derived from mesenchyme. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can arise from myeloid, lymphoid, or erythroid lineages or their precursor cells.

[0069] As used herein, the term "bone marrow-associated autoimmune disease" refers to a group of diseases affecting the bone marrow. Some of the pathogenic cells in these diseases can reside in, develop in, and / or invade the bone marrow, thereby affecting its function. Bone marrow-associated autoimmune diseases include B cell-regulated autoimmune diseases. The term "B cell-regulated autoimmune disease" refers to an autoimmune disorder involving misregulation of B cells. B cell-regulated autoimmune diseases can be autoimmune disorders associated with self-reactive plasma cells and / or self-reactive memory B cells.

[0070] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human, or non-human, to which treatment is administered according to the methods disclosed herein. This disclosure contemplates both veterinary and non-veterinary applications. Human patients may be adults or adolescents (e.g., humans under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, this includes non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0071] As used herein, the term "donor" refers to an organism from which a biological sample is derived, such as a human from whom cells can be obtained. Organisms include mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term "donor" also covers any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. "Donor" can also refer to more than one donor, such as one or more humans or non-human animals or non-human mammals. However, advantageously, the donor is a mammal, such as a human. A donor can be a cancer patient to be treated with a cell population generated by the methods described herein (i.e., an autologous donor), or it can be an individual who donates a sample that, after generating a cell population generated by the methods described herein, will be used to treat a different individual or cancer patient (i.e., an allogeneic donor).

[0072] As used herein, the term "derived from" indicates a relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include limitations on the process or origin of the first molecule from which it is derived. For example, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ sequence / structure to enable it to perform the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include limitations on the specific process for generating the intracellular signaling domain; for example, it does not mean that, in order to provide the intracellular signaling domain, it is necessary to start with the CD3ζ sequence and delete unwanted sequences, or to apply mutations to obtain the intracellular signaling domain. Domains derived from a particular protein may have a sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the relevant functional portion of the particular protein.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including its definitions, shall prevail.

[0074] HvG response against engineered immune cells

[0075] Adoptive immunotherapy using T cells or NK cells engineered to express chimeric antigen receptors (CARs) via transient or stable gene transfer is a highly effective treatment for advanced chemotherapy- and radiotherapy-resistant malignancies in hematology and oncology. One of the biggest obstacles to the application of adoptive immunotherapy is host-vG (HvG) response to allogeneic products, which is caused by the patient's immune system's immune response to the transferred cells (e.g., CAR-NK cells). HvG responses significantly reduce the durability and efficacy of cell therapy. Furthermore, HvG responses can cause side effects (e.g., fever) and safety concerns.

[0076] Highly polymorphic HLA molecules in a population are a major cause of T cell-mediated HvG. Since β2-microglobulin (B2M) is essential for the stable presence of HLA class I molecules on the cell membrane, knocking out B2M can remove HLA class I molecules from the cell surface, thereby effectively mitigating the T cell-mediated HvG response. However, loss of B2M significantly increases the sensitivity of allogeneic cells to NK cell-mediated cytotoxicity, making them more susceptible to NK cell clearance.

[0077] Currently, three common strategies exist for addressing the HvG problem in allogeneic cell therapy (Table 1). First, lymphocyte-clearing agents, including antibody drugs and bispecific chimeric antigen receptors (CARs), can be used to eliminate immune cells (especially NK and T cells) in the subject, thereby attenuating or inhibiting the HvG response and prolonging the persistence of allogeneic cell therapy products in the subject. Second, allogeneic cell products can be modified with naturally occurring immunosuppressive molecules (through overexpression, knockdown, or knockout) to reduce the activation of the subject's immune system or suppress the subject's immune cells (especially NK and T cells), thereby reducing the rate at which the allogeneic cell therapy product is cleared by the subject's immune system. Finally, based on the frequency and distribution of polymorphic HLA alleles across populations in a specific region, multiple HLA haploidentical cells can be prepared, and a limited cell bank covering most HLA types in that population can be established. Using this cell bank, the immune response between HLA-incompatible donor and recipient cells can be reduced, and longer persistence of allogeneic cell therapy products in the subject can be achieved.

[0078] Table 1: Common strategies for resolving HvG reactions

[0079]

[0080] However, all three strategies described above have significant and insurmountable drawbacks. Using lymphocyte scavengers to clear or suppress the subject's immune system often increases the risk of infection, and antibody-mediated targeted cell clearance is expensive and usually accompanied by significant side effects. The effectiveness of modifying naturally occurring immunosuppressive molecules (e.g., HLA-E overexpression and HLA-A / B / C knockout) is uncertain due to a lack of clinical data. Finally, constructing cell banks only partially avoids, but does not resolve, the HvG problem in allogeneic cell therapy.

[0081] This disclosure provides methods for preventing, inhibiting, or reducing HvG responses associated with cell therapies (e.g., CAR-NK therapy).

[0082] Calcineurin inhibitors (CNI)

[0083] Calcineurin inhibitors (CNIs) are immunosuppressants used to manage autoimmune diseases, including but not limited to lupus nephritis, idiopathic inflammatory myositis, interstitial lung disease, and atopic dermatitis. Furthermore, they are used as a pillar of immunosuppression in solid organ transplantation.

[0084] Cyclosporin A (CsA)

[0085] Cyclosporin A (CsA) is a cyclic nonribosomal peptide composed of eleven amino acids; it is an immunosuppressive drug widely used after allogeneic organ transplantation to reduce the activity of the patient's immune system and thus reduce the risk of organ rejection. The structure, chemical, and physical properties of CsA are known in the art (see, for example, PubChem website, PubChem CID 5284373). Cyclosporin is used with other drugs to prevent the body from rejecting the transplanted organ (e.g., kidney, liver, or heart). Cyclosporin is also used to treat severe rheumatoid arthritis in patients who have failed methotrexate treatment. The drug is also used to treat adults with severe plaque psoriasis after failure of other treatments (e.g., PUVA, retinoids, methotrexate).

[0086] Cyclosporine's mechanism of action involves its role as a calcineurin inhibitor, a cytochrome P450 3A4 inhibitor, and a P-glycoprotein inhibitor. CsA inhibits the synthesis of interleukins (ILs), including IL-2, which are essential for the autoactivation and differentiation of T lymphocytes (LTs). Cyclosporine is effective due to its specific and reversible inhibition of immunocompetent lymphocytes during the G0 and G1 phases of the cell cycle. T helper cells are the primary target, although it may also inhibit T suppressor cells. LT-B lymphocyte (LB) synergy is essential for LB activation; the latter is also inhibited. Furthermore, studies have demonstrated that CsA has an inhibitory effect on CD4+CD25+ Tregs, which may block the host's immune tolerance potential.

[0087] The CNI used in the methods described herein can be CsA. CsA can be administered at any suitable dose. CsA can be administered at doses of approximately 1 to approximately 200 mg / kg / day, approximately 1 to approximately 175 mg / kg / day, approximately 1 to approximately 150 mg / kg / day, approximately 1 to approximately 125 mg / kg / day, approximately 1 to approximately 100 mg / kg / day, approximately 1 to approximately 75 mg / kg / day, approximately 1 to approximately 50 mg / kg / day, approximately 1 to approximately 25 mg / kg / day, approximately 1 to approximately 12.5 mg / kg / day, 5 to approximately 200 mg / kg / day, approximately 5 to approximately 175 mg / kg / day, approximately 5 to approximately 150 mg / kg / day, approximately 5 to approximately 125 mg / kg / day, approximately 5 to approximately 100 mg / kg / day, approximately 5 to approximately 75 mg / kg / day, approximately 5 to approximately 50 mg / kg / day, approximately 5 to approximately 25 mg / kg / day, 10 to approximately 200 mg / kg / day, approximately 10 to approximately 175 mg / kg / day, approximately 10 to approximately 150 mg / kg / day. mg / kg / day, about 10 to about 125 mg / kg / day, about 10 to about 100 mg / kg / day, about 10 to about 75 mg / kg / day, about 10 to about 50 mg / kg / day, about 10 to about 25 mg / kg / day, about 10 to about 12.5 mg / kg / day, about 12.5 to about 200 mg / kg / day, about 12.5 to about 175 mg / kg / day, about 12.5 to about 150 mg / kg / day, about 12.5 to about 125 mg / kg / day, about 12.5 to about 100 mg / kg / day, about 12.5 to about 75 mg / kg / day, about 12.5 to about 50 mg / kg / day, about 12.5 to 25 mg / kg / day, about 15 to 200 mg / kg / day, about 15 to 175 mg / kg / day, about 15 to 150 mg / kg / day, about 15 to 125 mg / kg / day, about 15 to 100 mg / kg / day, about 15 to 75 mg / kg / day, about 15 to 50 mg / kg / day, about 15 to 25 mg / kg / day, about 20 to 200 mg / kg / day, about 20 to 175 mg / kg / day, about 20 to 150 mg / kg / day, about 20 to 125 mg / kg / day, about 20 to 100 mg / kg / day, about 20 to 75 mg / kg / day, about 20 to 50 mg / kg / day, about 20 to 25 mg / kg / day, about 25 to 200 mg / kg / day, about 25 to 175 mg / kg / day, about 25 to 150 mg / kg / day mg / kg / day, approximately 25 to approximately 125 mg / kg / day, approximately 25 to approximately 100 mg / kg / day, approximately 25 to approximately 75 mg / kg / day, approximately 25 to approximately 50 mg / kg / day, approximately 50 to approximately 200 mg / kg / day, approximately 50 to approximately 175 mg / kg / day, approximately 50 to approximately 150 mg / kg / day, approximately 50 to approximately 125 mg / kg / day, approximately 50 to approximately 100 mg / kg / day, approximately 50 to approximately 75 mg / kg / day, approximately 75 to approximately 200 mg / kg / day, approximately 75 to approximately 175 mg / kg / day, approximately 75 to approximately 150 mg / kg / day, approximately 75 to approximately 125 mg / kg / day, approximately 75 to approximately 100 mg / kg / day, approximately 100 to approximately 200 mg / kg / day, approximately 100 to approximately 175 mg / kg / day, approximately 100 to approximately 150 mg / kg / day CsA can be administered at doses of approximately 1 to approximately 125 mg / kg / day, approximately 125 to approximately 200 mg / kg / day, approximately 125 to approximately 175 mg / kg / day, approximately 125 to approximately 150 mg / kg / day, approximately 150 to approximately 200 mg / kg / day, or approximately 150 to approximately 175 mg / kg / day. CsA can be administered at doses of approximately 1 to approximately 25 mg / kg / day. CsA can be administered at doses of approximately 5 to approximately 15 mg / kg / day. CsA can be administered at doses of approximately 0.5 to approximately 12.5 mg / kg twice daily. CsA can be administered at doses of approximately 2.5 to approximately 7.5 mg / kg twice daily. The trough blood concentration of CsA can be approximately 50 ng / ml to approximately 300 ng / ml.

