Lymphocyte populations and methods for producing same

Abstract

The present invention relates to novel populations of lymphocytes, methods for producing them, and their use in the treatment of disease.

Description

[Technical field]

[0001] The present disclosure relates to novel populations of lymphocytes, methods of producing them, and their use in the treatment of disease. More particularly, the present disclosure relates to methods of producing novel populations of natural killer T-cell-like cells (NKT-like cells) using high-dose glucocorticoids, glucocorticoid receptor agonists, and ICAM3 modulators. [Background technology]

[0002] The inventors previously found that high concentrations of glucocorticoids can be used to condition patients to enhance the efficacy of cellular immunotherapies such as adoptive T cell therapy. This is described in International Patent Application PCT / US2018 / 025517 (published as WO2018 / 183927). In that application, the inventors focused on the toxicity associated with chemotherapy and radiation mediated preconditioning, which is believed to non-selectively destroy splenic cellularity. The inventors provided acute doses of glucocorticoids (a subclass of steroids) and other non-toxic lymphodepleting drugs that will benefit cancer patients undergoing cellular immunotherapy.

[0003] In international patent application PCT / US2019 / 054395 (published as WO2020 / 072713), the inventors also described the use of high concentrations of glucocorticoids to cause lymphodepletion of peripheral blood lymphocytes without substantially affecting the cell count of other cells. In that application, the inventors reported that high concentrations of glucocorticoids can deplete peripheral blood lymphocytes, including, for example, islet-specific autoreactive T cells involved in diabetic autoimmunity, while sparing neutrophils, platelets, RBCs and stem cells (both HSCs and MSCs). The inventors provided glucocorticoids as a non-myeloablative regimen that can perform a safe immune reset with efficacy comparable to chemotherapy.

[0004] Reducing the use of cytotoxic chemotherapy is a top goal of the National Cancer Institute. Carcinomas, often referred to as solid tumors, account for 80–90% of all cancers, but have proven difficult to target in the development of new cancer therapies. Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable success in treating CD-19-expressing B-cell acute lymphoblastic leukemia. However, there are many obstacles that limit CAR T-cell therapy for solid tumors; namely, ineffective trafficking to tumors and the immunosuppressive microenvironment in solid tumors limit the efficacy of T cells. In addition, CAR T therapy has been associated with severe side effects, including cytokine release syndrome (CRS), neuroedema, and graft-versus-host disease (GvHD). Moreover, CAR T therapy is not curative, with up to 50% of subjects relapsing within 12 months even when minimal residual disease is negative (Nie et al., 2020). Conditioning or "preconditioning" prior to CAR T infusion is associated with improved survival, with preconditioning with high-dose chemotherapy associated with the best outcomes but also the most severe toxicity. Bispecific CAR T products designed to reduce recurrence, thought to be due to tumor escape due to loss of expression of CAR T target antigen or heterogeneous expression of antigen in tumors, do not appear to be more effective than first-generation CAR T (Gill et al, 2021). To address these limitations of CAR T, the field is turning to natural killer (NK), NK / NKT cell CARs, and gamma delta (γδ) T cell products.

[0005] Natural killer T cells (NKT) are a heterogeneous group of T cells that share characteristics of both T cells and natural killer (NK) cells. In contrast to conventional T cells, NKT are functionally mature upon exiting the thymus and primed for rapid cytokine production. NKT can directly kill CD1d-expressing cancer cells and tumor microenvironment macrophages, rapidly produce and release immune-activating cytokines such as IFN-gamma and IL-4, and activate other immune cells such as dendritic cells (DCs), NK cells, and B and T lymphocytes. Clinically, invariant NKT (iNKT) has been used against a variety of different cancers by injecting "autologous culture-activated iNKT", administering dendritic cells or monocytes loaded with αGal Cer (an NKT activator) to activate endogenous NKT, or administering NKT-activating antibodies or ligands such as KRN7000, a synthetic analog of αGal Cer.

[0006] However, none of these methods used to induce iNKT production have been shown to be effective in cancer patients. iNKT levels are reduced in cancer patients and clinical trials have been disappointing. iNKT levels are similarly low in older adults (Tarazona et al., 2003, incorporated herein by reference in its entirety). The use of “autologous culture-activated NKT” for melanoma was effective in 3 out of 9 cases, with outcomes directly related to tumor-infiltrating NKT numbers (Wolf et al., 2018 and Nair et al., 2017, incorporated herein by reference in their entirety). However, this approach was also limited by low NKT numbers in cancer patients and the plasticity of iNKT, which alternates between IFN gamma type 1 and tumor-promoting IL-4 type 2.

[0007] In cancer treatment, kinase inhibitors (KIs) are better tolerated compared to conventional cytotoxic chemotherapy. However, significant toxicities are still associated with kinase inhibitors, including fatigue, hypertension, rash, impaired wound healing, bone marrow suppression, and diarrhea, as well as abnormalities in thyroid function, bone metabolism, linear growth, gonadal function, fetal development, adrenal function, and glucose metabolism. Many patients require dose reduction due to the toxicity of KIs, which must be taken chronically (Lodisch et al., 2013, incorporated herein by reference in its entirety). Furthermore, resistance to KIs is common and time-dependent on treatment (Bhullar 2018, incorporated herein by reference in its entirety).

[0008] Despite efforts to reduce toxicities associated with cancer treatments, the physical burden and medical costs of managing these toxicities remain a significant concern. For example, up to 41% of patients with hematological cancers choose to discontinue taking novel kinase / proteasome inhibitors or biologics due to the physical and financial toxicities associated with these drugs (Mato 2018, Kadri 2017, Mato 2016, and Barrett 2010, each of which is incorporated herein by reference in its entirety).

[0009] T cells are a type of lymphocyte that play a key role in the immune response. T cells are distinguished from other types of lymphocytes by the presence of T cell receptors on their cell surface. The T cell receptor (TCR) is responsible for recognizing antigen fragments bound to major histocompatibility complex (MHC) molecules and is a heterodimer of two different protein chains. In humans, the TCRs of 95% of T cells consist of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), and the TCRs of 5% of T cells consist of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). This ratio changes depending on the disease state (e.g., leukemia).

[0010] In contrast to MHC-restricted alpha beta T cells, gamma delta T cells do not require major histocompatibility complex (MHC) presentation of peptide epitopes for activation, although some recognize MHC class Ib molecules. Some gamma delta T cells recognize markers of cellular stress resulting from infection and tumorigenesis. Gamma delta T cells are also thought to be involved in the recognition of lipid antigens.

[0011] Gamma delta T cells exhibit extensive functional plasticity after recognition of infected / transformed cells through production of cytokines (IFN-γ, TNF-α, IL-17) and chemokines (RANTES, IP-10, lymphotactin), cytolysis of infected or transformed target cells (perforin, granzymes, TRAIL), and interactions with other cells. Gamma delta T cells can recognize and lyse a variety of cancers in an MHC-nonrestricted manner, have protective functions in infectious diseases, and have been shown to be associated with the progression and prognosis of various infectious diseases (Gogoi et al., 2013; Pauza et al., 2018; Zheng et al., 2012; Dong et al., 2018; Zhao et al., 2018; all incorporated herein by reference in their entirety). Some gamma delta T cells also behave as antigen-presenting cells in some circumstances (Himoudi et al., 2012). Gamma delta T cells are therefore of great interest in the development of immunotherapies. Summary of the Invention

[0012] There is a need for additional treatments for cancer, autoimmune disorders, and infectious diseases (also called microbial diseases) that are safer, less toxic, and / or more effective than currently available treatments. Simpler, less toxic, and less costly treatments are desirable.

[0013] overview The present invention is based on the surprising discovery that high doses of glucocorticoids cause lymphodepletion of many types of peripheral blood lymphocytes in naive subjects, while also inducing the production / activation / mobilization of a novel population of natural killer T-cell-like cells (NKT-like cells). In addition to exhibiting known NKT cell properties, this novel population of NKT-like cells can directly phagocytose cancer cells, thus expanding the potential of high concentration glucocorticoids as a therapeutic treatment for solid cancers. As described in more detail elsewhere herein, these cells are CD3highCD49b+, are derived from the CD3highCD49b- population, and can be characterized by the pattern of surface proteins that they express.

[0014] In non-naive subjects, such as patients with cancer, autoimmune disease, or infectious disease, high doses of glucocorticoids deplete diseased / cancerous lymphocytes but spare normal lymphocytes. Without being bound by theory, the inventors hypothesize that lymphodepletion occurs in naive subjects but not in cancer patients because NKT-like cells induced / mobilized by high doses of glucocorticoids express gamma delta TCRs, which recognize phosphoantigens that are 100-1000 times more abundant or selectively expressed in stressed cells, including cancer, autoreactive lymphocytes, and infected cells. In a naive subject, a pathogen / disease-free environment, there are no stressed cells for the NKT-like cells to recognize, and in the absence of stressed cells, the NKT-like cells may recognize and deplete normal lymphocytes.

[0015] The inventors have also discovered that glucocorticoid molecules can bind and block intercellular adhesion molecules such as ICAM3 after administration of high doses. The binding is cooperative, and up to 26 molecules can bind to the first Ig domain of ICAM3. ICAM3 is expressed at significant levels on cells such as lymphocytes, monocytes and neutrophils, as well as on cancer cell types such as melanoma and osteosarcoma. Molecular modeling of the interaction between dexamethasone and ICAM3 confirms that they interact via low affinity hydrogen bonds. Without being bound by theory, the inventors hypothesize that the induction and / or recruitment of the novel NKT-like cells of the present invention may occur via these low affinity hydrogen bond interactions between ICAM3 and glucocorticoids, glucocorticoid receptor agonists, and ICAM3 modulating agents as described in more detail elsewhere herein.

[0016] Thus, in a first aspect, the present invention provides a method of generating a population of natural killer T-cell-like cells (NKT-like cells), the method comprising administering to a subject a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent (which may be a glucocorticoid such as dexamethasone) at a dose equivalent to at least about 6 mg / kg human equivalent dose (HED) of dexamethasone base, whereby the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent induces and / or mobilizes a population of NKT-like cells in the subject.

[0017] The NKT-like cells of the present invention exhibit a novel pattern of marker expression. In particular, the NKT-like cells generated / mobilized by the methods described herein express CD56 and TCR gamma / delta (γδTCR), as well as invariant TCR (iTCR). Thus, in some embodiments, a population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, and iTCR. The NKT-like cells generated / mobilized by the methods described herein also express CD16 and NKp44, which are markers of activated cells. Thus, in some embodiments, a population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD16 and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD, and / or TCR alpha / beta; and / or not expressing CD4. In some embodiments, the population of NKT-like cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34, and / or ICAM3; and / or do not express CD4.

[0018] In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, and iTCR. In some embodiments, the NKT-like cells express CD16 and NKp44. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, and NKp44. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, and CD19. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, CD19, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, and CD45. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, CD45, and TCR alpha / beta.

[0019] In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, and iTCR. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD16 and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, and CD19. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, CD19, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, and CD45.In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, CD45, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and TCR alpha / beta.

[0020] In some embodiments, the NKT-like cells do not express CD4. In some embodiments, the population of NKT-like cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells do not express CD4.

[0021] In some embodiments, the NKT-like cells of the present disclosure may express CD3. In some such embodiments, the NKT-like cells of the present disclosure may be CD3+ / dim. In some such embodiments, the NKT-like cells of the present disclosure may express CD8. In some such embodiments, the NKT-like cells of the present disclosure may be CD8+ / dim. The NKT-like cells may be described as CD3+ / dim and / or CD8+ / dim. NKT-like cells may be described as having these characteristics in naive subjects. NKT-like cells may be described as having these characteristics in tumor / cancerous or autoimmune conditions. The expression level of the cell markers may be determined relative to the average expression level in a population of reference NKT-like cells derived from a common source that have not been contacted with a glucocorticoid receptor (GR) modulator or an ICAM3 modulator. Marker expression may be measured by flow cytometry, for example, performed using the devices, reagents, and / or conditions (used in isolation or in combination) described herein.

[0022] The glucocorticoid receptor (GR) modulator or ICAM3 modulator may be a glucocorticoid. In some embodiments, the glucocorticoid is selected from the group consisting of dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide, betamethasone, flumethasone, and beclomethasone.

[0023] In a preferred embodiment, the glucocorticoid is selected from the group consisting of dexamethasone, betamethasone and methylprednisone (preferably dexamethasone or betamethasone).

[0024] In some embodiments, the glucocorticoid is dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicotinate, dexamethasone-21-acetate, dexamethasone phosphate, dexamethasone-21-phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate, dexamethasone palmitate, dexamethasone-21-palmitate, Dexamethasone 21-phosphate disodium salt, Dexamethasone mesylate, Dexamethasone linoleate, Dexamethasone glucoside, Dexamethasone 21-phenylpropionate, Dexamethasone 21-sulfobenzoate, Dexamethasone hemosulfate, Dexamethasone sulfate, Dexamethasone veroxyl, Dexamethasone acid, Dexamethasone aceflate, Dexamethasone carboximide, Dexamethasone cipesilate, Dexamethasone 21-phosphate disodium salt, Dexamethasone mesylate, Dexamethasone linoleate, Dexamethasone glucoside, Dexamethasone 21-phenylpropionate, Dexamethasone 21-sulfobenzoate, Dexamethasone hemosulfate, Dexamethasone sulfate, Dexamethasone veroxyl, Dexamethasone acid, Dexamethasone aceflate, Dexamethasone carboximide, Dexamethasone cypesilate, Dexamethasone 21-phosphate disodium salt, Dexamethasone mesylate, Dexamethasone linoleate, Dexamethasone glucoside, Dexamethasone 21-phenylpropionate, Dexamethasone 21-phenylpropionate, Dexamethasone 21-sulfobenzoate, Dexamethasone hemosulfate, Dexamethasone sulfate, Dexamethasone veroxyl, Dexamethasone acid, Dexamethasone aceflate, Dexamethasone carboximide, Dexamethasone cypesilate, Dexamethasone 21-phosphate disodium salt, Dexamethasone mesylate, Dexamethasone linoleate, Dexamethasone glucoside, Dexamethasone glucuronide, dexamethasone iodoacetate, dexamethasone oxetanone, carboxymethylthio-dexamethasone, dexamethazon bisethoxime, dexamethasone epoxide, dexamethasone linoleidate, dexamethasone methyl orthovalerate, dexamethasone spermine, 6-hydroxydexamethasone, dexamethasone tributyl acetate, dexamethasone aspartate, dexamethasone galactopyranose, dexamethasone hydrochloride, hydroxydexamethasone, carboxydexamethasone, dexox Cidexamethasone, dexamethasone butazone, dexamethasone cyclodextrin, dihydrodexamethasone, oxodexamethasone, propionyloxydexamethasone, dexamethasone galactose, dexamethasone isonicotinate, dexamethasone sodium hydrogen phosphate, dexamethasone aldehyde, dexamethasone pibrate, dexamethasone tridecyllate, dexamethasone crotonate, dexamethasone methanesulfonate, dexamethasone butyl acetate, dehydrodexamethasone, dexamethasone isothiocyanatoethyl thioether,Dexamethasone bromoacetate, dexamethasone hemiglutarate, deoxydexamethasone, dexamethasone chlorambucylate, dexamethasone melphalanate, formyloxydexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and any combination therapy containing a form of dexamethasone.