[0088] Tacrolimus (Tac)

[0089] Tacrolimus (anhydrous) is a macrolide lactone containing a 23-membered lactone ring, which was initially isolated from the fermentation broth of a Japanese soil sample containing the bacterium *Streptomyces tsukubaensis*. It has functions as an immunosuppressant and a bacterial metabolite. The structure, chemical, and physical properties of Tac are known in the art (see, for example, PubChem website, PubChem CID 445643).

[0090] Tacrolimus (also known as FK-506 or tebufenozide) is an immunosuppressive drug primarily used to reduce the activity of the immune system in patients after organ transplantation, thereby reducing the risk of organ rejection. It is also used in topical formulations to treat severe atopic dermatitis, severe refractory uveitis after bone marrow transplantation, and vitiligo. Tacrolimus is chemically known as a macrolide. It reduces peptidyl-prolyl isomerase activity by binding to the immunosuppressant FKBP-12 (FK506-binding protein) to form a new complex. This FKBP12-FK506 complex inhibits calcineurin, which suppresses T-lymphocyte signaling and IL-2 transcription.

[0091] Other uses of tacrolimus include: prevention of corneal, pancreatic, kidney, and small bowel transplant rejection; induction therapy for lupus nephritis; treatment of Crohn's disease, membranous glomerulonephritis, and myasthenia gravis; and reducing the incidence of pancreatitis in patients undergoing liver transplantation after endoscopic retrograde cholangiopancreatography.

[0092] The CNI used in the methods described herein can be Tac. Tac can be administered at any suitable dose. Tac can be administered at doses of approximately 0.01 to approximately 20 mg / kg / day, approximately 0.01 to approximately 17.5 mg / kg / day, approximately 0.01 to approximately 15 mg / kg / day, approximately 0.01 to approximately 12.5 mg / kg / day, approximately 0.01 to approximately 10 mg / kg / day, approximately 0.01 to approximately 7.5 mg / kg / day, approximately 0.01 to approximately 5 mg / kg / day, approximately 0.01 to approximately 2.5 mg / kg / day, approximately 0.01 to approximately 1.25 mg / kg / day, approximately 0.01 to approximately 1 mg / kg / day, 0.5 to approximately 20 mg / kg / day, approximately 0.5 to approximately 17.5 mg / kg / day, approximately 0.5 to approximately 15 mg / kg / day, approximately 0.5 to approximately 12.5 mg / kg / day, approximately 0.5 to approximately 10 mg / kg / day, approximately 0.5 to approximately 7.5 mg / kg / day, approximately 0.5 to approximately 5 mg / kg / day. mg / kg / day, about 0.5 to about 2.5 mg / kg / day, about 1 to about 20 mg / kg / day, about 1 to about 17.5 mg / kg / day, about 1 to about 15 mg / kg / day, about 1 to about 12.5 mg / kg / day, about 1 to about 10 mg / kg / day, about 1 to about 7.5 mg / kg / day, about 1 to about 5 mg / kg / day, about 1 to about 2.5 mg / kg / day, about 2.5 to about 20 mg / kg / day, about 2.5 to about 17.5 mg / kg / day, about 2.5 to about 15 mg / kg / day, about 2.5 to about 12.5 mg / kg / day, about 2.5 to about 10 mg / kg / day, about 2.5 to about 7.5 mg / kg / day, about 2.5 to about 5 mg / kg / day, about 5 to about 20 mg / kg / day, about 5 to about 17.5 mg / kg / day, about 5 to about 15 mg / kg / day Tac can be administered at doses of approximately 5 to approximately 12.5 mg / kg / day, approximately 5 to approximately 10 mg / kg / day, approximately 5 to approximately 7.5 mg / kg / day, approximately 7.5 to approximately 20 mg / kg / day, approximately 7.5 to approximately 17.5 mg / kg / day, approximately 7.5 to approximately 15 mg / kg / day, approximately 7.5 to approximately 12.5 mg / kg / day, approximately 7.5 to approximately 10 mg / kg / day, approximately 10 to approximately 20 mg / kg / day, approximately 10 to approximately 17.5 mg / kg / day, approximately 10 to approximately 15 mg / kg / day, or approximately 10 to approximately 12.5 mg / kg / day. Tac can also be administered at doses of approximately 0.01 to approximately 1 mg / kg / day. Tac can also be administered at doses of approximately 0.1 to approximately 0.3 mg / kg / day.

[0093] Pimecrolimus

[0094] Pimecrolimus is approved as a 1% topical formulation and is FDA approved for the treatment of mild to moderate atopic dermatitis. Other uses of pimecrolimus include oral lichen planus, psoriasis of the face and intertriginous areas, vitiligo, vulvar lichen sclerosus, and seborrheic dermatitis (see, for example, Ayer J, Young HS. Pimecrolimus for psoriasis. Expert Opin Pharmacother. 2013 Apr;14(6):767-74).

[0095] The CNI used in the methods described herein can be pimecrolimus. Pimecrolimus can be administered at any suitable dose.

[0096] · vorciclosporin

[0097] Voclosporin is a novel drug in this class and was approved by the FDA in 2021 for oral administration in combination with other immunosuppressants to treat active lupus nephritis in adults (see, for example, Rovin BH et al., Efficacy and safety of voclosporin versus placebo for lupus nephritis (AURORA1): a double-blind, randomised, multicentre, placebo-controlled, phase 3trial. Lancet. May 29, 2021;397(10289):2070-2080; and Ponticelli C et al., Old and New Calcineurin Inhibitors in Lupus Nephritis. J Clin Med. October 21, 2021;10(21)).

[0098] The CNI used in the methods described herein can be vorticocporin. Vorticocporin can be administered at any suitable dose.

[0099] Vocyclosporine can be administered at doses of approximately 0.1 to approximately 200 mg / kg / day, approximately 0.1 to approximately 175 mg / kg / day, approximately 0.1 to approximately 150 mg / kg / day, approximately 0.1 to approximately 125 mg / kg / day, approximately 0.1 to approximately 100 mg / kg / day, approximately 0.1 to approximately 75 mg / kg / day, approximately 0.1 to approximately 50 mg / kg / day, approximately 0.1 to approximately 25 mg / kg / day, approximately 0.1 to approximately 12.5 mg / kg / day, approximately 0.1 to approximately 1 mg / kg / day, approximately 1 to approximately 200 mg / kg / day, approximately 1 to approximately 175 mg / kg / day, approximately 1 to approximately 150 mg / kg / day, approximately 1 to approximately 125 mg / kg / day, approximately 1 to approximately 100 mg / kg / day, approximately 1 to approximately 75 mg / kg / day, approximately 1 to approximately 50 mg / kg / day, approximately 1 to approximately 25 mg / kg / day, and approximately 1 to approximately 12.5 mg / kg / day. mg / kg / day, 1 to 10 mg / kg / day, 10 to 200 mg / kg / day, about 10 to 175 mg / kg / day, about 10 to 150 mg / kg / day, about 10 to 125 mg / kg / day, about 10 to 100 mg / kg / day, about 10 to 75 mg / kg / day, about 10 to 50 mg / kg / day, about 10 to 25 mg / kg / day, about 10 to 12.5 mg / kg / day, about 12.5 to 200 mg / kg / day, about 12.5 to 175 mg / kg / day, about 12.5 to 150 mg / kg / day, about 12.5 to 125 mg / kg / day, about 12.5 to 100 mg / kg / day, about 12.5 to 75 mg / kg / day, about 12.5 to 50 mg / kg / day, about 12.5 to 25 mg / kg / day, about 15 to 200 mg / kg / day, about 15 to 175 mg / kg / day, about 15 to 150 mg / kg / day, about 15 to 125 mg / kg / day, about 15 to 100 mg / kg / day, about 15 to 75 mg / kg / day, about 15 to 50 mg / kg / day, about 15 to 25 mg / kg / day, about 20 to 200 mg / kg / day, about 20 to 175 mg / kg / day, about 20 to 150 mg / kg / day, about 20 to 125 mg / kg / day, about 20 to 100 mg / kg / day, about 20 to 75 mg / kg / day, about 20 to 50 mg / kg / day, about 20 to 25 mg / kg / day, about 25 to 200 mg / kg / day, about 25 to 175 mg / kg / day, about 25 to 150 mg / kg / day mg / kg / day, approximately 25 to approximately 125 mg / kg / day, approximately 25 to approximately 100 mg / kg / day, approximately 25 to approximately 75 mg / kg / day, approximately 25 to approximately 50 mg / kg / day, approximately 50 to approximately 200 mg / kg / day, approximately 50 to approximately 175 mg / kg / day, approximately 50 to approximately 150 mg / kg / day, approximately 50 to approximately 125 mg / kg / day, approximately 50 to approximately 100 mg / kg / day, approximately 50 to approximately 75 mg / kg / day, approximately 75 to approximately 200 mg / kg / day, approximately 75 to approximately 175 mg / kg / day, approximately 75 to approximately 150 mg / kg / day, approximately 75 to approximately 125 mg / kg / day, approximately 75 to approximately 100 mg / kg / day, approximately 100 to approximately 200 mg / kg / day, approximately 100 to approximately 175 mg / kg / day, approximately 100 to approximately 150 mg / kg / day Fibrosporine can be administered at doses of approximately 100 to approximately 125 mg / kg / day, approximately 125 to approximately 200 mg / kg / day, approximately 125 to approximately 175 mg / kg / day, approximately 125 to approximately 150 mg / kg / day, approximately 150 to approximately 200 mg / kg / day, or approximately 150 to approximately 175 mg / kg / day. Fibrosporine can be administered at any suitable or recommended amount or dose, for example, approximately 27.3 mg twice daily in human subjects.