[0025] In some embodiments, the glucocorticoid is dexamethasone, which may be dexamethasone sodium phosphate.

[0026] The methods of the invention may involve administration of a specific glucocorticoid dose. In some embodiments, the glucocorticoid is administered at a dose of about: · Human equivalent dose (HED) of dexamethasone base of 6–12 mg / kg; · Human equivalent dose (HED) of dexamethasone base of at least 6 mg / kg; · Human equivalent dose (HED) of dexamethasone base of at least 12 mg / kg; · Human equivalent dose (HED) of dexamethasone base of at least 15 mg / kg; · Human equivalent dose (HED) of dexamethasone base of at least 18 mg / kg; · Human equivalent dose (HED) of dexamethasone base of at least 21 mg / kg; · Human equivalent dose (HED) of dexamethasone base of at least 24 mg / kg; Human equivalent dose (HED) of dexamethasone base up to 45 mg / kg is administered at a dose equivalent to In some preferred embodiments, the glucocorticoid is administered at a dose equivalent to at least about 18 mg / kg human equivalent dose (HED) of dexamethasone base. In some other preferred embodiments, the glucocorticoid is administered at a dose equivalent to at least about 6-18 mg / kg human equivalent dose (HED) of dexamethasone base. In some other preferred embodiments, the glucocorticoid is administered at a dose equivalent to at least about 15-18 mg / kg human equivalent dose (HED) of dexamethasone base.

[0027] In some embodiments, the glucocorticoid has a dosage of about: · Human equivalent dose (HED) of dexamethasone phosphate 6–12 mg / kg; · Human equivalent dose (HED) of dexamethasone phosphate of at least 6 mg / kg; · A human equivalent dose (HED) of dexamethasone phosphate of at least 12 mg / kg; · Human equivalent dose (HED) of dexamethasone phosphate of at least 15 mg / kg; · A human equivalent dose (HED) of dexamethasone phosphate of at least 18 mg / kg; · A human equivalent dose (HED) of at least 21 mg / kg dexamethasone phosphate; · A human equivalent dose (HED) of at least 24 mg / kg dexamethasone phosphate; Human equivalent dose (HED) of dexamethasone phosphate up to 45 mg / kg is administered at a dose equivalent to In some preferred embodiments, the glucocorticoid is administered at a dose equivalent to at least about 18 mg / kg human equivalent dose (HED) of dexamethasone phosphate. In some other preferred embodiments, the glucocorticoid is administered at a dose equivalent to at least about 6-18 mg / kg human equivalent dose (HED) of dexamethasone phosphate. In some other preferred embodiments, the glucocorticoid is administered at a dose equivalent to at least about 15-18 mg / kg human equivalent dose (HED) of dexamethasone phosphate.

[0028] A glucocorticoid dose can be defined as a human equivalent dose (HED) of dexamethasone having a mg / kg value from a range of mg / kg values, the range being defined by two of the mg / kg values ​​above. For example, a glucocorticoid dose can be defined as a dexamethasone HED of 6-45 mg / kg. In another example, a glucocorticoid dose can be defined as a dexamethasone HED of 12-24 mg / kg.

[0029] The glucocorticoid may be administered as a single acute dose or as a total dose administered over about 72 hours. Additionally, the method may include administering one or more additional doses of glucocorticoid. In some embodiments, the one or more additional doses are administered 24 hours to 120 hours after the preceding glucocorticoid administration; 24 hours to 48 hours after the preceding glucocorticoid administration; 72 hours to 120 hours after the preceding glucocorticoid administration; every 24, 48, 72, 96, 120, 144, or 168 hours after the first glucocorticoid administration; once weekly after the first glucocorticoid administration; once every two weeks after the first glucocorticoid administration; once monthly after the first glucocorticoid administration; or twice weekly after the first glucocorticoid administration.

[0030] The disclosed method may include activating the NKT-like cells of the present disclosure. Thus, in some embodiments, the method may further include administering to the subject an NKT cell activator, a T cell activator, and / or an NK cell activator. The NKT cell activator may be selected from the group consisting of alphaGalCer, sulfatide, or an NKT-activating antibody. The NKT cell activator may be an alphaGalCer-loaded dendritic cell or monocyte. The T cell activator may be selected from the group consisting of zoledronate, mevastatin, or a T cell activating antibody. The NK cell activator may be selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, or an NK cell activating antibody. The NKT cell activator, T cell activator, and / or NK cell activator may be administered within 1-48 hours or around the time of administration of the glucocorticoid.

[0031] In some embodiments, the subject is a human. In some embodiments, the subject is a mammalian subject having a humanized immune system, such as a human immune system (HIS) mouse. Preferably, the subject is a human.

[0032] The subject may have or be suspected of having (or diagnosed with) cancer, an autoimmune disease, or an infectious disease (also called a microbial disease). The cancer may be a solid tumor. Alternatively, the cancer may be a lymphoma, preferably a B-cell lymphoma or a T-cell lymphoma. In some preferred embodiments, the cancer may be a non-Hodgkin's lymphoma.

[0033] The cancer may be selected from the group consisting of squamous cell carcinoma (such as epithelial squamous cell carcinoma); lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung); peritoneal cancer; hepatocellular carcinoma; gastric cancer including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver cancer; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocellular carcinoma; anal cancer; penile cancer; head and neck cancer.

[0034] The NKT-like cells of the present invention can treat cancer through tumor infiltration. The NKT-like cells of the present invention can treat cancer through the release of immune-activating cytokines. The NKT-like cells of the present invention can treat cancer by phagocytosing and killing cancer cells. The NKT-like cells of the present invention can treat cancer by promoting the infiltration of other immune cells into tumors. The NKT-like cells of the present invention can treat cancer through CD1d-induced apoptosis. The NKT-like cells of the present invention can treat cancer through tumor necrosis. The NKT-like cells of the present invention can treat cancer by recognizing high levels of phosphoantigens produced by tumor cells through expression of gamma-delta T cell receptors on the NKT-like cells of the present invention. Thus, in some embodiments, the present invention provides methods of causing tumor necrosis by inducing, recruiting, or administering the NKT-like cells of the present invention. In some embodiments, the present invention provides methods of causing CD1d-induced apoptosis of cancer cells by inducing, recruiting, or administering the NKT-like cells of the present invention. In some embodiments, the present invention provides methods of phagocytosing and / or killing cancer cells using the NKT-like cells of the present invention. In some embodiments, the present invention provides methods for activation of gamma-delta expressing NKT-like cells by cancer cell phosphoantigens, which recognize and kill cancer cells via the NK receptor(s) on the NKT-like cells.

[0035] In embodiments where the subject has or is suspected of having (or has been diagnosed with) an autoimmune disease, the autoimmune disease can be multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes (T1D), scleroderma, pemphigus, or lupus. In embodiments where the subject has or is suspected of having (or has been diagnosed with) an infectious disease, the infectious disease can be HIV, herpes, hepatitis, or human papillomavirus. In some embodiments, the infectious disease is HIV. In some preferred embodiments, the infectious disease can be COVID-19 (coronavirus 2019; a disease caused by severe acute respiratory syndrome coronavirus 2 SARS-CoV-2).

[0036] The methods of the invention may include an isolation and / or expansion step. For example, the methods may include a step of isolating a population of NKT-like cells from a subject or from a sample derived from a subject. Optionally, the isolating step may be performed at least 48 hours after glucocorticoid administration; 48 hours to 13 days after glucocorticoid administration; or 6 to 48 hours after glucocorticoid administration. In some embodiments (e.g., embodiments in which the subject has cancer, an infectious or microbial disease, or an autoimmune disease), the isolating NKT-like cells may be performed within 3 hours after glucocorticoid administration, preferably within 1 hour after glucocorticoid administration. In some embodiments, the isolating step may be performed 30 to 60 minutes after glucocorticoid administration.

[0037] The sample may be selected from the group consisting of blood, plasma, tumor biopsy or surgically resected tumor, bone marrow, liver, spleen biopsy, and fat or adipose tissue. In some embodiments, the method further comprises expanding the isolated NKT-like cells. In some embodiments, the method comprises activating the isolated NKT-like cells with an NKT cell activator, a T cell activator, and / or an NK cell activator. The NKT cell activator, the T cell activator, and / or the NK cell activator may be as described elsewhere herein.

[0038] The isolated NKT-like cells of the present invention can be further engineered, for example, by transfecting the cells with a nucleic acid. Thus, in some embodiments, the method further comprises introducing a nucleic acid encoding a protein into the isolated NKT-like cells and culturing the cells under conditions promoting expression of the protein. The protein can be one or more of a T cell receptor (TCR), a chimeric antigen receptor (CAR), a split, universal and programmable CAR (SUPRA-CAR). The CAR and / or TCR comprises an antigen-binding domain that binds to an antigen selected from the group consisting of CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, Her2.

[0039] The NKT-like cells of the present invention find use in medicine. For example, the isolated NKT cells of the present invention can be used medically, for example, in the treatment of cancer, autoimmune disease, or infectious disease (also called microbial disease) in a subject. In these embodiments, the method can include administering a therapeutically effective dose of the NKT-like cells isolated via the methods disclosed herein to a subject suffering from one of the aforementioned diseases. In some embodiments, the subject to which the isolated NKT-like cells are administered is the same as the subject from which the NKT-like cells were isolated. Alternatively, the subject to which the isolated NKT-like cells are administered is different from the subject from which the NKT-like cells were isolated.

[0040] The NKT-like cells are administered to the subject by a method selected from the group consisting of intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, injection into the cerebrospinal fluid (CSF), direct injection into the tumor, and as a gel that is placed on or near the solid tumor.

[0041] The present invention also extends to the use of a glucocorticoid in the manufacture of a medicament for use in the methods of treatment disclosed herein.

[0042] The invention further extends to the use of dexamethasone or other glucocorticoids to induce a population of NKT-like cells, wherein the population of NKT cells is induced by a method according to any one of statements 101 to 148.

[0043] The invention further extends to the use of dexamethasone or other glucocorticoids to mobilize a population of NKT-like cells, wherein the population of NKT cells is mobilized by a method according to any one of statements 101 to 148.

[0044] Also provided are induced pluripotent stem cells derived from the NKT-like cells of the invention. Thus, in one aspect, the invention provides a method of generating induced pluripotent stem cells (iPSCs), comprising reprogramming NKT-like cells isolated by the methods disclosed herein to generate iPSCs. Reprogramming may comprise introducing one or more nucleic acids encoding Oct3 / 4, Klf4, Sox2, and C-myc into the NKT-like cells. The nucleic acids may be DNA (e.g., DNA expression cassettes) or RNA molecules. Reprogramming may further comprise introducing one or more expression cassettes encoding one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and / or LIN28 into the NKT-like cells. Reprogramming may further include introducing one or more of mRNAs encoding Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and / or LIN28 into the NKT-like cells. The iPSCs may then be induced to differentiate, for example, into NKT-like cells or into the NKT cell lineage.

[0045] The present invention also provides isolated natural killer T-cell-like cells (NKT-like cells) or populations of NKT-like cells produced by the methods disclosed herein. Relatedly, the NKT-like cells of the present invention may be defined by an expression profile (multiples) as described elsewhere herein. For example, the present invention provides isolated natural killer T-cell-like cells (NKT-like cells) characterized in that they express CD56, TCR gamma / delta and iTCR, and optionally express one or more of CD16, NKp44, CD3, CD8, CD14, CD19, CD45 and / or TCR alpha / beta, and / or do not express CD4. The isolated NKT-like cells may be from a non-diseased subject.

[0046] The present invention also provides isolated populations of natural killer T-cell-like cells (NKT-like cells). The isolated population of NKT-like cells may be defined by an expression profile (multiples) as described elsewhere herein. For example, the isolated population of NKT-like cells may be characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD56, TCR gamma / delta, and iTCR, and / or express one or more of CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta, and / or do not express CD4.

[0047] The present invention provides a glucocorticoid for use in a method of treating cancer, an autoimmune disease, or an infectious disease (also referred to as a microbial disease) in a subject. The method comprises administering a glucocorticoid to the subject at a dose equivalent to about 6-45 mg / kg human equivalent dose (HED) of dexamethasone, where the glucocorticoid induces / activates / mobilizes a population of NKT-like cells of the present invention as defined herein. For example, the present invention provides a glucocorticoid for use in a method of inducing tumor necrosis, causing tumor infiltration, releasing immune activating cytokines, phagocytosis and killing of tumor cells, promoting infiltration of other immune cells into the tumor, and / or causing CD1d-induced apoptosis in a cancer patient. The method comprises administering a glucocorticoid to the subject at a dose equivalent to about 6-45 mg / kg human equivalent dose (HED) of dexamethasone to induce a population of NKT-like cells of the present invention as defined herein. For example, the invention provides a glucocorticoid for use in a method of inducing tumor necrosis, causing tumor infiltration, releasing immune activating cytokines, phagocytosing and killing tumor cells, promoting infiltration of other immune cells into tumors, and / or causing CD1d-induced apoptosis in a cancer patient, the method comprising administering the glucocorticoid to the patient at a dose equivalent to about 6-45 mg / kg human equivalent dose (HED) dexamethasone to mobilize a population of NKT-like cells of the invention, as defined herein. For example, the invention provides a glucocorticoid for use in a method of inducing viral death, releasing immune activating cytokines, phagocytosing and killing virally infected cells, and promoting infiltration of other immune cells into virally infected organs, the method comprising administering the glucocorticoid to the subject at a dose equivalent to about 6-45 mg / kg human equivalent dose (HED) dexamethasone to induce a population of NKT-like cells of the invention, as defined herein. The HED of dexamethasone can be any value within the range of values ​​disclosed herein.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present disclosure are described below with reference to the accompanying figures. [Brief description of the drawings]

[0049] [Figure 1] Acute high-dose dexamethasone reduces murine lymphocyte counts in naive mice. Absolute lymphocyte counts (ALC minus NK and NKT cells) measured by complete blood count (cells / ul = absolute counts obtained from CBC) are significantly reduced 6, 24, 48, 7, 13, and 21 days after high-dose dexamethasone (18 mg / kg HED dexamethasone phosphate (DP)) administration compared to placebo. Near-complete lymphodepletion is observed 6 and 48 hours after administration, an effect comparable to that achieved with standard Cy / Flu chemotherapy (cyclophosphamide at 13 mg / kg HED and fludarabine at 0.8 mg / kg HED).

[0050] [Diagram 2] Acute high-dose dexamethasone reduces murine B-lymphocyte numbers in naive mice. B-lymphocyte numbers measured by complete blood count (cells / ul = absolute count obtained from CBC) are significantly reduced 6, 24, 48, 7, 13, and 21 days after administration of high-dose dexamethasone (DP at 18 mg / kg HED) compared to placebo. The lymphodepletion effect on B-lymphocytes is comparable to that achieved with standard Cy / Flu chemotherapy (cyclophosphamide at 13 mg / kg HED and fludarabine at 0.8 mg / kg HED).

[0051] [Diagram 3]Acute high-dose dexamethasone reduces murine monocyte numbers in naive mice. Monocyte numbers measured by complete blood count (cells / ul = absolute count obtained from CBC) are significantly reduced 6, 24 and 48 hours after high-dose dexamethasone (DP at 18 mg / kg HED) compared to placebo. The depletion effect on monocytes is superior to that obtained with standard Cy / Flu chemotherapy (cyclophosphamide at 13 mg / kg HED and fludarabine at 0.8 mg / kg HED).

[0052] [Figure 4] Acute high-dose dexamethasone reduces murine neutrophil counts in naive mice. Neutrophil counts measured by complete blood count (cells / ul = absolute count obtained from CBC) are significantly reduced in mice treated with high-dose dexamethasone (DP at 18 mg / kg HED) compared to placebo at 6, 24, and 48 hours.

[0053] [Diagram 5] Acute high-dose dexamethasone spares mouse platelets. Acute high-dose dexamethasone (DP at 18 mg / kg HED) does not affect platelet counts measured by complete blood count (cells / ul=absolute count obtained from CBC). Thus, acute high-dose dexamethasone eliminates the need for blood transfusions and provides a safer, non-toxic alternative to chemotherapy regimens. Because platelets express the glucocorticoid receptor (GR), the absence of effects on platelets suggests a glucocorticoid receptor-independent mechanism of action.

[0054] [Figure 6]Acute high-dose dexamethasone preserves hematopoietic stem cells. The numbers of viable hematopoietic stem cells measured at 6 hours to 35 days after naive mice were treated with placebo (Placebo) or acute high-dose dexamethasone are shown. Acute high-dose dexamethasone (DP at 18 mg / kg HED) did not significantly change the number of viable hematopoietic stem cells. Thus, a nonmyeloablative regimen represented by acute high-dose dexamethasone can eliminate the need for stem cell infusion for hematopoietic recovery after immune reset.

[0055] [Figure 7] Acute high-dose dexamethasone induces NKT upregulation (Figure 7) and the production of a new population of NKT cells (AVM-NKT). Total NKT cell numbers (cells / ul=absolute counts obtained from CBC) measured by complete blood count are decreased 6 and 24 hours after high-dose dexamethasone (18X mg / kg HED DP) compared to placebo (Placebo). Surprisingly, by 48 hours after high-dose dexamethasone, total NKT cell numbers measured by complete blood count increase, then gradually decrease until approximately 13 days after high-dose dexamethasone treatment. No such increase in NKT cell numbers was observed 48 hours after treatment with standard Cy / Flu chemotherapy (13 mg / kg HED cyclophosphamide and 0.8 mg / kg HED fludarabine).

[0056] [Figure 8]Following treatment with high-dose dexamethasone, two NKT cell populations can be identified in peripheral blood. Flow cytometric examination of peripheral blood following acute high-dose dexamethasone identified two NKT cell populations: NKT cells (middle rectangular gate) defined as CD3medCD49b+ (CD56 in humans) and corresponding to previously described NKT cells, and a novel NKT cell population defined as CD3highCD49b+ (CD56 in humans; AVM-NKT cells; center right rectangular gate). AVM-NKT cells are CD49b+ (CD56 in humans) and CD3 very bright, in contrast to known NKT cells that express CD3 at a half- to one-log lower mean fluorescence intensity (MFI) than AVM-NKT.

[0057] [Figure 9] Time course of AVM-NKT upregulation. Quantification of AVM-NKT cells per microliter of blood using CBC and flow cytometry results. AVM-NKT cells are evident in the blood of naïve mice 48 hours to 13 days after a single high-dose dexamethasone (HED 18.1 mg / kg DP PO) treatment. *=statistically significant.

[0058] [Figure 10A] Changes in the A20 tumor environment induced by high-dose dexamethasone (HED 18 mg / kg DP) treatment. After 48 hours, increased necrosis is evident in tumors from high-dose dexamethasone-treated mice compared to placebo.

[0059] [Figure 10B]AVM-NKT cells maximally eradicated A20 lymphomas implanted in the flanks of mice within 3 hours of administration of 18 mg / kg HED dexamethasone phosphate (left panel), maximally eradicated A20 metastases in blood and thymus at 24 hours (middle panel), and maximally eradicated A20 metastases in bone marrow at 48 hours (right panel). Mice were inoculated in the flanks with 2 x 106 A20 lymphoma cells in 100 μL PBS mixed with 100 μL chilled Matrigel. After 3, 24 and 48 hours, mice were euthanized and tumors, blood, bone marrow and thymus were harvested and single cell suspensions were prepared for detection of live A20 cells by flow cytometry.

[0060] [Figure 11] Acute high-dose dexamethasone (AVM0703; HED 18.1 mg / kg orally) significantly delays the growth of A20 B-cell lymphoma compared to placebo. Days of high-dose dexamethasone or placebo administration are indicated by arrows.

[0061] [Figure 12] CD45 / CD56 scatter plot of osteoarthritis patients treated with 3-6 mg / kg DSP. AVM-NKT cells (indicated by rectangular boxes) were identified that were CD45 dim and CD56 very bright (CD49b in mouse) similar to those in mouse.

[0062] [Figure 13] Flow cytometry data from a healthy blood donor and a prostate cancer patient 1 hr post and 3 hr post administration of 6 mg / kg AVM0703. In the prostate cancer patient, a novel CD45dim CD56bright cell population (circled) is evident 1 hr post infusion. These data indicate that human patients mobilize cells that correspond to the AVM-NKT cells identified in mice.

[0063] [Figure 14] Lysed whole blood flow cytometry results for an AVM0703 treated patient show double positive γδTCR and iTCR cells from the live cell CD56+ gate. CD56+ cells are shown on a γδTCR and iTCR scattergram (left). 51% of CD56+ cells are positive for both γδTCR and iTCR. The γδTCR+ iTCR+ quadrant (AVM_NKT) was then assessed for CD16 and NKp44 expression (right). CD16 and NKp46 were expressed in nearly 100% of AVM_NKT cells, indicating an activated state.

[0064] [Figure 15] Size (FSC) and complexity (SSC) scatter plots of AVM_NKT cells from 101-001. One hour after 6 mg / kg AVM0703 (DP) infusion IV over 1 hour, whole blood was collected and shipped at room temperature to AVM Biotechnology. Blood was stained with the based panel antibodies for 15 minutes in the dark at room temperature. RBC lysis was performed by addition of 1 mL of 1X BD FACS lysis solution (BD Bioscience) diluted in MilliQ water for 10 minutes in the dark at room temperature. Samples were washed with 2 mL of 1X DPBS CMF (Gibco) and resuspended in 300uL of 1X DPBS CMF, after which 5uL of 7AAD (Biolegend) was added to each sample for live / dead cell determination and incubated for 5 minutes in the dark at room temperature. 250 uL of sample was loaded for data acquisition on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells that co-express gamma delta and invariant TCRs and were labeled red in the forward vs. side scattergram. The red labeled cells are within the forward vs. side scattergram where neutrophils and large granular cells such as large granular lymphocytes are known to reside.

[0065] [Figure 16]Flow cytometry data for CD56+γδTCR+invTCR+ cells for patient 101-001. Top left scattergram: pre-infusion; top right scattergram: 1 hour post-infusion; bottom left scattergram: 3 days post-infusion; bottom right scattergram: 14 days post-infusion. Whole blood was processed as described for FIG. 15 and samples acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells, which were then broadcast onto a scattergram showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56, gamma delta TCR and invariant TCR are found in the top right quadrant of each scattergram. This patient showed evidence of significant numbers of CD56+gdTCR+invTCR+ cells (79 cells / microliter blood) 14 days after AVM0703 6 mg / kg infusion.

[0066] [Figure 17] Flow cytometry data for CD56+γδTCR+invTCR+ cells for patient 103-002. Top left scatter plot: pre-infusion; top right scatter plot: 1 hour post-infusion; bottom left scatter plot: 3 days post-infusion; bottom right scatter plot: 14 days post-infusion. Whole blood was processed as described in Figure 15 and samples acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells and these CD56+ cells were then broadcast onto a scatter plot showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56 and gamma delta TCR and invariant TCR are found in the upper right quadrant of each scatter plot. This patient had baseline evidence of circulating CD56+gdTCR+invTCR+ cells (89 cells / microliter), consistent with observations in tumor-bearing mice that the tumor environment can induce and mobilize these NKT-like cells. Three days after a 6 mg / kg dose, these CD56+gdTCR+invTCR+ cells reached circulating levels of 366 cells / microliter.

[0067] [Figure 18] Flow cytometry data for CD56+γδTCR+invTCR+ cells for patient 103-005. Top left scatter plot: pre-infusion; top right scatter plot: 1 hour post-infusion; bottom: 3 days post-infusion. Whole blood was processed as described in Figure 15 and samples acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells, and these CD56+ cells were then broadcast onto a scattergram showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56, gamma delta TCR and invariant TCR are found in the upper right quadrant of each scattergram. One hour after infusion of 9 mg / kg, baseline CD56+gdTCR+invTCR+ cells (347 cells / μL) were significantly reduced to 22 cells / μL. This suggests that circulating cells were activated and homing to the tumor in response to 9 mg / kg DP, which is consistent with the clinical observations of the tumor flare in this patient and with the observations in mice bearing A20 lymphoma that the tumor environment can induce the production of these NKT-like cells, but that dexamethasone phosphate and other glucocorticoids are required to optimally activate these cells and allow them to home to and eradicate tumor cells.

[0068] [Figure 19]Flow cytometry data for CD56+γδTCR+invTCR+ cells for the first infusion of patient 108-001. Top left scatter plot: pre-infusion; top right scatter plot: 1 hour post-infusion; bottom: 3 days post-infusion. Whole blood was processed as described in FIG. 15 and samples acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells, which were then broadcast onto a scatter plot showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56, gamma delta TCR and invariant TCR are found in the upper right quadrant of each scatter plot. One hour after 9 mg / kg AVM0703 infusion, blood CD56+gdTCR+iTCR+ cells increased approximately 20-fold from baseline (5.8 to 112 cells / uL) and remained elevated (36 cells / uL) on day 3. The patient demonstrated a significant clinical response with recovery of vision on day 3 after AVM0703 infusion.

[0069] [Figure 20]Flow cytometry data for CD56+γδTCR+invTCR+ cells for patient 108-003. Top left scatter plot: pre-infusion; top right scatter plot: 1 hour post-infusion; bottom left scatter plot: 3 days post-infusion; bottom right scatter plot: 14 days post-infusion. Whole blood was processed as described in Figure 15 and samples were acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live white blood cells were gated for CD56+ cells and these CD56+ cells were then broadcast onto a scatter plot showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56, gamma delta TCR and invariant TCR are found in the upper right quadrant of each scatter plot. CD56+γδTCR+invTCR+ cells increased over time from 24 cells / microliter blood at baseline to 94 cells / microliter 14 days after a 12 mg / kg infusion. This patient demonstrated a tumor flare response and dramatic immune homing to the cervical lymph nodes, which were the site of a pharyngitis that required hospitalization on day 14. This patient's clinical response and the detection of CD56+γδTCR+invTCR+ cells by flow cytometry are consistent with preferential homing of these cells to the tumor site.

[0070] [Figure 21]Flow cytometry data for CD56+γδTCR+invTCR+ cells for patient 108-004. Top left scatter plot: pre-infusion; top right scatter plot: 1 hour post-infusion; bottom: 3 days post-infusion. Whole blood was processed as described in Figure 15 and samples acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells and these CD56+ cells were then broadcast onto a scatter plot showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56 and gamma delta TCR and invariant TCR are found in the upper right quadrant of each scatter plot. Patient 108-004 did not show evidence of CD56+γδTCR+invTCR+ cells at any time point and was the only patient treated with acute high-dose DP at ≥6 mg / kg who did not have a clinical response to treatment consistent with the antitumor activity being mediated by the induction and mobilization of these NKT-like cells.