[0100] engineered receptors

[0101] This disclosure relates to methods for treating diseases or disorders in subjects, reducing immune clearance of modified immune cells, or increasing in vivo infiltration of modified immune cells into tissues (e.g., tumor tissue), methods including administering an effective amount of modified immune cells and CNI to the subject.

[0102] Modified immune cells can express chimeric antigen receptors (CARs). A chimeric antigen receptor (CAR) typically comprises an extracellular domain capable of binding to an antigen and an intracellular domain containing one or more intracellular signaling domains derived from signal transduction proteins. These intracellular signaling domains are typically distinct from the polypeptides that derive the extracellular domains. The extracellular domain can be any protein molecule or portion thereof capable of specifically binding to a predetermined antigen. The extracellular domain may contain an antibody or its antigen-binding fragment. The intracellular signaling domain can be any known oligopeptide or polypeptide domain that functions to transmit signals that induce activation or inhibition of biological processes within the cell, such as the activation of immune cells like NK cells.

[0103] Chimeric antigen receptors (CARs) integrate many facets of normal T cell or NK cell activation into a single protein. They link an extracellular antigen recognition domain with an intracellular signal transduction domain, thereby activating T cells or NK cells upon binding to an antigen. CARs typically contain the following regions: an antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular domain. The intracellular region contains the intracellular signal transduction domain or intracellular signal transduction region.

[0104] Detailed reviews of CAR and CAR-expressing cells can be found in, for example, J Exp Clin Cancer Res, March 31, 2022;41(1):119; Mol Cancer, January 30, 2023;22(1):20; and Br J Haematol, April 2021;193(2):216-230. doi: 10.1111 / bjh.17186. Electronic publication on November 20, 2020, each of these references is incorporated herein by reference in its full text. Exemplary structures of antigen receptors, including hinges, transmembrane domains, and intracellular signaling domains, as well as methods for engineering and introducing such receptors into cells, are described in, for example, Chandran et al., "T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance." Immunological reviews 290.1 ​​(2019):127-147; Cartellieri, Marc et al., "Chimeric antigen receptor-engineered T cells for immunotherapy of cancer." BioMed Research International 2010 (2010); and PCT publication number WO 2017173256A1; US ​​2002 / 131960, US 2013 / 287748, US 2013 / 0149337, US6,451,995, US 7,446,190, US 8,252,592; each of these references is incorporated herein by way of citation in its entirety.

[0105] The CAR expressed on modified immune cells (e.g., CAR-NK cells) disclosed herein may comprise an extracellular domain, a transmembrane region, and an intracellular signaling domain. The extracellular domain may include at least one antigen-binding domain that specifically binds to at least one target antigen. The intracellular signaling domain may generate signals that promote the immune effector function of CAR-containing cells (e.g., CAR-NK cells). "Immune effector function or immune effector response" refers to a function or response of immune effector cells that enhances or promotes the immune attack of target cells. For example, an immune effector function or response may refer to the property of NK cells to promote the killing of target cells or inhibit the growth or proliferation of target cells. The intracellular signaling domain may generate signals that promote the proliferation and / or survival of CAR-containing cells. CARs may comprise one or more intracellular signaling domains. The signaling domain of a naturally occurring molecule may comprise the entire intracellular (or cytoplasmic) portion of the molecule or the entire natural intracellular signaling domain, or a fragment or derivative thereof.

[0106] The intracellular signaling domains of CARs can include primary intracellular signaling domains. A "primary intracellular signaling domain" refers to an intracellular signaling sequence that acts in a stimulatory manner to induce the function of immune effectors. Primary intracellular signaling domains can contain signaling motifs known as tyrosine-based activation motifs of immune receptors or ITAMs. Primary intracellular signaling domains can contain functional signaling domains of proteins selected from the following groups: CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. Primary intracellular signal transduction domains can contain non-functional or attenuated signal transduction domains of proteins selected from the following groups: CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. Primary intracellular signal transduction domains can also contain the functional signal transduction domain of CD3ζ.

[0107] The intracellular signaling domain of a CAR may contain one or more (such as any one of 1, 2, 3 or more) costimulatory signaling domains. A "costimulatory signaling domain" can be the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a homologous binding partner on an immune cell (such as an NK cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the immune cell, such as, but not limited to, proliferation and survival. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an efficient immune response. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activated NK cell receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, DAP12, DAP10, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, and LFA-1. ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0108] A CAR may contain a single costimulatory signaling domain. A CAR may contain two or more costimulatory signaling domains. Intracellular signaling domains may contain a functional primary intracellular signaling domain and one or more costimulatory signaling domains. One or more costimulatory signaling domains may be derived from one or more molecules selected from the group consisting of: CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. Costimulatory signaling domains may be derived from CD137.

[0109] The CAR may further include a DAP12 intracellular signal transduction domain, and the DAP12 intracellular signal transduction domain may be located at the C-terminus of the intracellular signal transduction domain. The intracellular signal transduction domain may include a functional primary intracellular signal transduction domain and one or more co-stimulatory signal transduction domains, as well as the DAP12 intracellular signal transduction domain. The intracellular signal transduction domain may include the following domains from the N-terminus to the C-terminus: a CD137 co-stimulatory signal transduction domain, a CD3ζ primary intracellular signal transduction domain, and a DAP12 intracellular signal transduction domain.

[0110] The antigen-binding domain of a CAR can be an antibody or antibody fragment, such as scFv, Fv, Fab, (Fab')2, single-domain antibody (sdAb), or V. H The H domain. The antigen-binding domain of a CAR may contain a ligand or extracellular portion of the receptor that specifically binds to the target antigen. CARs can be monospecific, bispecific, or multispecific. The antigen-binding domain of a CAR can specifically bind to a single target antigen. The antigen-binding domain of a CAR can bind to two or more target antigens.

[0111] Target antigens can be selected from tumor antigens, autoimmune disease antigens, inflammatory disease antigens, neuronal disorder antigens, HIV / AIDS antigens, diabetes antigens, cardiovascular disease antigens, and infectious disease antigens (including viral antigens, protozoan antigens, bacterial antigens, and allergens), or combinations thereof.

[0112] Target antigens can be selected from the following groups: BCMA, CLL1, CD4, GPC3, GPRC5D, GUCY2C, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, Le Y, L1 cell adhesion molecules, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survival protein, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, dentin 18.2, dentin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1 and PD-L2, and other clinically significant target antigens and combinations thereof.

[0113] Target antigens can be tumor antigens. Tumor antigens can contain one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecular group, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is oncoemulsification antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the true tumor-specific immunoglobulin antigens unique to the individual tumor. B-cell differentiation antigens (such as CD19, CD20, and CD37) are other candidates for target antigens in B-cell lymphomas.

[0114] Tumor antigens can be tumor-specific antigens (TSA) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present on other cells in the body. TAA-associated antigens are not specific to tumor cells; instead, they are also expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during embryonic development (when the immune system is immature and unable to respond), or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0115] Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Many TCRs specific to tumor antigens, including tumor-associated antigens, have been described, including, for example, the NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigen, and TCRs targeting tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1).

[0116] Target antigens can be autoimmune disease antigens. As used herein, an autoimmune disease antigen refers to a self-antigen or an antigen that cross-reacts with a self-antigen, against which the body produces a dysfunctional immune response that causes disease. Autoimmune disease antigens can be nucleic acids, proteins, and / or peptides encoding the autoimmune disease antigen. Autoimmune disease antigens can be associated with any autoimmune disease.

[0117] Many CARs targeting different target antigens have been widely disclosed in the art, such as CD19 CARs or BCMA CARs. The extracellular antigen-binding domain of a CD19 CAR may be or contain a CD19-binding fragment (e.g., FMC63, SJ25C1, or those disclosed in various patents or patent publications such as PCT Publication No. WO 2022 / 012683). BCMA CARs have also been well described, and relevant patents include, but are not limited to, PCT Publication Nos. WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, and WO 2018 / 028647.

[0118] The transmembrane region of CARs may contain or be selected from the α, β, or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane regions of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. Transmembrane regions may include transmembrane regions of CD8α.

[0119] Extracellular domains can connect to transmembrane regions via hinge regions. Hinge regions can contain the hinge regions of CD8α.

[0120] CARs can contain signal peptides (SPs), such as the CD8α signal peptide.

[0121] CARs can be BCMA CARs. Various antigen-binding domain sequences can be used as antigen-binding domains for CARs. CARs can specifically bind to BCMA-positive tumor cells.

[0122] CARs can be single CARs, dual CARs, tandem CARs, or separate CARs.

[0123] Modified immune cells

[0124] This disclosure relates to methods for treating a disease or disorder in a subject, reducing immune clearance of modified immune cells (e.g., CAR-NK cells), or increasing in vivo infiltration of modified immune cells into a tissue (e.g., tumor tissue), including administering an effective amount of CNI (e.g., CsA or Tac) to a subject who is receiving or will receive treatment with modified immune cells.

[0125] Modified immune cells may include the CAR described herein. Modified immune cells may be NK cells, NKT cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, or combinations thereof.

[0126] Modified cells may contain one or more polynucleotides encoding the CAR described herein. Therefore, such modified immune cells (e.g., CAR-NK cells) possess specificity directed by an engineered receptor (e.g., CAR) expressed therein. For example, modified cells of this disclosure containing CAR are specific for one or more antigens on target cells (e.g., one or more tumor antigens on cancer cells).