[0071] [Figure 22] Flow cytometry data for CD56+γδTCR+invTCR+ cells for patient 108-002. Top left scatter plot: pre-infusion; top right scatter plot: 1 hour post-infusion; bottom left scatter plot: 3 days post-infusion; bottom right scatter plot: 14 days post-infusion. Whole blood was processed as described in FIG. 15 and samples acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live leukocytes were gated for CD56+ cells, which were then broadcast onto a scatter plot showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56, gamma delta TCR and invariant TCR are found in the upper right quadrant of each scatter plot.

[0072] [Figure 23A-C]Scatter plots of γδTCR+invTCR+ bispecific CD56+ cells from apparently healthy blood donors. Scatter plots of γδTCR+iTCR+ cells from total CD56+ WBCs for 12 healthy blood donors are shown. Whole blood was processed as described in FIG. 15 and samples were acquired on a Macsquant 16 flow cytometer (Miltenyi, serial number 40150). Live white blood cells were gated for CD56+ cells and these CD56+ cells were then broadcast onto a scatter plot showing gamma delta TCR vs. invariant TCR staining. Cells expressing CD56, gamma delta TCR and invariant TCR are found in the upper right quadrant of each scatter plot. Some "healthy" blood donors have circulating levels of CD56+γδTCR+invTCR+ cells, in contrast to naive mice that do not have these cells at all. Naive mice are maintained essentially pathogen-free and free of infection, whereas "healthy" blood donors may not have asymptomatic or undiagnosed infections, or autoimmune diseases or cancers that have induced the expression of these NKT-like cells.

[0073] [Figure 24]AVM0703 induces mobilization of γδTCR+ invTCR+ bispecific CD56+ cells into humanized mouse blood. AVM0703 induces CD56+TCRγδ+ (12% of hCD45+ cells) that are CD16+, suggesting an activated state (mouse 10 Taconic NOG-EXL). Mouse leukocytes are gated on the LIVE population and then gated on human CD45+ cells. Human CD45+dim cells (top left panel, labeled and boxed in blue), identified from other experiments as the population containing novel AVM-NKT cells, are broadcast onto a scatter plot of CD56 and gdTCR (top right panel). CD56+gdTCR+ cells are in the top right quadrant. Nearly all of the blue-stained human CD45+dim cells are in the top right quadrant of CD56+gdTCR+. Human CD45+dim, CD56+, gdTCR+ cells are then identified in blue on histograms of mean fluorescence intensity (MFI) for CD8 (bottom left panel) and CD16 (bottom right panel) expression. More than 80% of the cells are CD8 positive, indicative of a cytotoxic cell type, and CD16 positive, indicative of an activated cell type.

[0074] [Diagram 25] >18 mg / kg AVM0703 HED induces bispecific immune cell recruitment of 2-12% of hCD45+ cells (top panel: mouse M5 5.14%; bottom panel mouse M7 2.37%; CRL-NCG humanized mice). Leukocytes were gated for LIVE cells and then for human CD45+ cells, which are then broadcast onto a scatter plot for CD56 and gdTCR expression (left panel). hCD45+, CD56+, gdTCR+ cells are found in the top right quadrant and are labeled in red. Human CD45+CD56+gdTCR+ cells are then broadcast onto a histogram showing MFI for invariant TCR (right panel). Over 97% of these cells also express invariant TCR.

[0075] [Figure 26]>18 mg / kg AVM0703 HED induces bispecific immune cell recruitment of 2-12% of hCD45+ cells (top panel: mouse M1 3.35%; bottom panel mouse M3 4.13%; CRL-NCG humanized mice). Leukocytes were gated for LIVE cells, then gated for human CD45+ cells, then broadcast onto a scatter plot for CD56 and gdTCR expression (left panel). hCD45+, CD56+, gdTCR+ cells are found in the top right quadrant and labeled in red. Human CD45+CD56+gdTCR+ cells are then broadcast onto a histogram showing MFI for invariant TCR (right panel). Over 97% of these cells also express invariant TCR.

[0076] [Figure 27] AVM0703 induces γδTCR+invTCR+ bispecific activated CD56+ bone marrow cells in humanized mice. >18 mg / kg AVM0703 HED treated humanized mice have bispecific immune cells that are 0.3-8.5% of hCD45+ cells in bone marrow (top: mouse M90; bottom: mouse M88; Taconic-NOG-EXL humanized mice). Approximately 90% of bispecific γδTCR+invTCR+ cells are CD16+, indicating an activated state. In the top left panel, iTCD is iTCR. Bone marrow cells were gated on LIVE cells and then CD56 expressing human CD45+ cells were broadcast onto a scatter plot for gdTCR and invTCR expression (left panel). Human CD45+CD56+gdTCR+invTCR+ cells were then broadcast onto a histogram showing MFI for CD16 (right panel). Approximately 90% of these cells express CD16, indicating an activated state.

[0077] [Figure 28]AVM0703 induces γδTCR+invTCR+ bispecific activated CD56+ bone marrow cells in humanized mice. >18 mg / kg AVM0703 HED treated humanized mice have bispecific immune cells that are 0.3-8.5% of hCD45+ cells in the bone marrow (top panel: mouse M5; bottom panel: mouse M7; CRL-NCG humanized mice). Approximately 90% of the bispecific γδTCR+invTCR+ cells are CD16+, indicating an activated state. Bone marrow cells are gated for LIVE cells and then CD56 expressing human CD45+ cells are broadcast onto a scatter plot for gdTCR and inv TCR expression (left panel). Human CD45+CD56+gdTCR+invTCR+ cells are then broadcast onto a histogram showing MFI for CD16 (right panel). Approximately 90% of these cells express CD16, indicating an activated state.

[0078] [Figure 29] AVM0703 induces myeloid cell production in humanized mice. Data from two placebo mice (top plot) and one AVM0703-treated mouse (bottom plot) are shown after the first dose of AVM0703 or placebo. Top left: M12 placebo mouse; Top right: M90 placebo mouse; Bottom: M88 32 mg / kg AVM0703-treated mouse. Forward and side scatter plots are shown, with lymphocytes circled on the scatter plot. In the placebo-treated humanized mouse (top), non-lymphoid cells appear randomly with no clear population events. The AVM0703-treated mouse (bottom plot) has evidence of a clear non-lymphoid population after the first dose, showing a higher side scatter than the lymphoid population. Repeated doses, as shown in Figure 30, further induce this non-lymphocyte population, which has a forward vs. side scatter signal similar to that expected for myeloid cells and large granular lymphocytes.

[0079] [Diagram 30]AVM0703 induces myeloid cell production in humanized mice. Data are shown for two placebo mice (top plots) and one AVM0703-treated mouse (bottom plots) after a second AVM0703 or placebo dose. Top left: M12 placebo mouse; Top right: M90 placebo mouse; Bottom: M88 32 mg / kg AVM0703-treated mouse. Repeated doses further induced non-lymphoid populations in AVM0703-treated mice, which have forward vs. side scatter signals similar to those expected for myeloid cells and large granular lymphocytes.

[0080] [Diagram 31] Humanized mice have mostly human lymphoid cells. In M12 placebo mice, lymphocytes after the first dose are mostly human CD45+ (top plot) and minority myeloid cells are mostly mCD45+ (bottom plot). Forward vs. side scatter plots for placebo treated mice are shown, showing that in humanized mice, lymphocytes are mostly human CD45+ (labeled green in top plot) and minority myeloid cells are mostly mCD45+ (labeled red in bottom plot).

[0081] [Diagram 32] AVM0703 treatment induces bone marrow cell production in humanized mice. M12 placebo: Mouse lymphocytes are 13% of mouse total WBCs (top left); human lymphocytes are 60% of human total WBCs (top right); total lymphocytes are 45% of total WBCs.

[0082] [Diagram 33] AVM0703 treatment induces the production of myeloid cells in humanized mice. M90 placebo: Mouse lymphocytes are only 12.5% ​​of total WBCs; human lymphocytes are 31.5% of total human WBCs; total lymphocytes are 30% of total WBCs.

[0083] [Diagram 34]AVM0703 treatment induces the production of myeloid lineage cells in humanized mice. M88 AVM0703: Mouse lymphocytes are only 5.7% of total WBCs; human lymphocytes are 58% of total human WBCs; total lymphocytes are 32% of total WBCs. The data in this figure shows that the total lymphocyte population is reduced from approximately 45% to approximately 32% of total WBCs after AVM0703 treatment compared to placebo-treated humanized mice. Induction of myeloid cell production reduces the percentage of WBCs that are lymphocytes.

[0084] [Diagram 35] AVM0703 treatment induces myeloid cell production in humanized mice. M01 AVM0703: Mouse lymphocytes are only 6.7% of total WBCs; human lymphocytes are 67% of total human WBCs; total lymphocytes are 35% of total WBCs. The data in this figure shows that the total lymphocyte population is reduced from approximately 45% of total WBCs to approximately 35% of total WBCs after AVM0703 treatment compared to placebo-treated humanized mice. Induction of myeloid cell production reduces the percentage of WBCs that are lymphocytes.

[0085] [Diagram 36] AVM0703 treatment induces myeloid cell production in humanized mice. M03 AVM0703: Mouse lymphocytes are only 23.7% of total WBCs; human lymphocytes are 47% of total human WBCs; total lymphocytes are 40% of total WBCs. The data in this figure shows that the total lymphocyte population is reduced from approximately 45% of total WBCs to approximately 40% of total WBCs after AVM0703 treatment compared to placebo-treated humanized mice. Induction of myeloid cell production reduces the percentage of WBCs that are lymphocytes.

[0086] [Figure 37]AVM0703 treatment induces myeloid cell production in humanized mice. M05 AVM0703: Mouse lymphocytes are only 2.0% of total WBCs; human lymphocytes are 50.1% of total human WBCs; total lymphocytes are 20.9% of total WBCs. The data in this figure shows that the total lymphocyte population is reduced from approximately 45% of total WBCs to approximately 21% of total WBCs after AVM0703 treatment compared to placebo-treated humanized mice. Induction of myeloid cell production reduces the percentage of WBCs that are lymphocytes.

[0087] [Figure 38] AVM0703 treatment induces myeloid cell production in humanized mice. M07 AVM0703: Mouse lymphocytes are only 20.4% of total WBCs; human lymphocytes are 58.2% of total human WBCs; total lymphocytes are 41.9% of total WBCs.

[0088] [Figure 39] AVM0703 treatment induces myeloid cell production in humanized mice. M10 AVM0703: Mouse lymphocytes are only 5.2% of total WBCs; human lymphocytes are 37.5% of total human WBCs; total lymphocytes are 28.1% of total WBCs. The data in this figure shows that total lymphocyte counts are reduced from approximately 45% of total WBCs to approximately 30% of total WBCs after AVM0703 treatment compared to placebo-treated humanized mice. Induction of myeloid cell production reduces the percentage of WBCs that are lymphocytes.

[0089] [Diagram 40]ACT AVM-NKT cells from AVM0703-treated mice significantly reduced the total number of viable MOPC315 cells in the tumor (top left) and spleen (top right) of AVM0703-preconditioned mice. ACT after AVM0703 preconditioning also showed a trend towards reduced viable MOPC315 cells in the blood (bottom left) and bone marrow (bottom right). Distribution of different populations of viable MOPC315 cells in subcutaneous (top left) tumor, (top right) spleen, (bottom left) bone marrow and (bottom right) blood of BALB / c mice analyzed after single cell processing and flow cytometry (CD138+CD4+). Cell recipient groups (n=8) were preconditioned with 18 mg / kg HED of AVM0703 (oral gavage) 48 hours prior to adoptive cell transfer (ACT). Forty-nine naïve donor BALB / c mice were orally administered 45 mg / kg AVM0703 to induce bispecific γδTCR+invTCR+NKT-like cells for ACT, whereas eight naïve donor BALB / c mice were orally administered placebo (3.3 million splenocytes per mouse intravenously; splenocytes from AVM0703- or placebo-treated mice were pooled) 96 hours prior to ACT. The first part of the group name indicates the preconditioned (PC) recipient group, and the second part indicates whether the donor cells were from AVM0703 mice or placebo (e.g., AVM18 PC-AVM ACT is a group that received intravenous splenocytes from mice preconditioned with 18 mg / kg AVM0703 48 hours prior to ACT and 1x 45 mg / kg 96 hours prior to harvest). All mice were sacrificed approximately 18 hours after ACT. The mean tumor volume at preconditioning was approximately 130 mm3. (*) P<0.05 (Kruskal-Wallis test - comparing each group to the "Placebo PC-Placebo ACT" group). ACT cells from AVM0703-treated mice significantly reduced the total number of viable MOPC315 cells in the tumors and spleens of AVM0703-preconditioned mice.Furthermore, ACT of cells from placebo-treated mice following AVM0703 preconditioning showed a trend towards a decrease in viable MOPC315 cells, although the decrease was not statistically significant, which was expected based on the ability of AVM0703 preconditioning to induce / mobilize endogenous bispecific NKT-like cells in MOPC315-vaccinated mice. AVM0703 preconditioning followed by ACT also showed a trend towards a decrease in viable MOPC315 in blood and bone marrow, although the results were not statistically significant.

[0090] [Diagram 41] Patient 103-007 (mantle cell lymphoma). Absolute lymphocyte count (ALC) showed lymphodepletion with AVM0703 in only one patient with baseline lymphocytosis.

[0091] [Diagram 42] In patient 103-007, monocytes, platelets, hematocrit and RBCs were not decreased after AVM0703 administration.

[0092] [Diagram 43] Ex Vivo / In Vitro Dexamethasone CRC Showing Lack of GCR Activation at Concentrations Representing Suprapharmacological Doses. Mouse whole blood (WB) and splenocytes (spl) were incubated with increasing concentrations of dexamethasone base for 6 hours, then cell counts (WB) or apoptosis (spl) were measured by CBC analysis (WB) or flow cytometry (spl) after co-staining for live / dead cells with Viobility™ (Miltenyi Biotec) and eBioscience™ Calcein AM Viability Dye (Invitrogen, ThermoFisher Scientific). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0093] The present disclosure relates to a method for generating / activating / mobilizing a population of natural killer T-cell-like cells (NKT-like cells), an isolated NKT-like cell or a population of isolated NKT-like cells generated by such a method, and a method of treatment in which NKT-like cells are induced in a subject or administered to a subject.The present disclosure is based on the authors' discovery that high doses of glucocorticoid receptor modulating agents, such as glucocorticoid dexamethasone, can induce the generation and mobilization of γδ natural killer T-like cells (CD56+γδTCR+) that also express invariant TCR (iTCR+).These newly discovered cells and populations of these cells are referred to herein as natural killer T-cell-like cells (NKT-like cells), but may also be referred to, for example, as natural killer T cells (NKT cells), CD56+γδTCR+iTCR+NKT cells, or AVM-NKT cells. As used herein, the term "population of cells" may refer to a collection or group of cells that share similar properties, e.g., a collection or group of multiple cells that share a characteristic pattern of surface protein expression. By way of example, a population of cells may refer to a group or collection of cells that all express CD56, TCR gamma / delta and iTCR.

[0094] As used herein, "mobilizing" such cells may mean promoting their movement from lymphoid organs / tissues (e.g., thymus and spleen) to the systemic circulation, which may then migrate to other sites, e.g., tumor sites. The disclosed methods may include more than one of the above aspects. For example, the disclosed methods may induce the generation of a population of NKT-like cells as described herein in the thymus and / or spleen and / or bone marrow, and may mobilize a population of NKT-like cells as described herein from the thymus and / or spleen and / or bone marrow.

[0095] As disclosed herein, the method for generating a population of NKT-like cells comprises administering a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent to a subject. The ICAM3 modulating agent may activate ICAM3 signaling in a subject or cause ICAM3 shedding, so that NKT-like cells are induced. The glucocorticoid receptor (GR) modulating agent or the ICAM3 modulating agent induces a population of NKT-like cells in a subject. The glucocorticoid receptor (GR) modulating agent or the ICAM3 modulating agent may mobilize a population of NKT-like cells in a subject.

[0096] Also disclosed are isolated populations of NKT-like cells and isolated NKT-like cells that can be produced by the disclosed methods.

[0097] The disclosed NKT-like cells can be characterized by the pattern of surface proteins they express. In some embodiments, the disclosed NKT-like cells can express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34 and / or ICAM3. In some embodiments, the disclosed NKT-like cells can express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45 and / or TCR alpha / beta. In some embodiments, the disclosed NKT cells may not express CD4.

[0098] In some embodiments, the NKT-like cells express TCR gamma / delta and iTCR. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta and iTCR. In some embodiments, the NKT-like cells express CD45, TCR gamma / delta and iTCR. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta and iTCR.

[0099] In some embodiments, the NKT-like cells express CD16. In some embodiments, the NKT-like cells express TCR gamma / delta, iTCR and CD16. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR and CD16. In some embodiments, the NKT-like cells express CD45, TCR gamma / delta, iTCR and CD16. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR and CD16. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR and CD16.

[0100] In some embodiments, the NKT-like cells express CD16 and NKp44. In some embodiments, the NKT-like cells express TCR gamma / delta, iTCR and CD16. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16 and NKp44. In some embodiments, the NKT-like cells express CD45, TCR gamma / delta, iTCR, CD16 and NKp44. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR, CD16 and NKp44.

[0101] In some embodiments, the NKT-like cells express TCR alpha / beta. In some embodiments, the NKT-like cells express TCR gamma / delta, iTCR and TCR alpha / beta. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR and TCR alpha / beta. In some embodiments, the NKT-like cells express CD45, TCR gamma / delta, iTCR and TCR alpha / beta. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR and TCR alpha / beta. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR and TCR alpha / beta. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR, TCR alpha / beta and CD16. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR, TCR alpha / beta, CD16 and NKp44.

[0102] In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, and CD45. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, CD45, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, and CD19. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, CD19, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, and iTCR. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the NKT-like cells express CD45, CD56, TCR gamma / delta, iTCR, TCR alpha / beta, and CD8. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, and CD8. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD8, and CD3. In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, CD34, and ICAM3.In some embodiments, the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, CD34, ICAM3, and NKp44.

[0103] In embodiments relating to the disclosed populations of NKT-like cells, the populations of NKT-like cells may be characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the cells express a marker or combination of markers outlined above.

[0104] Thus, in embodiments related to the disclosed populations of NKT-like cells, the population of NKT-like cells may be characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34, and / or ICAM 3. In embodiments related to the disclosed populations of NKT-like cells, the population of NKT-like cells may be characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta. In some embodiments, a population of NKT-like cells may be characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells do not express CD4.

[0105] In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing TCR gamma / delta and iTCR. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, and iTCR. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, TCR gamma / delta, and iTCR. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, and iTCR.

[0106] In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD16. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing TCR gamma / delta, iTCR, and CD16. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, and CD16. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, TCR gamma / delta, iTCR, and CD16. In some embodiments, the population of NKT-like cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD45, CD56, TCR gamma / delta, iTCR, and CD16.

[0107] In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD16 and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing TCR gamma / delta, iTCR, and CD16. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, TCR gamma / delta, iTCR, CD16, and NKp44. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, iTCR, CD16, and NKp44.

[0108] In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, iTCR, TCR alpha / beta, and CD 16. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, iTCR, TCR alpha / beta, CD 16, and NKp44.

[0109] In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, and CD45. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD19, CD45, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, and CD19. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD8, CD14, CD19, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, and iTCR. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD45, CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, the population of NKT-like cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD45, CD56, TCR gamma / delta, iTCR, TCR alpha / beta, and CD8.In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, and CD8. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD8, and CD3. In some embodiments, the population of NKT-like cells is characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, iTCR, CD16, CD34, and ICAM3. In some embodiments, the population of NKT-like cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD56, TCR gamma / delta, iTCR, CD16, CD34, ICAM3, and NKp44.

[0110] The expression of surface proteins on cells can be easily determined using techniques well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA), magnetic activated cell sorting (MACS) or flow cytometry techniques. Flow cytometry uses the characteristics of light scattered from cells bound by fluorescently tagged antibodies to identify cells expressing a surface protein of interest. Flow cytometry can not only determine whether a cell expresses a protein of interest, but can also indicate the amount of protein expressed by a cell based on the intensity of fluorescence. In flow cytometry readings, as used herein, "+" (or "positive") indicates expression of a given surface protein, "-" (or "negative") indicates the absence of expression of a given surface protein, and "+ / -" indicates bimodal expression of a given surface protein. Expressions such as "bright" (sometimes referred to as "high" or "++"), "dim" (sometimes referred to as "low"), and "moderate" are used to indicate the relative amount of a particular cell surface protein.

[0111] [CD3] CD3 (cluster of differentiation 3) is a T cell coreceptor that serves to activate cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). Because CD3 is required for T cell activation, drugs (e.g., monoclonal antibodies) that target it have been investigated as immunosuppressive therapies (e.g., otelixizumab) for type 1 diabetes and other autoimmune diseases. The NKT-like cells of the present invention lose CD3 expression after activation (a known phenomenon of T cell activation, see, e.g., Valle et al, J Immunol. 2015 Mar 1;194(5):2117-27), and thus CD3 fluorescence intensity (e.g., whether the cells are CD3+ / dim or CD3+ / bright) may vary depending on whether the cells are activated or not.

[0112] In some embodiments, the NKT-like cells of the present disclosure express CD3. In some embodiments, the NKT cells of the present disclosure are CD3+ / dim. In embodiments related to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD3. In some embodiments, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may be CD3+ / dim. In some embodiments, the NKT cells of the present disclosure are CD3+ / bright. In some embodiments, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may be CD3+ / bright.

[0113] [CD4] CD4 (cluster of differentiation 4) is a glycoprotein found on the surface of immune cells, including helper T cells and monocytes. CD4 is a co-receptor for the T cell receptor (TCR) and aids in communication with antigen-presenting cells for antigen-induced T cell activation. Cross-linking of CD4 can induce T cell apoptosis via the Fas ligand pathway.

[0114] In some embodiments, the NKT-like cells of the disclosure do not express CD4. In embodiments relating to populations of NKT-like cells of the disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may not express CD4.

[0115] [CD8] CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). It is expressed primarily on the surface of cytotoxic T cells, but also on natural killer cells. On T cells, it plays a role in T cell-antigen interaction and T cell signaling.

[0116] In some embodiments, the NKT-like cells of the present disclosure express CD8. In some embodiments, the NKT-like cells of the present disclosure are CD8+ / dim. In some embodiments, the NKT-like cells of the present disclosure are CD8+ / moderate. In some embodiments, the NKT-like cells of the present disclosure are CD8+ / bright. In embodiments relating to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells can express CD8. In some embodiments, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells can be CD8+ / dim. In some embodiments, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells can be CD8+ / moderate. In some embodiments, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells can be CD8+ / bright.

[0117] [CD14] CD14 (cluster of differentiation 14) is a protein expressed primarily by macrophages as part of the innate immune system. It helps detect bacteria in the body by binding to lipopolysaccharides and was the first pattern recognition receptor described.

[0118] In some embodiments, the NKT-like cells of the disclosure express CD 14. In embodiments relating to populations of NKT-like cells of the disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD14.

[0119] [CD19] CD19 (also known as cluster of differentiation 19, B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, and CVID3) is a transmembrane protein expressed in all B-lineage cells. In human B cells, it acts as an adaptor protein that recruits cytoplasmic signaling proteins to the cell membrane and, within the CD19 / CD21 complex, acts to lower the threshold of the B-cell receptor signaling pathway.

[0120] In some embodiments, the NKT-like cells of the present disclosure express CD 19. In embodiments relating to populations of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD19.

[0121] [CD34] CD34 (cluster of differentiation 34) is a cell surface glycoprotein that functions as an intercellular adhesion molecule and is required for T cell entry into lymph nodes. Cells expressing CD34 are normally found in the umbilical cord and bone marrow as hematopoietic cells, or in endothelial precursor cells of blood vessels, endothelial cells, but not in lymphatic vessels (except thoracic lymphatic vessels), mast cells in the stroma and dermal appendages of the skin, a subpopulation of dendritic cells (which are factor XIIIa negative), and cells in soft tissue tumors.

[0122] In some embodiments, the NKT-like cells of the present disclosure express CD34. In embodiments relating to populations of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD34.

[0123] [CD45] CD45 (also known as cluster of differentiation 45, protein tyrosine phosphatase, receptor type, PTPRC) is an essential regulator of T-cell and B-cell antigen receptor signaling and a marker for all white blood cells. CD45 expression is essential for T-cell activation by the TCR. CD45 may be a receptor for CD26.

[0124] In some embodiments, the NKT-like cells of the present disclosure express CD45. In embodiments relating to populations of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD45.

[0125] CD45 can be any isoform of CD45, for example, CD45RA, CD45RO and / or CD45RABC (also known as CD45R, also known as B220).

[0126] [CD56] CD56 (cluster of differentiation 56, also known as neural cell adhesion molecule, NCAM) is a homophilic binding glycoprotein expressed on the surface of neurons, glia, and skeletal muscle. CD56 expression is associated with natural killer cells.

[0127] In some embodiments, the NKT-like cells of the present disclosure express CD56. In embodiments relating to populations of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD56.

[0128] [ICAM3] ICAM-3 (intercellular adhesion molecule 3, also known as CD50) is expressed by lymphocytes, monocytes, eosinophils and neutrophils (as well as on bronchioles and by lymphoma cells and some melanoma, sarcoma and other cancer cells).

[0129] In some embodiments, the NKT-like cells of the present disclosure express ICAM3. In embodiments relating to populations of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express ICAM3.

[0130] [Major histocompatibility complex; MHC]

[0131] The MHC was discovered by Goreer and Snell et al. in 1936. Their skin grafting experiments with mice revealed that self and non-self recognition differed depending on the genetic background. Sell et al. named the group of mouse genes that determine self / non-self as histocompatibility-2 (H-2). The genomic locus of the MHC encodes polymorphic cell membrane-bound glycoproteins known as MHC classical class I and class II molecules (antigens), which regulate immune responses by presenting peptides of fragmented proteins to circulating cytotoxic and helper T lymphocytes, respectively. Classical MHC class I proteins are subdivided as HLA-A, HLA-B and HLA-C (Nakamura et al., 2019, which is incorporated herein by reference in its entirety). On the other hand, HLA-E, HLA-F, HLA-G, MHC class I polypeptide-related sequence A (MICA) and FcRn, etc., are classified as non-classical MHC class I.

[0132] MHC classical class I molecules are expressed in most tissues, where they non-covalently associate with b2-microglobulin to present intracellularly processed peptide antigens (8-11 amino acids long) to the T cell receptors of specific CD8+ T cells to induce their activation and / or cytotoxicity (Shiina et al. 2016, incorporated herein by reference in its entirety). Processed peptides can originate from the cell's own proteome or from foreign intracellular pathogens. Mature dendritic cells use the MHC class I system to present peptides derived from antigens captured by endocytosis. This process, called cross-presentation, plays a crucial role in initiating the response of specific T CD8+ lymphocytes in peripheral lymphoid organs (Shiina et al. 2016). In addition, MHC classical class I proteins can act as ligands for killer cell immunoglobulin-like receptors, which regulate the cytotoxic activity of cytotoxic T cells and natural killer cells, as well as leukocyte immunoglobulin-like receptors expressed on myeloid monocytes and other leukocyte lineages. In contrast to classical class I antigens, classical class II antigens form heterodimeric structures specialized for the presentation of exogenous peptides (15-25 amino acids long) on ​​the surface of lymphoid cells to CD4+ helper T lymphocytes of the immune system. Class II gene expression is primarily restricted to lymphoid cells, e.g., B cells, monocytes, macrophages, endothelial cells, dendritic cells and activated T cells. MHC class II proteins are identified as HLA-DR, HLA-DP and HLA-DQ. MHC class II genes include HLA-DRA1, HLA-DQA1, HLA-DPA1, which code for the α chain, and HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5 (HLA-DRB3 / 4 / 5), HLA-DQB1 and HLA-DPB1, which code for the β chain. HLA-DRA1 forms heterodimers with HLA-DRB1 or HLA-DRB3 / 4 / 5 (Nakamura et al.) Similarly, HLA-DQA1 and HLA-DPA1 also associate with HLA-DQB1 and HLA-DPB1, respectively.HLA-DR is divided into five groups, consisting of DR1, DR51, DR52, DR53 and DR8, according to antigen group. Both DR1 and DR8 groups consist of only DRB1 as expressed gene. On the other hand, DR51, DR52 and DR53 groups contain DRB1 in common, and further consist of DRB5, DRB3 and DRB4, which are thought to be generated from the DRB1 gene by gene duplication, respectively, as expressed genes (Nakamura et al.).