[0127] The modified immune cells can be NK cells expressing CAR (i.e., CAR-NK cells). Human natural killer cells (NK cells) play an important role in the innate immune defense against malignant lymphoma cells and are therefore suitable for adoptive immunotherapy (i.e., adoptive cellular immunotherapy). CAR-NK cells can express an armor protein. The armor protein can be secreted IL-15 (e.g., wild-type IL-15, proprotein IL-15, or mutant IL-15, see, for example, PCT Publication No. WO2023280307 A1, the entire contents of which are incorporated herein by reference). CARs can fuse with secreted IL-15 via P2A.

[0128] NK cells are part of the innate immune system, providing the first line of defense against pathogens and cancer cells. They produce cytokines and mediate cytotoxicity without prior sensitization, and they have the ability to interact with and activate other immune cells. NK cells used in immunotherapy can be generated from multiple sources, such as expanded autologous or allogeneic peripheral blood, umbilical cord blood, hematopoietic stem cells, induced pluripotent stem cells, and cell lines.

[0129] NK cell activation and effector function are complex processes that depend on the integration of signals from two different types of receptors—activating receptors and inhibitory receptors. Normal, healthy cells express MHC class I molecules on their surface, which act as ligands for inhibitory receptors and contribute to NK cell self-tolerance. Cellular stresses associated with viral infection or tumor development (such as DNA damage, senescence, or tumor suppressor genes) upregulate ligands for activating receptors. This causes a shift in the balance toward NK cell activation. Transmembrane and intracellular stimulatory / activating molecules in NK cells can influence NK cell differentiation pathways, the metabolic cycle, apoptosis, and activation-induced cell death.

[0130] Modified immune cells (e.g., CAR-NK cells) can be autologous cells, syngeneic cells, allogeneic cells, or xenogeneic cells, depending on the individual receiving them.

[0131] The modified immune cells (e.g., CAR-NK cells) described herein can be eukaryotic cells, such as mammalian cells. Modified immune cells (e.g., CAR-NK cells) can be human cells. Modified immune cells (e.g., CAR-NK cells) can be horse, cow, mouse, sheep, dog, or cat cells.

[0132] Modified immune cells (e.g., CAR-NK cells) may be allogeneic cells obtained from a donor (e.g., a human) that will be administered to a human subject receiving them. Modified immune cells may be modified allogeneic NK cells (e.g., allogeneic CAR-NK cells) obtained from a donor (e.g., a human) that will be administered to a human subject receiving them.

[0133] The modified immune cells can be modified CAR-NK cells. CAR expression in modified immune cells (e.g., CAR-NK cells) can be determined by flow cytometry (FACS). Modified immune cells (e.g., CAR-NK cells) can have CAR positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Modified immune cells (e.g., CAR-NK cells) can also have CAR positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Modified immune cells (e.g., CAR-NK cells) can have CAR positivity rates of 70%-80%, 70%-90%, 75%-85%, or 75%-90%.

[0134] The purity of modified immune cells can be determined by flow cytometry (FACS). Modified immune cells can have a purity greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Modified immune cells can also have a purity less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Finally, modified immune cells can have a purity of 90%-100%, 95%-100%, 98%-100%, or 98%-99%.

[0135] Modified immune cells (e.g., CAR-NK cells) may not contain any modifications that confer resistance to CNIs (e.g., CsA or Tac). Modifications that confer resistance to CNIs may include gene editing or knockout of the FKBP12 or PPIA genes. Modifications that confer resistance to CNIs may include overexpression of at least one miRNA from the miR-17-92 cluster or its paralogs, or inactivation of at least one miR-17-92 cluster target gene.

[0136] Allogeneic cells or allogeneic cells

[0137] The modified immune cells (e.g., CAR-NK cells) described herein can be allogeneic cells (or allogenic cells). The terms "allogeneic cell," "allogeneic immune cell," or "allogeneic engineered immune cell" are used interchangeably herein to refer to cells obtained from an allogeneic donor. Allogeneic cells can be NK cells or CAR-NK cells.

[0138] The allogeneic CAR-NK cells described herein may not have gene modifications affecting their HLA complex, including, for example, B2M mutations (e.g., B2M gene knockout) or HLA allele mutations. Compared to allogeneic CAR-NK cells with MHC or B2M mutations (e.g., knockout), the allogeneic CAR-NK cells described herein may have reduced host NK cell recognition, for example, reduced host NK cell proliferation or killing by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%. Administration of allogeneic CAR-NK cells in the presence of the CNI described herein may not induce host T cell killing compared to administration of allogeneic CAR-NK cells in the absence of a CNI.

[0139] Treatment

[0140] In one aspect, this disclosure provides a method for treating a subject’s disease or disorder, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to a subject in need, wherein the subject is receiving or will receive treatment with modified immune cells.

[0141] In one aspect, this disclosure provides methods for increasing the in vivo infiltration of modified immune cells into the tissues of a subject, methods including administering an effective amount of a calcineurin inhibitor (CNI) to the subject.

[0142] As used herein, the term "effective dose" means the amount that is effective at the necessary dosage and duration to achieve the desired result.

[0143] The disease or disorder can be cancer, an autoimmune disease, or an infection.

[0144] On the other hand, this disclosure provides a method for treating cancer, comprising administering to a subject in need an effective amount of the modified immune cells described herein (e.g., CAR-NK cells) and the calcineurin inhibitor (CNI) described herein. Examples of cancers that can be treated include, but are not limited to, leukemias (including chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and T-cell and B-cell leukemias), lymphomas (Hodgkin's and non-Hodgkin's), lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, solid tissue cancers, hypoxic tumors, squamous cell carcinomas, genitourinary cancers (such as cervical and bladder cancer), hematopoietic cancers, head and neck cancers, and nervous system cancers.

[0145] This disclosure further includes the use of the modified immune cells (e.g., CAR-NK cells) and CNIs (e.g., CsA or Tac) described herein in the manufacture of medicaments or pharmaceutical compositions for modulating immune responses or treating diseases or disorders as described above.

[0146] This disclosure further includes the use of the CNIs described herein (e.g., CsA or Tac) in the manufacture of a medicament or pharmaceutical composition intended for use in: (a) reducing immune clearance of modified immune cells in a subject; (b) increasing in vivo infiltration of modified immune cells into the tissues of a subject; and / or (c) preventing and / or reversing subject exhaustion of modified immune cells in a subject who is receiving or will receive treatment with the modified immune cells described herein. This disclosure further includes the use of the CNIs described herein (e.g., CsA or Tac) in the manufacture of a medicament or pharmaceutical composition intended for use in the prevention or mitigation of host anti-graft (HvG) responses resulting from the administration of the modified immune cells described herein to a patient with cancer, wherein these modified immune cells are used to treat the patient’s cancer or autoimmune disease, and wherein these HvG responses are mediated by host T cells. Modified allogeneic cells may be isolated from a donor for administration to a subject. Modified immune cells can be modified natural killer (NK) cells. Modified NK cells can be CAR-NK cells expressing chimeric antigen receptors (CARs).

[0147] The CNIs (e.g., CsA or Tac) described herein can increase the in vivo infiltration of modified immune cells (e.g., CAR-NK cells) into tissues. Tissues can be tumor tissue. One limitation of cell therapy is the low ability of cells to infiltrate tumor tissues. The CNIs (e.g., CsA or Tac) described herein can increase the in vivo infiltration of modified immune cells (e.g., CAR-NK cells) into tumor tissues. The infiltration of modified immune cells (e.g., CAR-NK cells) into tissues can be determined by the number of modified immune cells (e.g., CAR-NK cells) in tumor tissues or tumor regions. Tissues can be organs or tissues affected by autoimmune diseases, such as the kidneys of patients with lupus nephritis, a class of kidney diseases caused by systemic lupus erythematosus-associated (SLE or lupus). The CNIs (e.g., CsA or Tac) described herein can increase the in vivo infiltration of modified immune cells (e.g., CAR-NK cells) described herein into these organs or tissues. Compared to modified immune cells without CNI (e.g., CsA or Tac), the tissue infiltration (e.g., the number of modified immune cells in tumor tissue or tumor regions) of modified immune cells can be increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. The modified immune cells (e.g., CAR-NK cells) described herein can have an increased tissue infiltration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more compared to the tissue infiltration of modified immune cells (e.g., CAR-NK cells) without CNI (e.g., CsA or Tac).

[0148] The increased tissue infiltration capacity of modified immune cells described herein (e.g., tumor tissue infiltration, kidney infiltration in patients with lupus nephritis) may be due to increased expression of the tumor infiltration marker CX3CR1 on the modified immune cells and increased interaction between CX3CL1 and CX3CR1. The increased tissue infiltration capacity of modified immune cells described herein (e.g., tumor tissue infiltration, kidney infiltration in patients with lupus nephritis) can be achieved via the interaction between CX3CR1 expressed on modified immune cells and CX3CL1 expressed on cells in tissues (e.g., T cells, NK cells, and macrophages in tumor tissue, and T cells, NK cells, and macrophages in kidney infiltration in patients with lupus nephritis).

[0149] CX3CL1 is a transmembrane protein from which a soluble form can be generated via proteolytic shedding. The membrane-bound and soluble forms of CX3CL1 exhibit distinct functions, although both bind to the CX3CR1 chemokine receptor. The CX3CL1-CX3CR1 axis mediates leukocyte adhesion and is also involved in cell survival and recruitment of immune cell subsets. CX3CL1 function is finely regulated by cytokines and transcription factors that modulate its expression and post-translational modifications. Under homeostasis, the CX3CL1-CX3CR1 axis is involved in the removal of damaged neurons and neurogenesis, and it is also involved in several pathological contexts. The CX3CL1-CX3CR1 axis induces several cancer-related cellular responses, such as resistance to proliferation, migration, invasion, and apoptosis. The CX3CL1-CX3CR1 axis is also involved in many autoimmune diseases, including systemic lupus erythematosus (SLE), kidney disease, and rheumatoid arthritis (RA). CX3CL1 is also produced by the renal tubular epithelium. CX3CR1 and its ligand are highly regulated in human kidney diseases (such as IgA nephritis and systemic lupus erythematosus) and inflammatory conditions (such as transplant rejection) (von Vietinghoff S, Kurts C. Regulation and function of CX3CR1 and its ligand CX3CL1 in kidney disease. Cell Tissue Res. Aug 2021; 385(2): 335-344).

[0150] Compared to modified immune cells (e.g., CAR-NK cells) without CNI (e.g., CsA or Tac), the expression of CX3CR1 on modified immune cells (e.g., CAR-NK cells) can be increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. The expression of CX3CR1 on modified immune cells (e.g., CAR-NK cells) can be increased by about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more compared to the expression of modified immune cells (e.g., CAR-NK cells) without CNI (e.g., CsA or Tac).

[0151] Administration of CNIs (e.g., CsA or Tac) can promote the enrichment of modified immune cells (e.g., CAR-NK cells) in the bone marrow of the subject. Subjects described herein may have bone marrow-related diseases or disorders, or blood cancers such as lymphoma or leukemia. Subjects may have a disease or disorder selected from: acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenström macroglobulinemia, follicular lymphoma, B-cell lymphoma, cutaneous T-cell lymphoma, multiple myeloma, plasmacytoma, and amyloidosis. Subjects may have bone marrow-related diseases. Subjects may have bone marrow-related autoimmune diseases, such as B-cell regulated autoimmune diseases. Subjects may have systemic lupus erythematosus (SLE), rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, ulcerative colitis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), systemic sclerosis / scleroderma (SSc), idiopathic inflammatory myopathy (IIM), antiphospholipid syndrome (APS), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis (MG), ANCA (anti-neutrophil cytoplasmic antibody)-associated vasculitis, diabetes, Raynaud's syndrome, Sjögren's syndrome, neuromyelitis optica (NMO) and glomerulonephritis, and myelin oligodendrocyte glycoprotein-IgG-associated disorder (MOGAD).

[0152] Compared to modified immune cells (e.g., CAR-NK cells) without CNI (e.g., CsA or Tac), the distribution of modified immune cells (e.g., CAR-NK cells) in the bone marrow (e.g., the number of modified immune cells in the subject's bone marrow) can be increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. The distribution of modified immune cells (e.g., CAR-NK cells) in the bone marrow (e.g., the number of modified immune cells in the subject's bone marrow) can be increased to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 times or more compared to the distribution of modified immune cells (e.g., CAR-NK cells) in the bone marrow (e.g., the number of modified immune cells in the subject's bone marrow) without the administration of CNI (e.g., CsA or Tac).

[0153] Administration of CNIs (e.g., CsA or Tac) can promote the expansion of modified immune cells (e.g., CAR-NK cells). Compared to cells without CNI (e.g., CsA or Tac) administration, the expansion of modified immune cells (e.g., CAR-NK cells) can be increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%. Compared to cells without CNI (e.g., CsA or Tac) administration, the expansion of modified immune cells (e.g., CAR-NK cells) can be increased by approximately 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. Cell expansion or proliferation can be measured by in vitro cell proliferation assays. Compared to cells without CNI (e.g., CsA or Tac), the modified immune cells (e.g., CAR-NK cells) described herein may have a cell number that is more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% higher. Compared to cells without CNI (e.g., CsA or Tac), the modified immune cells (e.g., CAR-NK cells) described herein may have a cell number that is less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90% higher.

[0154] Modified immune cells (e.g., CAR-NK cells) can kill tumor cells. The cytotoxicity of modified immune cells (e.g., CAR-NK cells) against tumor cells can be determined by long-term in vitro cytotoxicity assays. The effector cell:target cell (E:T) ratio can be approximately 1:10, 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, or 10:1. The E:T ratio can be 1:4. Following one, two, three, four, or five rounds of stimulation in a re-challenge assay, the modified immune cells (e.g., CAR-NK cells) exhibited cytotoxicity greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Following one, two, three, four, or five rounds of stimulation in a re-challenge assay, the modified immune cells (e.g., CAR-NK cells) exhibited cytotoxicity less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90%. After one, two, three, four, or five rounds of stimulation in the re-excitation assay, the modified immune cells (e.g., CAR-NK cells) can exhibit cytotoxicity of 10%-100%, 10%-50%, 20%-100%, 20%-60%, 20%-40%, 30%-70%, 40%-80%, 50%-90%, 70%-100%, 80%-100%, or 90%-100%. Compared to the cytotoxicity of modified immune cells (e.g., CAR-NK cells) without CNI (e.g., CsA or Tac), the cytotoxicity of modified immune cells (e.g., CAR-NK cells) increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1,000% after 1, 2, 3, 4, or 5 rounds of stimulation in a re-challenge assay.

[0155] In one aspect, this disclosure provides methods for reducing the immune clearance of modified immune cells (e.g., CAR-NK cells) in a subject, methods including administering to the subject an effective amount of these modified immune cells (e.g., CAR-NK cells) and a calcineurin inhibitor (CNI (e.g., CsA or Tac)).

[0156] Administration of a CNI (e.g., CsA or Tac) can protect the modified immune cells described herein (e.g., CAR-NK cells) from immune clearance (e.g., host T cell-mediated cytotoxicity or host immune cell-mediated cytotoxicity). Compared to cytotoxicity without CNI (e.g., CsA or Tac), T cell or immune cell-mediated killing by modified immune cells (e.g., CAR-NK cells) can be reduced by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%. Compared to cells without CNI (e.g., CsA or Tac), T cell or immune cell-mediated killing by modified immune cells (e.g., CAR-NK cells) can be reduced to about 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10 or less.

[0157] Modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can also be used in experimental models, for example, to further study and elucidate cell function.

[0158] Modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be administered via any suitable method. One or more of the modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) described herein can be administered to the subject in a single uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). Modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be administered via different methods. Modified immune cells (e.g., CAR-NK cells) can be administered via one or more injections, and CNIs (e.g., CsA or Tac) can be administered via injection alone or orally, or in combination thereof (e.g., CNIs (e.g., CsA or Tac) are injected during the subject's hospitalization and administered orally after the subject's discharge).

[0159] In some cases, modified immune cells (e.g., CAR-NK cells) can be expanded in the subject after administration. In some cases, modified immune cells (e.g., CAR-NK cells) have already been expanded in vitro before administration to the subject. Modified immune cells (e.g., CAR-NK cells) can be frozen to provide cells for multiple treatments with the same cell preparation. The modified immune cells (e.g., CAR-NK cells) disclosed herein and pharmaceutical compositions comprising these modified immune cells and any additional therapeutic agents (e.g., CNIs (e.g., CsA or Tac)) can be packaged as kits (reagents). Kits may include instructions (e.g., written instructions) regarding the use of the modified cells and CNIs (e.g., CsA or Tac) and the compositions comprising them.

[0160] In one aspect, this disclosure provides a treatment method comprising administering to a subject a therapeutically effective amount of the modified immune cells (e.g., CAR-NK cells) described herein and the CNI (e.g., CsA or Tac) described herein. The therapeutically effective amount of the modified immune cells (e.g., CAR-NK cells) may be administered in a single dose. The therapeutically effective amount of the modified immune cells (e.g., CAR-NK cells) may be administered multiple times (such as any one of 2, 3, 4, 5, 6, or more doses). The CNI (e.g., CsA or Tac) may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. By monitoring the patient's disease signs and adjusting the treatment accordingly, those skilled in the art can readily determine the optimal dosage and treatment regimen for a particular patient.

[0161] In the methods described herein, CNIs (e.g., CsA or Tac) may be administered before, during, and / or after administration of modified immune cells (e.g., CAR-NK cells). For example, CNIs (e.g., CsA or Tac) may be administered before administration of modified immune cells (e.g., CAR-NK cells) to provide an immunosuppressive environment in the subject to enhance the efficacy of the modified immune cells (e.g., CAR-NK cells). During administration of modified immune cells (e.g., CAR-NK cells), CNIs (e.g., CsA or Tac) may be continued throughout the course of administration of the modified immune cells (e.g., CAR-NK cells). After administration of modified immune cells (e.g., CAR-NK cells), CNIs (e.g., CsA or Tac) may be continued to maintain the subject's immunosuppressive condition to reduce, for example, HvG responses. The frequency and dosage of CNIs (e.g., CsA or Tac) may vary depending on the specific attributes of the disease or disorder and / or the patient and / or other treatments. For example, CNI (e.g., CsA or Tac) can be administered daily, every two days, every three days, every four days, every five days, every six days, weekly, every two weeks, or for longer periods.

[0162] In the methods described herein, a CNI (e.g., CsA or Tac) may be administered prior to the administration of modified immune cells (e.g., CAR-NK cells). The CNI (e.g., CsA or Tac) may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, or longer prior to the administration of modified immune cells (e.g., CAR-NK cells). The CNI may be administered up to 7 days prior to the administration of modified immune cells (e.g., CAR-NK cells).

[0163] CNIs (e.g., CsA or Tac) can be administered during and throughout the administration of modified immune cells (e.g., CAR-NK cells). In the methods described herein, CNIs (e.g., CsA or Tac) may be administered concurrently with modified immune cells (e.g., CAR-NK cells).

[0164] CNIs (e.g., CsA or Tac) can be administered after the administration of modified immune cells (e.g., CAR-NK cells). CNIs (e.g., CsA or Tac) can be administered approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or longer after the administration of modified immune cells (e.g., CAR-NK cells).

[0165] CNIs (e.g., CsA or Tac) can be administered after the administration of modified immune cells (e.g., CAR-NK cells) for approximately or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 1 month, or 2 months. CNIs (e.g., CsA or Tac) can be administered after the administration of modified immune cells (e.g., CAR-NK cells) for approximately 15 days, at least 20 days, at least 1 month, 2 months, or 3 months. CNIs (e.g., CsA or Tac) can be administered after the administration of modified immune cells (e.g., CAR-NK cells) for approximately 20 days.

[0166] CNIs (e.g., CsA or Tac) can be administered for approximately or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 month, 2 months, or 3 months. CNIs (e.g., CsA or Tac) can also be administered for 27 days.

[0167] In the methods described herein, lymphocyte clearance may have already been performed in the subject prior to administration of a CNI (e.g., CsA or Tac). In the methods described herein, lymphocyte clearance may also be performed in the subject after administration of a CNI (e.g., CsA or Tac).