[0133] Both classical class I and class II genes are often highly polymorphic, presumably to maintain inter-individual variability in antigen-presenting capacity and help species defend and survive natural selection pressures from various infectious pathogens. Non-classical class I and class II antigens are structurally similar to their classical class I or class II counterparts, but are usually less polymorphic, have variable or restricted tissue expression, and functions that are often distinct from those of classical class I or class II antigens. In addition, some non-classical MHC class I genes are located outside the MHC (Shiina et al.).

[0134] The loci of the HLA complex (e.g., HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ and HLA-DP) have a large number of polymorphisms, so the combinations (haplotypes) are very large. However, the MHC shows strong linkage disequilibrium, which is the occurrence of non-random association of alleles at multiple loci. This linkage disequilibrium in the MHC region often causes specific combinations of each locus of the MHC. When two genetic polymorphisms exist on the same chromosome, the two polymorphisms are classified as linked (Nakamura et al.). Considering that gene recombination occurs in a biologically conventional way, polymorphisms at separate sites cannot be determined to be in a linked state. However, linkage disequilibrium is a state in which a certain gene polymorphism can be predicted with a very high probability based on information on polymorphisms at separate sites. In the MHC, the loci are concentrated in a narrow region of chromosome 6, so the recombination between each gene is unlikely to occur. Thus, genes such as HLA-A, HLA-B, HLA-C and HLA-DRB1 are often inherited in a state of linkage disequilibrium. As HLA gene polymorphism analysis progresses, haplotypes that are frequently found in certain ethnic groups and associated with certain diseases have been elucidated. These ethnic-specific haplotypes are thought to be involved in the process of forming ethnic groups. Therefore, these haplotypes are often used to search for ethnic roots.

[0135] In humans, MHC classical class I genes are clinically implicated in organ transplant rejection and graft-versus-host disease after hematopoietic stem cell transplantation. Various associations have been demonstrated between HLA class I molecules and numerous autoimmune and infectious diseases and adverse drug reactions. Apart from their important role in generating adaptive immune responses, the role of MHC class I genes has been demonstrated in various steps of reproduction, such as pregnancy maintenance, mate selection and kin identification. The MHC has also been considered to be primarily a system for sexual selection and avoiding inbreeding, with histocompatibility playing a secondary role. MHC class I gene products also influence central nervous system development and plasticity, neurological cell interactions, synaptic function and behavior, cerebral hemisphere specialization and neurological and neuromedical disorders. Thus, the human MHC class I region is one of the most biomedically diverse and important genomic regions (Shiina et al.).

[0136] [TCR Gamma / Delta] The T cell receptor gamma delta (TCR gamma / delta, TCRγδ) is a T cell receptor composed of one gamma (gamma) chain and one delta (delta) chain. T cells expressing TCR gamma / delta (gamma delta T cells) are important recognizers of lipid antigens expressed by cancer cells and stressed cells such as cancer cells, microbial and viral infected cells and autoreactive lymphocytes. Gamma delta T cells exhibit several features that place them at the border between the more evolutionarily primitive innate immune system, which allows rapid beneficial responses against a variety of foreign substances, and the adaptive immune system, where B and T cells orchestrate slow but highly antigen-specific immune responses and provide long-lasting memory against subsequent exposure with the same antigen. Gamma delta T cells can be considered a component of adaptive immunity in that they can rearrange TCR genes to provide junctional diversity and develop a memory phenotype.

[0137] The most common human gamma delta variant is the V gamma 9 / V delta 2 variant in blood, while V delta 1 type gamma delta T cells in tumors were associated with poor prognosis. V delta 3 variants have also been described, as have V delta 2 negative variants that reduced cancer risk after CMV infection. In contrast to MHC-restricted alpha beta T cells, gamma delta T cells do not require antigen processing and MHC presentation of peptide epitopes, although some can recognize MHC class Ib. As a result, tumor cells cannot escape detection by downregulating MHC, and thus gamma delta T cells also have an equal chance of killing tumors with low mutational load and are less likely to be affected by resistance problems. Gamma delta T cell tumor infiltration is also most highly correlated with survival and lower incidence of graft-versus-host disease. Gamma delta T cells naturally home to a variety of tissues to detect tumors and are more favorable for allogeneic therapy than alpha beta T cells.

[0138] In some embodiments, the NKT-like cells of the present disclosure express TCR gamma / delta. In embodiments relating to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express TCR gamma / delta. The NKT-like cells may express TCR gamma / delta including delta 1 (δ1), delta 2 (δ2), delta 3 (δ3), or delta 5 (δ5) delta chains. That is, the NKT-like cells of the present disclosure may be delta 1 or delta 2 or delta 3 or delta 5 positive. In embodiments relating to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may be delta 1 or delta 2 or delta 3 or delta 5 positive.

[0139] [Invariant TCR (iTCR)] The invariant TCR (iTCR) is a highly conserved invariant receptor consisting of the Vα24-Jα18 chain coupled to the Vβ11 chain in humans and the Vα14-Jα18 chain preferentially pairing with the Vβ2, Vβ7 or Vβ8.2 chain in mice. The iTCR is expressed by invariant natural killer T cells (iNKT), a unique innate T lymphocyte with characteristics of both conventional T cells and natural killer cells. These cells directly kill tumor cells and transactivate the antitumor functions of dendritic cells (DCs), natural killer (NK) cells, and T and B cells. iNKT cell activation generally requires engagement of the iTCR by CD1d, which presents glycolipid antigens.

[0140] In some embodiments, the NKT-like cells of the present disclosure express an iTCR. In embodiments relating to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express an iTCR.

[0141] [TCR Alpha / Beta] The T cell receptor alpha beta (TCR alpha / beta, TCRαβ) is the predominant TCR heterodimer composed of one α (alpha) chain and one β (beta) chain.

[0142] In some embodiments, the NKT-like cells of the present disclosure may express TCR alpha / beta. In embodiments relating to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express TCR alpha / beta.

[0143] [CD16] CD16 (cluster of differentiation 16, also known as FcγRIII) is a transmembrane protein present on activated natural killer cells and a marker of cell activation.

[0144] In some embodiments, the NKT-like cells of the present disclosure may express CD 16. In embodiments relating to populations of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express CD16.

[0145] [NKp44] NKp44 (natural cytotoxicity-inducing receptor 2, also known as cluster of differentiation 336) is a cell surface receptor selectively expressed on activated NK cells and a marker of cell activation.

[0146] In some embodiments, the NKT-like cells of the present disclosure express NKp44. In embodiments relating to a population of NKT-like cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells may express NKp44.

[0147] The NKT-like cells of the present disclosure may express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45 and / or TCR alpha / beta. The NKT-like cells of the present disclosure may not express CD4. In some preferred embodiments, the NKT-like cells of the present disclosure express CD56, TCR gamma / delta and iTCR. In some preferred embodiments, the NKT-like cells of the present disclosure express CD16 and NKp44. In some preferred embodiments, the NKT-like cells of the present disclosure express CD56, TCR gamma / delta, iTCR, CD16 and NKp44. In some preferred embodiments, the NKT-like cells of the present disclosure express CD56, TCR gamma / delta, iTCR and TCR alpha / beta. In some preferred embodiments, the NKT-like cells of the present disclosure express CD56, TCR gamma / delta, iTCR, CD16, NKp44 and TCR alpha / beta. In some embodiments, the NKT-like cells of the present disclosure express one or more of CD56, TCR gamma / delta, iTCR, CD16 and NKp44, and CD3, CD8, CD14, CD19, CD45 and / or TCR alpha / beta. In some embodiments, the NKT-like cells of the present disclosure express one or more of CD56, TCR gamma / delta, iTCR, CD16, NKp44 and TCR alpha / beta, and CD3, CD8, CD14, CD19 and / or CD45.

[0148] In some particularly preferred embodiments, the NKT-like cells of the present disclosure express CD56, TCR gamma / delta and / or iTCR.

[0149] In embodiments relating to populations of NKT-like cells of the present disclosure, the population of NKT-like cells may be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta. In some such embodiments, the population of NKT-like cells may be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells not expressing CD4. In some embodiments, the population of NKT-like cells may be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells expressing CD56, TCR gamma / delta, and iTCR. In some embodiments, a population of NKT-like cells may be characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells expressing CD16 and NKp44. In some embodiments, a population of NKT-like cells may be characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells expressing CD56, TCR gamma / delta, iTCR, CD16, and NKp44. In some embodiments, a population of NKT-like cells may be characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells expressing CD56, TCR gamma / delta, iTCR, and TCR alpha / beta. In some embodiments, a population of NKT-like cells may be characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells express CD56, TCR gamma / delta, iTCR, CD16, NKp44, and TCR alpha / beta. In some embodiments, a population of NKT-like cells may be characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT-like cells express one or more of CD56, TCR gamma / delta, iTCR, CD16, and NKp44, as well as CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta.In some embodiments, a population of NKT-like cells may be characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the NKT-like cells express one or more of CD56, TCR gamma / delta, iTCR, CD16, NKp44 and TCR alpha / beta, as well as CD3, CD8, CD14, CD19 and / or CD45.

[0150] In some particularly preferred embodiments, the population of NKT-like cells may be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the NKT-like cells expressing CD56, TCR gamma / delta and iTCR.

[0151] [Gamma delta T cells] Gamma delta T cell surface marker characteristics may include (but are not limited to) CD3, CD4, CD8, CD69, CD56, CD27, CD40, CD40L, CD45RA, CD45, CD83, CD16, CD16a, CD16b, ICOS, CD161, Fas, CLEC7A / Dectin-1, FasL, E-cadherin, IL-18Ralpha, IL-23R, NKG2D / CD314, NKG2E, occludin, TKR2, TRAIL, TCR-Vg9, TCR-Vd2, TCR-Vd1, TCR-Vd3, TCR-pan g / d, NKG2D, monoclonal chemokine receptor antibodies CCR5, CCR6, CCR7, CXCR3, CXCR4 or CXCR5 or combinations thereof. Surface marker characteristics of the NKT-like cells of the present invention may include one / more of these. Gamma delta T cells may secrete (including but not limited to) CCL2 / JE / MCP-1, CXCL13 / BLC / BCA-1, beta-defensin 2, beta-defensin 3, alpha-defensin 1, EGF, KGF / FGF-7, FGF-10, GM-CSF, granulysin, granzyme A, granzyme B, IFN-gamma, IGF-I / IGF-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-12 / IL-23 p40, IL-12 p70, IL-13, IL-17 / IL-17A, IL-22, IL-6 / IL-6R alpha complex, LAP (TGF-beta 1), TGF-beta and / or TNF-alpha. The NKT-like cells of the present invention may secrete one / more of these.

[0152] The NKT-like cells and populations of NKT-like cells of the present disclosure may be characterized in that they express CD56, TCR gamma / delta and iTCR. Although cells expressing both TCR gamma / delta and iTCR can be produced (e.g., by transduction of TCR gamma / delta positive cells with iTCR), to the best of the authors' knowledge, they have not been described as naturally occurring (i.e., one or both of them are not recombinantly introduced). Thus, the NKT-like cells of the present disclosure are unique in that they are naturally occurring, generated and / or mobilized in a subject following administration of high doses of glucocorticoids. That is, the NKT-like cells of the present disclosure are unique in that they express both TCR gamma / delta and iTCR without the need for recombinant expression of one or both of them, advantageously avoiding the drawbacks associated with the use of manufactured γδTCR / iTCR cell lines. Thus, the isolated NKT-like cells and populations of NKT-like cells of the present disclosure may be described as naturally occurring. The cells and populations of cells of the present disclosure have not been transfected, transduced or otherwise genetically modified to express TCR gamma / delta. The cells and populations of cells of the present disclosure have not been modified by introducing a nucleic acid encoding TCR gamma / delta into the cell(s). The cells and populations of cells of the present disclosure have not been transfected, transduced or otherwise genetically modified to express iTCR. The cells and populations of cells of the present disclosure have not been modified by introducing a nucleic acid encoding iTCR into the cell(s). In some embodiments, the cells and populations of cells of the present disclosure may not be transfected, transduced or otherwise genetically modified to express TCR alpha / beta. In some embodiments, the cells and populations of cells of the present disclosure may not be modified by introducing a nucleic acid encoding TCR alpha / beta into the cell(s).In some embodiments, the cells and populations of cells of the present disclosure may not be transfected, transduced or otherwise genetically modified to express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34 and / or ICAM 3. In some embodiments, the cells and populations of cells of the present disclosure may not be transfected, transduced or otherwise genetically modified to express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45 and / or TCR alpha / beta. In some embodiments, the cells and populations of cells of the disclosure may not have been modified by introducing into the cell(s) a nucleic acid encoding CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34, and / or ICAM3. In some embodiments, the cells and populations of cells of the disclosure may not have been modified by introducing into the cell(s) a nucleic acid encoding CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta. In some embodiments, the cells and populations of cells of the present disclosure may not be transfected, transduced or otherwise genetically modified to express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34 or ICAM 3. In some embodiments, the cells and populations of cells of the present disclosure may not be transfected, transduced or otherwise genetically modified to express CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45 or TCR alpha / beta.In some embodiments, the cells and populations of cells of the present disclosure may not be modified by introducing a nucleic acid encoding CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34 or ICAM3 into the cell(s). In some embodiments, the cells and populations of cells of the present disclosure may not be modified by introducing a nucleic acid encoding CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45 or TCR alpha / beta into the cell(s). In some embodiments, the cells and populations of cells of the present disclosure may be isolated from a subject. The subject may be a subject as defined elsewhere herein. In some embodiments, the cells and populations of cells of the present disclosure may be isolated from the subject to which they are derived / mobilized. In some embodiments, the cells and populations of cells of the present disclosure can be isolated from a subject from which they were derived / mobilized following administration of a glucocorticoid to the subject. In some embodiments, the cells and populations of cells of the present disclosure can be isolated from a subject from which they were derived / mobilized via methods as disclosed elsewhere herein. In some embodiments, the cells and populations of cells of the present disclosure can be generated and isolated by methods as disclosed elsewhere herein.