[0168] In the methods described herein, lymphocyte depletion may have already been performed in the subject prior to administration of the modified immune cells (e.g., CAR-NK cells). Lymphocyte depletion may have been performed in the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or longer prior to administration of the modified immune cells (e.g., CAR-NK cells). Lymphocyte depletion may have been performed in the subject approximately 3 or 4 days prior to administration of the modified immune cells (e.g., CAR-NK cells). Lymphocyte depletion may be performed multiple times (such as any of 2, 3, 4, 5, 6 or more times). Lymphocyte depletion may be performed up to 3 times.

[0169] Lymphocyte clearance may have occurred in the subject after administration of modified immune cells (e.g., CAR-NK cells). Lymphocyte clearance may have occurred in the subject approximately or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration of modified immune cells (e.g., CAR-NK cells).

[0170] CNIs can be administered 7 days prior to the administration of modified immune cells (e.g., CAR-NK cells). CNIs (e.g., CsA or Tac) can be administered for up to 27 days. Lymphocyte clearance may have already been performed in the subject approximately 3 or 4 days prior to the administration of modified immune cells (e.g., CAR-NK cells). Lymphocyte clearance can be performed up to 3 times.

[0171] In the methods described herein, CNI (e.g., CsA or Tac) may be administered orally after the subject is discharged from the hospital.

[0172] In the method described herein, the blood concentration of CNI (e.g., CsA or Tac) can be monitored throughout the treatment process.

[0173] The trough blood concentration of CsA can be approximately 10-500 ng / ml, 10-400 ng / ml, 10-300 ng / ml, 10-200 ng / ml, 10-100 ng / ml, 10-50 ng / ml, 100-500 ng / ml, 100-400 ng / ml, 100-300 ng / ml, 100-200 ng / ml, 200-500 ng / ml, 200-400 ng / ml, or 200-300 ng / ml. At the start of treatment (e.g., immediately before, during, or after administration of modified immune cells), the trough blood concentration of CsA can be approximately 100-300 ng / ml. At later time points in the treatment (e.g., at least 1, 2, 3, or 4 weeks after administration of modified immune cells), the trough blood concentration of CsA may be less than 300 ng / ml, less than 200 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 10 ng / ml.

[0174] The trough blood concentration of Tac can be approximately 1-50 ng / ml, 1-40 ng / ml, 1-30 ng / ml, 1-20 ng / ml, 1-10 ng / ml, 1-5 ng / ml, 5-50 ng / ml, 5-40 ng / ml, 5-30 ng / ml, 5-20 ng / ml, or 5-10 ng / ml. At the start of treatment (e.g., immediately before, during, or after administration of modified immune cells), the trough blood concentration of CsA can be approximately 5-15 ng / ml. At later time points in treatment (e.g., at least 1, 2, 3, or 4 weeks after administration of modified immune cells), the trough blood concentration of CsA can be approximately less than 30 ng / ml, less than 20 ng / ml, less than 10 ng / ml, less than 5 ng / ml, or less than 1 ng / ml.

[0175] This article also provides a method for enhancing the degranulation of modified immune cells (e.g., CAR-NK cells) in a subject, which involves administering an effective amount of a calcineurin inhibitor (CNI) to the subject who is receiving or will receive treatment with modified immune cells.

[0176] NK cells kill cells through mechanisms involving the release of small cytolytic granules containing granzyme B and perforin, which induce cell death in target cells. While NK cell numbers can be counted using immunophenotypic analysis on flow cytometry, a significant limitation of NK cell research is the lack of availability of high-throughput assays for detecting NK cell functional activity.

[0177] During degranulation, cytolytic granules in NK cells are released, and lysosome-associated membrane protein-1 (LAMP-1, CD107a) present on the surface of the cytolytic granules is transported to the cell surface and becomes available for antibody binding. This allows for the identification of activated NK cells, making them an attractive biomarker for assessing the integrity of the granule exocytosis mechanism.

[0178] In the method described herein, administration of CNI can enhance the degranulation of modified immune cells (e.g., CAR-NK cells). Degranulation can be enhanced by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0179] In the method described herein, administration of CNI can increase the expression of CD107a on modified immune cells (e.g., CAR-NK cells). The expression of CD107a on modified immune cells (e.g., CAR-NK cells) can be increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold.

[0180] The modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the modified cells can vary. For example, modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be used as a prophylactic agent and can be administered continuously to subjects with a condition or predisposition to reduce the likelihood of the disease or condition occurring. Modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be administered to subjects as soon as possible during or after the onset of symptoms. Administration of modified cells can begin immediately upon the onset of symptoms, within 3 hours before the onset of symptoms, within 6 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 48 hours before the onset of symptoms, or at any time after the onset of symptoms. Initial administration can be via any practical route (e.g., intravenous infusion or injection), such as any of the routes described herein using any of the formulations described herein. The administration of the modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) disclosed herein may be intravenously administered. Following the onset of cancer or an infectious disease, one or more doses of the modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) may be administered as soon as feasible and for the duration required to treat the disease, such as approximately 24 hours to approximately 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, or approximately 1 month to approximately 3 months. For the treatment of cancer, one or more doses of the modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) may be administered several years after the onset of cancer and before or after other treatments. Modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) may be administered for at least approximately 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary for each subject.

[0181] Methods of administering modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) for adoptive cell therapy are known and can be used in combination with the methods and compositions provided. For example, adoptive T-cell or NK-cell therapy methods are described in the following literature: for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) NatRev Clin Oncol. 8(10):577-85. See, for example, Themeli et al., (2013) Nat Biotechnol.31(10): 928-933; Tsukahara et al., (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al., (2013) PLoS ONE 8(4): e61338.

[0182] Cell therapy (e.g., adoptive NK cell therapy) can be performed via allogeneic transfer, where cells are isolated and / or otherwise prepared from a subject other than the one to be or ultimately receive the cell therapy (e.g., a first subject). The cells can then be administered to a different subject of the same species, such as a second subject. The first and second subjects can be genetically identical. The first and second subjects can be genetically similar. The second subject can express the same HLA class or supertype as the first subject.

[0183] Prior to administration of cells or cell-containing compositions and / or CNIs (e.g., CsA or Tac), the subject may have been treated with a therapeutic agent targeting a disease or condition (e.g., tumor). In some respects, the subject may be refractory or unresponsive to other therapeutic agents. The subject may have a persistent or relapsing disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even though the subject has become resistant to another therapy, administration may still be effective in treating the subject.

[0184] The subject may respond to another treatment agent, and treatment with that agent reduces the disease burden. In some cases, the subject initially responds to the treatment agent but exhibits a relapse of the disease or condition over time. The subject may not experience a relapse. It can be determined that the subject is at risk of relapse, such as being at high risk of relapse, and therefore cells are administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse. In some cases, the subject has not previously received treatment with another treatment agent.

[0185] Subjects may have persistent or recurrent diseases, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even if a subject has become resistant to another therapy, administration can still effectively treat the subject.

[0186] The modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) described herein can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. Furthermore, the modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be used to treat any condition associated with cancer, particularly a cell-mediated immune response against one or more tumor cells, where treatment or ablation of the disease is desired. Types of cancer for which the modified cell or pharmaceutical composition may be used include carcinoma, blastoma, and sarcoma, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, etc. Cancer can be a non-solid tumor (such as a hematologic malignancy) or a solid tumor. Adult oncology / cancer and pediatric oncology / cancer are also included. Cancer can be a solid tumor or a blood cancer. Cancer can be carcinoma. Cancer can be a sarcoma. Cancer can be leukemia. Cancer can be a solid tumor.

[0187] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the types of cells that form them (such as sarcoma, epithelial carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, seminomas, bladder cancer, melanomas, and CNS tumors (such as gliomas (such as brainstem gliomas and mixed gliomas), glioblastomas (also known as multimorphic glioblastomas), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0188] Cancers suitable for treatment using the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (various tissues), bladder cancer, including transitional cell carcinoma (a malignant tumor of the bladder), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell carcinoma and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, osteoblastoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0189] Sarcomas suitable for treatment using the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0190] The modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) described herein can be administered to animals, preferably mammals, and even more preferably humans, to treat bone marrow-related autoimmune diseases. Furthermore, the modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be used to treat any condition associated with bone marrow-related autoimmune diseases. Bone marrow-related autoimmune diseases are a group of diseases affecting the bone marrow. Some of the pathogenic cells in these diseases can be located in, develop in, and / or invade the bone marrow, thereby affecting its function. Bone marrow-related autoimmune diseases include B cell-regulated autoimmune diseases. B cell-regulated autoimmune diseases are autoimmune disorders involving misregulation of B cells. B cell-regulated autoimmune diseases can be autoimmune disorders associated with self-reactive plasma cells and / or self-reactive memory B cells.

[0191] Examples of bone marrow-related autoimmune diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, ulcerative colitis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenic purpura, multiple sclerosis (MS), systemic sclerosis / scleroderma (SSc), idiopathic inflammatory myopathy (IIM), antiphospholipid syndrome (APS), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis (MG), ANCA (anti-neutrophil cytoplasmic antibody)-associated vasculitis, diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome, neuromyelitis optica (NMO), glomerulonephritis, and myelin oligodendrocyte glycoprotein-IgG-related disorder (MOGAD).

[0192] Bone marrow-related autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, ulcerative colitis, Sjögren's syndrome, graft-versus-host disease, multiple sclerosis, systemic sclerosis / scleroderma (SSc), systemic sclerosis, myasthenia gravis, rheumatoid arthritis, juvenile rheumatoid arthritis, idiopathic inflammatory myopathy (IIM), antiphospholipid syndrome (APS), psoriasis, ANCA-associated vasculitis, diabetes mellitus, neuromyelitis optica (NMO), osteoarthritis, psoriatic arthritis, dermatitis, atopic dermatitis, chronic autoimmune urticaria, and polymyositis. Dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, myelin oligodendrocyte glycoprotein-IgG related disorder (MOGAD), allergic diseases, eczema, asthma, atherosclerosis, autoimmune myocarditis, leukocyte adhesion defect, lupus (nephritic, non-nephrogenic, discoid, alopecia type), allergic encephalomyelitis, tuberculosis, sarcoidosis, granulomatous disease, Wegener's granulomatosis, agranulocytosis, vasculitis, aplastic anemia, Coombs-positive anemia. Positive anemia, Diamond Blackfan anemia, immune hemolytic anemia, hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte exudation, multiple organ injury syndrome, antiglomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome. Solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus, autoimmune polyendocrine disorders, Reiter's disease.Diseases including stiff-person syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune orchitis, autoimmune oophoritis, primary hypothyroidism; autoimmune endocrine diseases, autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, polyendocrine syndrome, Sheehan's syndrome. Syndrome, autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), non-transplantable bronchiolitis obliterans, Guillain-Barré syndrome, large vessel vasculitis, polymyalgia rheumatica, giant cell (Gau's) arteritis, medium vessel vasculitis, Kawasaki disease, polyarteritis nodosa, ankylosing spondylitis, Berger's disease, rapidly progressive glomerulonephritis, primary biliary cirrhosis, stomatitis, cryoglobulinemia, ALS, and coronary artery disease.

[0193] Since CNIs (e.g., CsA or Tac) can promote the infiltration of modified immune cells (e.g., CAR-NK cells) into the bone marrow, the methods described herein may be helpful in treating bone marrow-related autoimmune diseases.

[0194] The modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) described herein may be included in compositions for immunotherapy. The compositions may include pharmaceutical compositions and further include pharmaceutically acceptable carriers. A therapeutically effective amount of the pharmaceutical composition containing the modified cells may be administered.

[0195] The modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) can be used immediately for the above-described treatments, experimental or commercial applications, or the cells can be cryopreserved for later use. The pharmaceutical composition may be included in a container, package or dispenser along with the instructions for use.

[0196] The modified immune cells (e.g., CAR-NK cells) and / or CNIs (e.g., CsA or Tac) described herein can be formulated into unit dosage forms suitable for precise single-dose administration. In some cases, the unit dosage form includes additional lymphocytes. In a unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit doses can be in the form of packages containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose, non-resealable containers. Multi-dose, resealable containers can be used, for example, with or without preservatives. In some instances, the pharmaceutical composition does not contain preservatives. Formulations for parenteral injection can be presented in unit dosage forms, such as in ampoules or in multi-dose containers with preservatives.

[0197] Example

[0198] The disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0199] Example 1. Calcineurin inhibitors suppress the tumor-killing effect of CAR-T cells.

[0200] To investigate the effect of plasma-concentrated CNI on CAR-T cell killing of target cells, PBMCs were isolated from human peripheral blood. CAR-T cells specifically targeting BCMA were prepared. The CAR used in this experiment was the 32M CAR described in PCT application number PCT / CN2023 / 096342.

[0201] CAR-T cells were incubated with target H929 cells at a ratio of 1:4. Target cells were replenished every 24 hours at the same ratio to establish a repetitive stimulation system for evaluating the cytotoxic effect of CAR-T cells in a continuous killing assay. Various concentrations of calcineurin inhibitors, such as Tac (MJedChemExpress) or CsA (Selleck), were added. Figure 2 As shown. After 72 hours, the percentage of viable H929 cells was determined by flow cytometry. Figure 2 In the study, Ctrl was the control group without any calcineurin inhibitors; BCMA showed residual tumor marker levels; and sdAb showed CAR-T cell levels. Figure 2 As shown, the calcineurin inhibitors tacrolimus (Tac) and cyclosporine A (CsA) inhibited the killing of target cells by CAR-T cells at clinically relevant concentrations.

[0202] Example 2: Calcineurin inhibitors enhance the tumor-killing effect of CAR-NK cells

[0203] The CAR-NK cells used in this experiment were the 32M CAR-NK cells described in PCT application number PCT / CN2023 / 096342. CAR-NK cells were incubated with target H929 cells at a ratio of 1:4. Target cells were replenished every 24 hours at the same ratio to establish a repetitive stimulation system to evaluate the cytotoxic effect of CAR-NK cells in a continuous killing assay. Various concentrations of calcineurin inhibitors, such as Tac (MJedChemExpress) or CsA (Selleck), were added. Figure 3A As shown. After 72 hours, the percentage of viable H929 cells was determined by flow cytometry.

[0204] Unlike the results observed with CAR-T cells, the calcineurin inhibitors tacrolimus (Tac) and cyclosporine A (CsA) did not inhibit the killing of target cells by CAR-NK cells at clinically relevant concentrations. Instead, the calcineurin inhibitors tacrolimus (Tac) and cyclosporine A (CsA) enhanced the killing of target cells by CAR-NK cells (see [link to relevant documentation]). Figure 3A ).

[0205] Furthermore, calcineurin inhibitors such as cyclosporine A (CsA) enhanced CD107a expression in CAR-NK cells. CAR-NK cells were incubated with target NCI-H929 cells at a 1:1 ratio for 2 hours with or without gradient concentrations of CsA. Cells were then collected, and the degranulation marker CD107a (LAMP-1) was stained and analyzed by flow cytometry. Figure 3B As shown in -C, CD107a expression on CAR-NK was higher in the group treated with CsA compared to the group without CsA.

[0206] Example 3: Calcineurin inhibitors enhance the tumor-killing effect of CAR-NK cells in animal models.

[0207] To further investigate the effects of CsA on the function of CAR-NK cells in an animal model, we constructed an NCG tumor mouse model and performed CAR-NK cell infusion. Specifically, luciferase-labeled H929 cells were inoculated into NCG mice via tail vein injection. Twelve days post-injection, tumor-bearing mice were randomly assigned to either the CsA treatment group (CsA) or the control group (Ctrl), and inoculated with the same number of CAR-NK cells. Three days after CAR-NK infusion, mice were administered either CsA or plain buffer. Total throughput was recorded twice weekly after CsA or buffer treatment. Figure 4AAs shown, 10 days after CAR-NK infusion, mice treated with 50 mg / kg CsA (Selleck) had significantly lower flux levels compared to the control group. More importantly, this effect on tumor progression was not due to CsA directly killing tumor cells. Figure 4B As shown, CsA alone (without CAR-NK cells) did not inhibit the growth of tumor cells.

[0208] These data demonstrate that CsA enhances the tumor-killing effect of CAR-NK cells in animal models.

[0209] Example 4: Calcineurin inhibitors upregulate CX3CR1 expression in CAR-NK cells

[0210] Interestingly, through phenotypic analysis of CAR-NK cells treated with CsA or Tac, we found that CAR-NK cells treated with CsA or Tac had upregulated expression of the tissue infiltration marker CX3CR1. Figures 5A-5B ).

[0211] One limitation of cell therapy products is their low tumor invasiveness. Furthermore, the CX3CL1-CX3CR1 axis plays a crucial role in the chemotaxis of immune cells towards the tumor microenvironment. Therefore, the upregulation of CX3CR1 expression on CAR-NK cells suggests that CAR-NK cells possess enhanced in vivo tissue / tumor invasiveness.

[0212] Example 5: Calcineurin inhibitors promote CAR-NK cell infiltration in bone marrow

[0213] We further investigated the effect of CNI on the distribution of CAR-NK cells in bone marrow and peripheral blood in a mouse model of tumors. An NCG H929 tumor mouse model was constructed, and CAR-NK cells were infused into mice as described in Example 3. Mice were administered 50 mg / kg CsA (CsA group) or plain buffer (Ctrl) for three days. Bone marrow and peripheral blood samples were obtained from the mice, and the number of CAR-NK cells was analyzed using flow cytometry. Figure 6A and 6B ).like Figure 6A As shown, treatment with CsA increased the distribution and number of CAR-NK cells in the bone marrow, indicating that CsA promotes the infiltration of CAR-NK cells in the bone marrow.

[0214] Furthermore, CX3CR1 expression on CAR-NK cells isolated from bone marrow was evaluated. Consistent with in vitro observations, 21-day CsA treatment also increased CX3CR1 expression in CAR-NK cells isolated from bone marrow. Figure 6C ).

[0215] Example 6: Calcineurin inhibitors suppress the host immune system and promote the expansion of CAR-NK cells.

[0216] To investigate whether CNI can suppress the host immune system and promote the expansion of CAR-NK cells, we established a mixed lymphocyte response (MLR) system. Using CsA and Tac as an example, in the allogeneic 3-dimensional MLR of the CsA experimental system, a ratio of PBMCs : allo-CAR-NK cells : tumor cells = 50 : 1 : 4 was used for co-culture. Simultaneously, 200 ng / ml of CsA was added to the CsA group. Figure 7A As shown, the addition of CsA significantly promoted the expansion of CAR-NK cells, eliminating rejection mediated by allogeneic PBMCs. Similarly, in the allogeneic 3-way MLR in the Tac experimental group, a PBMC:allo-CAR-NK:tumor cell ratio of 50:1:4 was used for co-culture. Simultaneously, 10 ng / ml Tac was added to the Tac group. Figure 7B As shown, the addition of Tac significantly promoted the expansion of CAR-NK cells and eliminated rejection mediated by allogeneic PBMCs.

[0217] Example 7: Calcineurin inhibitors enhance the tumor-killing effect of CAR-NK cells in animal models.

[0218] To further investigate the effects of CsA or Tac on the function of CAR-NK cells in an animal model, we constructed an NCG tumor mouse model and performed CAR-NK cell infusion. Specifically, luciferase-labeled H929 cells were injected into NCG mice via tail vein injection. Twelve days post-injection, tumor-bearing mice were randomly assigned to a CsA treatment group (CAR-NK, CsA), a Tac treatment group (CAR-NK, Tac), or a control group (CAR-NK, vector), and inoculated with the same number of CAR-NK cells. One day prior to CAR-NK infusion, mice were administered CsA, Tac, or plain buffer. Total throughput was recorded twice weekly after CsA, Tac, or buffer treatment. Figure 8A As shown, mice treated with 50 mg / kg CsA or 5 mg / kg Tac were more effective in controlling tumor progression. More importantly, peripheral CAR-NK cell expansion was unaffected, as... Figure 8B As shown in the image.