[0153] In the context of the present disclosure, an ICAM3 modulating agent is one that binds to ICAM3 and promotes the induction and / or recruitment of NKT-like cells of the present invention. An ICAM3 modulating agent that binds to ICAM3 may alternatively be referred to as an ICAM3 binding molecule, an ICAM3 binding agent, etc. An ICAM3 modulating agent may be an ICAM3 antagonist / ICAM3 inhibitor or may be an ICAM3 agonist / activator.

[0154] Such ICAM3 modulating agents may include, for example, ICAM3 antibodies or portions thereof raised against anti-ICAM3, small molecule modulators of ICAM3 (e.g., activators or inhibitors of ICAM3) and peptide agents / proteins that bind to ICAM3. Suitable means for identifying ICAM3 modulating agents will be well known in the art. For example, anti-ICAM3 antibodies can be identified by a method that may include contacting a library of antibody molecules and an ICAM3 epitope and selecting one or more specific antibody molecules of the library that can bind to said epitope. Alternatively, they may be identified using competitive binding assays using known anti-ICAM3 antibodies, with competition being determined, for example, using ELISA or flow cytometry. Similarly, small molecule modulators of ICAM3 can be identified by routine screening experiments such as radioligand binding assays and functional assays.

[0155] As already described above, the present authors have discovered the surprising ability of glucocorticoid receptor modulating agents (e.g., dexamethasone and other glucocorticoids) to bind to ICAM3 and exert a modulating effect on ICAM3. Thus, in some embodiments, the ICAM3 modulating agent can be a glucocorticoid receptor (GR) modulating agent. In some embodiments, the ICAM3 modulating agent can be a glucocorticoid, e.g., dexamethasone or betamethasone. The ICAM3 modulating agent can be a molecule that binds to the same region of ICAM3 as glucocorticoids, such as dexamethasone. The ICAM3 modulating agent can be a molecule that binds to ICAM3 through interaction with SER31 and / or MET49 residues in ICAM3. The ICAM3 modulating agent can be a molecule that binds to ICAM3 through interaction with THR38, LEU40, LEU56, VAL59 and / or ILE65 residues in ICAM3. The ICAM3 modulating agent can be a molecule that binds to ICAM3 through interactions with PHE21, VAL22, GLU32, LYS33, TRP51 and / or ALA52 residues in ICAM3. The ICAM3 modulating agent can be a molecule that binds to ICAM3 through interactions with SER25, ASN23, GLU37, PHE54 and / or GLN75 residues in ICAM3. The ICAM3 modulating agent can be a molecule that binds to ICAM3 through interactions with PHE21, VAL22, ASN23, SER25, SER31, GLU32, LYS33, GLU37, THR38, LEU40, MET49, TRP51, ALA52, PHE54, LEU56, VAL59, ILE65 and / or GLN75 residues in ICAM3. The ICAM3 modulating agent can be any molecule such as an anti-ICAM3 antibody, a small molecule modulator of ICAM3 (including activators and inhibitors of ICAM3) or a peptide agent / protein that binds to ICAM3 and competes with glucocorticoids such as dexamethasone for binding to ICAM3. The ICAM3 modulating agent can be an anti-ICAM3 antibody, e.g., ICR 8.1 or a humanized version thereof.Those skilled in the art are aware of suitable techniques by which binding to the same region of ICAM3 can be determined, for example, by molecular modeling or competitive binding assays.

[0156] As used herein, the term glucocorticoid receptor (GR) modulating agent includes glucocorticoids, glucocorticoid receptor agonists and any compound that binds to glucocorticoid receptor.Glucocorticoid receptor (GR) modulating agents, such as glucocorticoids, exert their effects through both membrane GR and cytoplasmic GR, which activate or repress gene expression.It is believed that some of the desirable lymphodepleting effects of glucocorticoids and GR modulating agents are mediated by their non-genomic effects in addition to membrane GR or other genomic effects.Glucocorticoids have been reported to have variable effects on lymphocyte levels depending on the concentration of glucocorticoid administered and the duration of treatment. In general, glucocorticoids at low doses, typically used for chronic therapy, have been reported to redistribute lymphocytes from the peripheral blood to the bone marrow, at moderate doses glucocorticoids have been reported to cause leukocytosis, believed to be a redistribution of leukocytes from the bone marrow, spleen and thymus to the peripheral blood, and at high doses glucocorticoids are lymphotoxic to lymphocytes by inducing apoptosis and necroptosis. The duration of effect also varies with dose level; for example, Fauci et al (1976) reported that a single oral 0.24 mg / kg dexamethasone dose suppressed peripheral blood T and B lymphocytes by 80%, with recovery beginning at 12 hours and normal levels by 24 hours. The authors have previously demonstrated (in International Patent Application PCT / US2019 / 054395) that an acute oral dose of 3 mg / kg or more of dexamethasone is necessary to reduce peripheral blood T and B cells 24-48 hours after administration, with recovery to baseline levels occurring approximately 5-14 days after dosing.

[0157] Glucocorticoid receptor (GR) modulating agents that can be used in the disclosed methods include, for example, selective glucocorticoid receptor modulators (SEGRMs) and selective glucocorticoid receptor agonists (SEGRAs). Glucocorticoids, selective glucocorticoid receptor modulators and selective glucocorticoid receptor agonists (SEGRAs) that can be utilized in the disclosed methods are well known to those skilled in the art.

[0158] Some such glucocorticoids include, but are not limited to, dexamethasone, dexamethasone-containing formulations, hydrocortisone, methylprednisone, prednisone, corticone, budesonide, betamethasone, and beclomethasone. Other glucocorticoids include prednisolone, mometasone furoate, triamcinolone acetonide, and methylprednisolone.

[0159] Therefore, in some embodiments of the method of the present disclosure, the glucocorticoid receptor (GR) modulating agent can be a glucocorticoid.In some such embodiments, the glucocorticoid can be selected from the group consisting of dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide, betamethasone, flumethasone and beclomethasone.In some preferred embodiments, the glucocorticoid can be selected from the group consisting of dexamethasone, betamethasone and methylprednisone.In some particularly preferred embodiments, the glucocorticoid can be dexamethasone or betamethasone.

[0160] In some embodiments of the methods of the present disclosure, the glucocorticoid is dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicotinate, dexamethasone-21-acetate, dexamethasone phosphate, dexamethasone-21-phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate. Dexamethasone palmitate, dexamethasone-21-palmitate, dexamethasone dipropionate, dexamethasone propionate, dexamethasone acetate anhydrous, dexamethasone-21-phenylpropionate, dexamethasone-21-sulfobenzoate, dexamethasone hemosulfate, dexamethasone sulfate, dexamethasone veroxyl, dexamethasone acid, dexamethasone aceflate, dexamethasone carboximide, dexamethasone cypesilate ... Samethasone 21-phosphate disodium salt, dexamethasone mesylate, dexamethasone linoleate, dexamethasone glucoside, dexamethasone glucuronide, dexamethasone iodoacetate, dexamethasone oxetanone, carboxymethylthiodexamethasone, dexamethasonebisethoximes, dexamethasone epoxide, dexamethasone linoleidinate, dexamethasone methylorthovalerate, dexamethasone Spermine, 6-hydroxydexamethasone, dexamethasone tributylacetate, dexamethasone aspartate, dexamethasone galactopyranose, dexamethasone hydrochloride, hydroxydexamethasone, carboxydexamethasone, desoxydexamethasone, dexamethasone butazone, dexamethasone cyclodextrin, dihydrodexamethasone, oxodexamethasone, propionyloxydexamethasone, dexamethasone galactodys, dexamethasone isonicotinate, dexamethasone sodium hydrogen phosphate, dexamethasone aldehyde, dexamethasone pibratepivlate), dexamethasone tridecylate, dexamethasone crotonate, dexamethasone methanesulfonate, dexamethasone butylacetate, dehydrodexamethasone, dexamethasone isothiocyanatoethyl thioether, dexamethasone bromoacetate, dexamethasone hemiglutarate, deoxydexamethasone, dexamethasone chlorambucillate, dexamethasone melphalanate, formyloxydexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and any combination therapy containing a form of dexamethasone. In some preferred embodiments, the glucocorticoid may be dexamethasone base or dexamethasone sodium phosphate.

[0161] In some embodiments of the present disclosure, the glucocorticoid receptor modulating agent may not be one or more of the agents listed above.

[0162] In the methods of the disclosure, the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent is administered at a dose equivalent to a human equivalent dose (HED) of about at least 6 mg / kg of dexamethasone base, or at a dose equivalent to a human equivalent dose (HED) of about at least 6 mg / kg of dexamethasone phosphate.

[0163] Equivalent doses of another glucocorticoid or glucocorticoid receptor modulating agent can be easily and simply calculated using publicly available corticoid conversion algorithms, preferably at http: / / www.medcalc.com. As an example, 3-12 mg / kg dexamethasone converts to 19-75 mg / kg prednisone. Since the biological half-life of dexamethasone is about 36-54 hours, while that of prednisone is about 20 hours, prednisone would be administered between 19-75 mg / kg every 24 hours for equivalent biological dosing. More specifically, a dose of 12 mg / kg dexamethasone corresponds to a dose of 75 mg / kg prednisolone, requiring repeated dosing of about 2 to about 3 doses every 24 hours. A 10 mg / kg dose of betamethasone is approximately 12 mg / kg of dexamethasone and has a similar pharmacodynamic (biological) half-life as dexamethasone.

[0164] The dexamethasone doses in the examples in this application are shown as human equivalent doses (HEDs). Methods for calculating human equivalent doses (HEDs) are known in the art. For example, the FDA's Center for Drug Evaluation and Research (CDER) issued a highly cited guidance document in 2005 (US Department of Health CDER, 2005), which describes in Table 1 on page 7 an established algorithm for converting animal doses to HEDs based on body surface area (a generally accepted method for estimating doses between species). For reference, Table 1 is reproduced below. Those skilled in the art will appreciate that the animal doses, HEDs in mg / kg, described below, are easily calculated using standard conversion factors in the right-hand column of Table 1: [Table 1]

[0165] In some embodiments of the disclosed method, the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent is administered at a dose equivalent to about at least 12 mg / kg of human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate. In other preferred embodiments, the glucocorticoid receptor (GR) modulating agent is administered at a dose equivalent to about at least 15 mg / kg or about at least 18 mg / kg of human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate. In other preferred embodiments, the glucocorticoid receptor (GR) modulating agent is administered at a dose equivalent to about at least 21 mg / kg or about at least 24 mg / kg of human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate. In some preferred embodiments, the glucocorticoid receptor (GR) modulating agent is administered at a dose equivalent to about 12 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate, about 15 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate, or about 18 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate, or about 21 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate, or about 24 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate, or about 30 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate, or about 45 mg / kg human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate.

[0166] In some embodiments of the disclosed methods, the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent is a human equivalent dose (HED) of about at least 6-45 mg / kg of dexamethasone base or dexamethasone phosphate, a human equivalent dose (HED) of about at least 15-24 mg / kg of dexamethasone base or dexamethasone phosphate, a human equivalent dose (HED) of about at least 6-12 mg / kg of dexamethasone base or dexamethasone phosphate, dexamethasone In embodiments where the infectious disease is a disease resulting from infection with a coronavirus, e.g., COVID-19, the glucocorticoid receptor (GR) modulating agent may be administered at a dose equivalent to between about 18-30 mg / kg of human equivalent dose (HED) of dexamethasone base or dexamethasone phosphate.

[0167] In the method of the present disclosure, the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent can be administered as a single acute dose or as a total dose given over a period of about 24, 48 or 72 hours. In some preferred embodiments, the glucocorticoid receptor (GR) modulating agent is administered as a single acute dose. In other preferred embodiments, the glucocorticoid receptor (GR) modulating agent is administered as a total dose given over a period of about 72 hours.

[0168] In some embodiments where a subject has, is suspected of having, or has been diagnosed with an infectious disease, e.g., a disease resulting from infection with a coronavirus (such as COVID-19), the glucocorticoid receptor modulating agent, which may preferably be dexamethasone or betamethasone, may be administered as a solution in an aqueous medium. In some such embodiments, the glucocorticoid receptor modulating agent may be provided at a concentration equivalent to about 24 mg / ml dexamethasone phosphate (20 mg / ml dexamethasone base, 26.2 mg / ml dexamethasone sodium phosphate), administered by intravenous (IV) infusion over a period of about 1-2 hours, with a final target dose of between about 18-30 mg / kg human equivalent dose (HED) of dexamethasone base. In other embodiments, the glucocorticoid receptor modulating agent may be provided as dexamethasone tablets dissolved in orange juice or citric acid (pH 3.3-4.2) and administered orally or by gastric tube, at a final target dose of between about 18-30 mg / kg human equivalent dose (HED) of dexamethasone base.

[0169] In some embodiments of the methods of the present disclosure, the methods may include administering to the subject one or more doses of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent.

[0170] In this context, one or more doses may be administered in addition to a first or preceding dose of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent, and thus may be referred to as a subsequent, or second, third, fourth, etc. dose. Thus, in some embodiments, one or more additional doses may be administered about 24, 48, 72, 96, 120, 144, or 168 hours after the preceding dose. In some embodiments, one or more additional doses may be administered about every 24, 48, 72, 96, 120, 144, or 168 hours after the preceding dose. In some other embodiments, one or more additional doses may be administered once a week, once every two weeks, once every three weeks, or once a month after the preceding dose. In some other embodiments, one or more additional doses may be administered twice a week after the preceding dose.

[0171] In some embodiments, the one or more additional doses may be administered between about 24 hours and 168 hours after the preceding dose. In other embodiments, the one or more additional doses may be administered between about 24 hours and 120 hours, between about 24 hours and 72 hours, or between about 24 hours and 48 hours after the preceding dose. In some other embodiments, the one or more additional doses may be administered between about 48 hours and 168 hours, between about 48 hours and 120 hours, or between about 48 hours and 72 hours after the preceding dose. In some other embodiments, the one or more additional doses may be administered between about 72 hours and 168 hours, or between about 72 hours and 120 hours after the preceding dose.