[0219] We also performed RNA sequencing to further characterize the RNA from sources such as... Figure 8A and 8B The gene expression profiles of CAR-NK cells isolated in the aforementioned experiment were analyzed. KEGG gene enrichment analysis showed that multiple genes positively controlling NK cell activity were upregulated.

[0220] In summary, these data support the conclusion that calcineurin inhibitors (including CsA and Tac) do not inhibit CAR-NK activity, but rather positively regulate it.

[0221] Example 8: CsA prevents in vivo CAR-NK rejection in a PBMC-based HvG model.

[0222] To further investigate whether CsA treatment suppresses host immune rejection and thus promotes graft-CAR-NK persistence, we established a human PBMC-based HvG model. For this purpose, NCG mice were treated with CsA or a carrier at doses of 50 mg / kg (once daily) or 25 mg / kg (twice daily). After 5 days, 20 × 10⁶ mice were inoculated. 6 One PBMC was used to rebuild the human immune system. Then, three days later, 1.5 × 10⁶ PBMCs were infused. 6 One HLA-A / B / C mismatched allogeneic CAR-NK cell, and peripheral CAR-NK counts were monitored by FACS. Figure 9A ).like Figure 9B As shown, CsA treatment prolonged CAR-NK persistence and increased Cmax, which supports the view that CsA suppresses the host's immune rejection of allogeneic CAR-NK and thus benefits CAR-NK amplification in this HvG model.

[0223] We also evaluated T cell counts and T cell phenotypes derived from PBMCs. Consistently, CsA treatment suppressed T cell remodeling ( Figure 9C It also downregulated the expression of T cell activation markers, including CD38 and CD71. Figure 9D-9E Furthermore, we investigated the effect of CsA discontinuation on T cell recovery. For this purpose, we discontinued CsA treatment 18 days after CAR-NK infusion, as shown by the dashed line. After CsA discontinuation, T cell expansion and activation markers largely recovered (…). Figure 9D-9E ).

[0224] Example 9: CsA prevents CAR-NK rejection in vivo in a dose-dependent manner.

[0225] In a clinical setting, clinicians can adjust the dosage of CsA to control the degree of rejection. To further investigate the effect of different CsA dosages on CAR-NK rejection, we established a similar HvG model as mentioned in Example 8, and in this case, we treated mice with gradient concentrations of CsA. Figure 10As shown, CsA at doses ranging from 20 mg / kg / day to 50 mg / kg / day effectively protected CAR-NK from clearance and promoted CAR-NK amplification. Increased CsA doses were associated with better CAR-NK persistence, as revealed by increased Cmax and prolonged amplification. Again, we discontinued CsA treatment on day 13 (dashed line) in the 50 mg / kg / day treatment group to investigate the effect of CsA discontinuation on CAR-NK clearance. Preliminary data suggest that CsA discontinuation tends to accelerate CAR-NK rejection.

[0226] These data indicate that CsA prolongs CAR-NK persistence in a dose-dependent manner.

[0227] Example 10: CsA treatment duration determines CAR-NK persistence

[0228] In a clinical setting, clinicians can adjust the duration of CsA treatment to control the degree of rejection. To investigate the impact of CsA treatment duration on CAR-NK durability, we established a similar HvG model as mentioned in Example 8. Here, CsA treatment was set to 4, 7, or 22 days after CAR-NK infusion. Figure 11A As shown, longer CsA treatment duration is associated with better CAR-NK persistence, as revealed by prolonged CAR-NK expansion and higher Cmax. Consistently, CsA treatment inhibits T cell expansion, and CsA discontinuation leads to significant T cell recovery. Figure 11B ).

[0229] These data suggest that CsA prolongs CAR-NK persistence in a treatment duration-dependent manner.

[0230] Example 11: CsA treatment improves CAR-NK efficacy in tumor-bearing HvG models

[0231] To investigate whether CsA treatment inhibits immune rejection and thus improves the tumor-killing efficacy of CAR-NK, we established a tumor-bearing HvG model, in which myeloma H929 tumor cells were additionally inoculated before establishing the HvG model as mentioned in Example 8. Figure 12A As shown, CsA treatment promoted the tumor-killing effect of CAR-NK cells compared to the mediator treatment group. Coordinated, CAR-NK amplification was also evaluated during CsA treatment, such as... Figure 12B As shown in the image.

[0232] These data support the conclusion that CsA inhibits PBMC-mediated rejection of allogeneic CAR-NK cells and enhances their antitumor efficacy. Furthermore, these data support the possibility that clinicians can adjust the dosage or duration of CsA treatment to achieve the desired CAR-NK durability.

[0233] Other embodiments

[0234] It should be understood that although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for treating a disease or disorder in a subject, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to a subject in need, wherein the subject is receiving or will receive treatment with modified immune cells.

2. The method of claim 1, wherein the disease or disorder is cancer, an autoimmune disease, or an infection.

3. A method for reducing immune clearance of modified immune cells in a subject, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to the subject, wherein the subject is receiving or will receive treatment with modified immune cells.

4. The method of claim 3, wherein the modified immune cells are allogeneic cells isolated from a donor for administration to a subject.

5. The method of claim 3 or 4, wherein the immune clearance of the modified immune cells is carried out through host immune cell-mediated cytotoxicity.

6. The method of any one of claims 3-5, wherein the immune clearance of the modified immune cells is carried out via host T cell-mediated cytotoxicity.

7. The method of any one of claims 3-6, wherein the application of CNI increases the amplification of the modified immune cells.

8. A method for increasing the in vivo infiltration of modified immune cells into the tissues of a subject, the method comprising administering an effective amount of a calcineurin inhibitor (CNI) to the subject.

9. The method of claim 8, wherein the tissue is tumor tissue.

10. The method of claim 8 or 9, wherein the modified immune cells have increased expression of CX3CR1.

11. The method of claim 10, wherein the increased in vivo infiltration of the modified immune cells into the tissue is achieved via an interaction between CX3CR1 expressed on the modified immune cells and CX3CL1 expressed on cells in the tissue.

12. The method of any one of claims 1-11, wherein the modified immune cell is a modified natural killer (NK) cell.

13. The method of claim 12, wherein the modified NK cell is a CAR-NK cell expressing a chimeric antigen receptor (CAR).

14. The method of any one of claims 1-13, wherein the CNI is cyclosporine A (CsA).

15. The method of claim 14, wherein the CsA is administered at a dose of about 1 to about 25 mg / kg / day or about 0.5 to about 12.5 mg / kg / day twice daily.

16. The method of any one of claims 1-13, wherein the CNI is tacrolimus.

17. The method of claim 16, wherein the tacrolimus is administered at a concentration of about 0.01 to 1 mg / kg / day.

18. The method of claim 13, wherein the CAR receptor specifically binds to the target antigen.

19. The method of claim 18, wherein the target antigen is selected from the group consisting of: BCMA, CLL1, CD4, GPC3, GPRC5D, GUCY2C, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL. -13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, duracin 18.2, duracin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1 and PD-L2.

20. The method of any one of claims 1-19, wherein the modified immune cells are not modified by gene editing or knockout of the FKBP12 or PPIA gene, the gene editing or knockout making the cells resistant to CNI.

21. The method of any one of claims 1-19, wherein the modified immune cells are not modified by overexpression of at least one miRNA of the miR-17-92 cluster or its paralogs, or by inactivation of at least one miR-17-92 cluster target gene, the overexpression or inactivation of which renders the cells resistant to CNI.

22. The method of any one of claims 1-19, wherein the modified immune cells do not contain any modifications that make the cells resistant to CNI.

23. The method of any one of claims 1-22, wherein the modified immune cells are enriched in the bone marrow of the subject.

24. The method of any one of claims 1-23, wherein the subject suffers from a bone marrow-related disease or disorder, or a blood cancer such as lymphoma or leukemia.

25. The method of claim 24, wherein the subject suffers from acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenström macroglobulinemia, follicular lymphoma, B-cell lymphoma, cutaneous T-cell lymphoma, multiple myeloma, plasmacytoma, or amyloidosis.

26. The method of any one of claims 1-23, wherein the subject suffers from a bone marrow-related disease.

27. The method of claim 26, wherein the subject suffers from a bone marrow-related autoimmune disease, such as a B-cell regulated autoimmune disease.

28. The method of claim 27, wherein the subject suffers from systemic lupus erythematosus (SLE), rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, ulcerative colitis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), systemic sclerosis / scleroderma (SSc), idiopathic inflammatory myopathy (IIM), antiphospholipid syndrome (APS), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis (MG), ANCA (anti-neutrophil cytoplasmic antibody)-associated vasculitis, diabetes, Raynaud's syndrome, Sjögren's syndrome, neuromyelitis optica (NMO), glomerulonephritis, and oligodendrocyte glycoprotein-IgG-associated disorder (MOGAD).

29. The method of any one of claims 1-28, wherein the CNI is administered before, during, and / or after the administration of the modified immune cells.

30. The method of any one of claims 1-29, wherein the CNI is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days prior to the administration of the modified immune cells.

31. The method of any one of claims 1-30, wherein the CNI is administered throughout the administration of the modified immune cells.

32. The method of any one of claims 1-31, wherein the CNI is administered for at least 15 days, at least 20 days, at least 1 month, 2 months, or 3 months after the administration of the modified immune cells.

33. The method of any one of claims 1-32, wherein lymphocyte depletion is performed in the subject before or after administration of the CNI and before administration of the modified immune cells.

34. The method of any one of claims 1-33, wherein the CNI is administered by intravenous injection and / or oral administration.

35. The method of claim 34, wherein the CNI is administered orally after the subject is discharged from the hospital.

36. The method of any one of claims 1-35, wherein the modified immune cells have been expanded in vitro prior to administration to the subject.

37. The method of any one of claims 1-36, wherein the degranulation of the modified immune cells is enhanced by administering the CNI.

38. The method of any one of claims 1-37, wherein the expression of CD107a on the modified immune cells is increased by administering the CNI.

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