[0172] In some embodiments, the subsequent dose is given 7 days after the first dose. In some embodiments, the subsequent dose is given 14 days after the first dose. In some embodiments, the subsequent dose is given 21 days after the first dose.

[0173] In some embodiments, in which the subject has, is suspected of having, or has been diagnosed with T-cell lymphoma, one or more additional doses may be administered every 21 days, or every 14 days, or every 5-7 days, for a period of time that may be determined by a physician.

[0174] In some embodiments, in which the subject has, is suspected of having, or has been diagnosed with B cell lymphoma, one or more additional doses may be administered every 21 days, or every 14 days, or every 5-7 days, for a period that may be determined by a physician.

[0175] In some embodiments of the methods of the present disclosure, the methods may further comprise administering to the subject an NKT cell activator, a T cell activator and / or an NK cell activator.

[0176] As used herein, the term NKT cell activator includes any agent or molecule that induces the activation of NKT cell.The activation of NKT cell is associated with the upregulation of activation markers and Th1 and Th2 cytokines and chemokines.The NKT cell activator that can be used in the disclosed method is well known to those skilled in the art.

[0177] Some such NKT cell activators include, but are not limited to, the adipokines leptin, adiponectin, apelin, chemerin, MCP-1, PAI-1, RBP4, visfatin, omentin, vaspin, progranulin, CTRP-4, and the cytokines IL-1α, IL-1β, IL-1RA. IL-18, IL-33, IL-36α, IL-36β, IL-36γ. IL-36RA, IL-37, IL-38, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-ω, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, IL-6, IL-11, IL-31, CLCF1, CNTF, leptin, LIF, OSM, iL-12, IL-17A, IL-17B, IL-17C, IL-17 D, IL-17E, IL-17F, 4-1BBL, BAFF, CD40LG, CD70, CD95L / CD178, EDA-A1, LTA / TNF-β, TNF-α, TNFSF4, TNFS8, TNFSF10, TNFS F11, TNFSF12, TNFSF13, TNFSF15, TGF-β1, TGF-β2, TGF-β3, IL-13, G-CSF, GM-CSF, CSF1. Chemokine, CXCL1-CXCL17, CC, CCL1-C CL28, CX3CL1, XCL1, XCL2, myokine, BDNF, decorin, irisin, myostatin, myonectin, osteonectin, prostaglandin, PGI2, PGD2, PGE2, PGF2α, prostamide, prostamide I2, prostamide D2, prostamide E2, prostamide F2α, Virokines, growth factors, adrenomedullin, angiopoietin , autocrine motility factor, bone morphogenetic protein, ciliary neurotrophic factor, leukemia inhibitory factor, M-CSF, EGF, ephrine A1-A5, ephrine B1-B3, erythropoietin, FGF1-FGF23, fetal bovine somatotrophin, GDNF, neurturin, persephin, artemin, growth differentiation factor-9, hepatocyte growth factor, hepatocyte-derived growth factor, insulin,Insulin-like growth factor 1 / 2, keratinocyte growth factor, migration stimulating factor, macrophage stimulating protein, neuregulin 1-4, neurotrophin 3 / 4, nerve growth factor, placental growth factor, platelet-derived growth factor, renalase, T cell growth factor, TGF-α, TGF-β, VEGF, Wnt signaling pathway, anti-NKG2D antibody or its ligand MICA (MHC class I chain-related sequence A), DNAM-1 binding, 4-1BB binding, PD-1 inhibitor, NKT activator, α-galactosylceramide, α-glucoronosylceramide, α-galcturonsylceramide, α-galactosyldiacylgylocerol, phosphatidylinositol-manosidase, Examples of such anti-inflammatory agents include α-glucosyldiacylglycerol, cholesterol α-glucoside, β-glaactocsylceramide, isoglobotrihexosylceramide, diasialoganglioside, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, house dust extract, GSL-1, NKp44L, ULBP, pathogen-derived molecular structures, PAMP, LPS, pathogen-derived RNA, pathogen-derived DNA, viral ligands, synthetic α-galacosylceramide, KRN7000, PBS44, PBS57, anti-inflammatory agents, IL-10, IL-19, IL-20, IL-22, IL-24, IL-28A, IL-28B, and IL-29.

[0178] In some embodiments of the present disclosure, the NKT cell activator may not be one or more of the agents listed above.

[0179] After activation, NKT cells express NKp44, downregulate CD3 and CD49b expression, and express IL-10, TGF-β, IFN gamma, IL-4 and several Th1 and Th2 cytokines, human class I restricted T cell associated molecule (CRTAM), CCL3 / MIP1a, CCL4 / MIP1h and CCL5 / Rantes and XCL1 / lymphotactin, granzymes, CD45RO+CD62L+, CD25, IL2R beta, GM-CSF, IL-2, IL-13, TNF alpha, IL-17, IL-21, CD44, CD69 and IL-22. Furthermore, in the tumor environment, NKT cells become organized into lines that migrate toward tumor cells from all directions.

[0180] In some preferred embodiments of the disclosed methods, the NKT cell activator may be selected from the group consisting of alphaGalCer (alpha-galactosylceramide, α-GalCer) sulfatide (3-O-sulfogalactosylceramide, SM4, sulfated galactocerebroside) or an NKT-activating antibody, or perforin, nitric oxide, IL-2, interferon alpha and gamma, TGF beta, TNF alpha, TNF beta, G-CSF, VEGF, FGF-18, IL-17, CXCL5, CXCR2, CXCR 5, CCR4-CCL17 / 22, CCR8-CCL1, CCR10-CCL28 and CXCR3-CCL9 / 10 / 11, CCL5, CXCR9, CCL2, CCL3, CCL4, CCL5, CXCL9 or CXCL10, interferon (IFN) gamma-inducible chemokines CXCL9, CXCL10 and CXCL11, CCL5 and CXCL9, CCR5, IL-32, IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG or MIP-1α. In some other preferred embodiments of the methods of the present disclosure, the NKT cell activator can be alphaGalCer-loaded dendritic cells or monocytes.

[0181] As used herein, the term T cell activator includes any agent or molecule that induces the activation of T cells. T cells can be activated through the interaction of TCR with antigenic peptides and MHC, and through non-antigen specific costimulatory molecules (such as cytokine interleukin 1). T cell activation is associated with increased cytokine and chemokine production, induction of dendritic cell maturation, macrophage recruitment and increased cytolytic activity. Gamma delta T cell activation may also be associated with increased production of growth factors that maintain epithelial integrity (such as IGF-1, VEGF and FGF-2) and antigen presentation of alpha beta T cells. T cell activation may also be associated with changes in the pattern of expression of surface markers. For gamma delta T cells, this may include one or more of the following marker phenotypes: CD5-, CD4- / CD8- (double negative), CD3+, CD69, CD56, CD27, CD45RA+, CD45, TCR-Vg9+, TCR-Vd2+, TCR-Vd1+ and / or TCR-Vd3+. T cell activators that can be utilized in the disclosed methods are well known to those of skill in the art.

[0182] Some such T cell activators include, but are not limited to, the adipokines leptin, adiponectin, apelin, chemerin, MCP-1, PAI-1, RBP4, visfatin, omentin, vaspin, progranulin, CTRP-4, and the cytokines IL-1α, IL-1β, IL-1RA. IL-18, IL-33, IL-36α, IL-36β, IL-36γ. IL-36RA, IL-37, IL-38, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-ω, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, IL-6, IL-11, IL-31, CLCF1, CNTF, leptin, LIF, OSM, iL-12, IL-17A, IL-17B, IL-17C, IL-17 D, IL-17E, IL-17F, 4-1BBL, BAFF, CD40LG, CD70, CD95L / CD178, EDA-A1, LTA / TNF-β, TNF-α, TNFSF4, TNFS8, TNFSF10, TNFS F11, TNFSF12, TNFSF13, TNFSF15, TGF-β1, TGF-β2, TGF-β3, IL-13, G-CSF, GM-CSF, CSF1. Chemokine, CXCL1-CXCL17, CC, CCL1-C CL28, CX3CL1, XCL1, XCL2, myokine, BDNF, decorin, irisin, myostatin, myonectin, osteonectin, prostaglandin, PGI2, PGD2, PGE2, PGF2α, prostamide, prostamide I2, prostamide D2, prostamide E2, prostamide F2α, Virokine, growth factor, adrenomedullin, angiopoietin, Autocrine motility factor, bone morphogenetic protein, ciliary neurotrophic factor, leukemia inhibitory factor, M-CSF, EGF, ephrine A1-A5, ephrine B1-B3, erythropoietin, FGF1-FGF23, fetal bovine somatotrophin, GDNF, neurturin, persephin, artemin, growth differentiation factor-9, hepatocyte growth factor, hepatocyte-derived growth factor, insulin,Insulin-like growth factor 1 / 2, keratinocyte growth factor, migration stimulating factor, macrophage stimulating protein, neuregulin 1-4, neurotrophin 3 / 4, nerve growth factor, placental growth factor, platelet-derived growth factor, renalase, T cell growth factor, TGF-α, TGF-β, VEGF, Wnt signaling pathway, NKT activator, α-galactosylceramide, α-glucoronosylceramide, α-galacturonsylceramide, α-galactosyldiacylgylocerol, phosphatidylinositol-manosidase, α-glucosyldiacylglycerol, cholesterol α-glucosyl Examples of the anti-inflammatory agents include β-glaactocsylceramide, isoglobotrihexosylceramide, diasialoganglioside, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, house dust extract, GSL-1, NKp44L, ULBP, pathogen-derived molecular structures, PAMP, LPS, pathogen-derived RNA, pathogen-derived DNA, viral ligands, synthetic α-galacosylceramide, KRN7000, PBS44, PBS57, anti-inflammatory agents, IL-10, IL-19, IL-20, IL-22, IL-24, IL-28A, IL-28B, and IL-29.

[0183] In some preferred embodiments of the methods of the present disclosure, the T cell activator may be selected from the group consisting of zoledronate, mevastatin, or a T cell activating antibody.

[0184] In some embodiments of the present disclosure, the T cell activator may not be one or more of the agents listed above.

[0185] As used herein, the term NK cell activator includes any agent or molecule that induces the activation of NK cells.

[0186] Some such NK cell activators include, but are not limited to, the adipokines leptin, adiponectin, apelin, chemerin, MCP-1, PAI-1, RBP4, visfatin, omentin, vaspin, progranulin, CTRP-4, and the cytokines IL-1α, IL-1β, IL-1RA. IL-18, IL-33, IL-36α, IL-36β, IL-36γ. IL-36RA, IL-37, IL-38, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-ω, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, IL-6, IL-11, IL-31, CLCF1, CNTF, leptin, LIF, OSM, iL-12, IL-17A, IL-17B, IL-17C, IL-17 D, IL-17E, IL-17F, 4-1BBL, BAFF, CD40LG, CD70, CD95L / CD178, EDA-A1, LTA / TNF-β, TNF-α, TNFSF4, TNFS8, TNFSF10, TNFS F11, TNFSF12, TNFSF13, TNFSF15, TGF-β1, TGF-β2, TGF-β3, IL-13, G-CSF, GM-CSF, CSF1. Chemokine, CXCL1-CXCL17, CC, CCL1-C CL28, CX3CL1, XCL1, XCL2, myokine, BDNF, decorin, irisin, myostatin, myonectin, osteonectin, prostaglandin, PGI2, PGD2, PGE2, PGF2α, prostamide, prostamide I2, prostamide D2, prostamide E2, prostamide F2α, Virokine, growth factor, adrenomedullin, angiopoietin, Autocrine motility factor, bone morphogenetic protein, ciliary neurotrophic factor, leukemia inhibitory factor, M-CSF, EGF, ephrine A1-A5, ephrine B1-B3, erythropoietin, FGF1-FGF23, fetal bovine somatotrophin, GDNF, neurturin, persephin, artemin, growth differentiation factor-9, hepatocyte growth factor, hepatocyte-derived growth factor, insulin,Insulin-like growth factor 1 / 2, keratinocyte growth factor, migration stimulating factor, macrophage stimulating protein, neuregulin 1-4, neurotrophin 3 / 4, nerve growth factor, placental growth factor, platelet-derived growth factor, renalase, T cell growth factor, TGF-α, TGF-β, VEGF, Wnt signaling pathway, NKT activator, α-galactosylceramide, α-glucoronosylceramide, α-galacturonsylceramide, α-galactosyldiacylgylocerol, phosphatidylinositol-manosidase, α-glucosyldiacylglycerol, cholesterol α-glucosyl Examples of the anti-inflammatory agents include β-glaactocsylceramide, isoglobotrihexosylceramide, diasialoganglioside, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, house dust extract, GSL-1, NKp44L, ULBP, pathogen-derived molecular structures, PAMP, LPS, pathogen-derived RNA, pathogen-derived DNA, viral ligands, synthetic α-galacosylceramide, KRN7000, PBS44, PBS57, anti-inflammatory agents, IL-10, IL-19, IL-20, IL-22, IL-24, IL-28A, IL-28B, and IL-29.

[0187] In some preferred embodiments of the methods of the present disclosure, the NK cell activator may be selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, or an NK cell activating antibody.

[0188] In some embodiments of the present disclosure, the NK cell activator may not be one or more of the agents listed above.

[0189] In some embodiments of the disclosed methods, the NKT cell activator, T cell activator and / or NK cell activator may be administered within 1, 3, 24, 48, 72, 96, 120, 144 or 168 hours of administration of a dose of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent. In some preferred embodiments, the NKT cell activator, T cell activator and / or NK cell activator may be administered within or about 1, 3 or 48 hours of administration of a dose of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent. In some particularly preferred embodiments, the NKT cell activator, T cell activator and / or NK cell activator may be administered within or about 1, 3 or 48 hours of administration of a dose of a glucocorticoid.

[0190] The terms "subject" and "patient" are used interchangeably herein and refer to a human or an animal. In some embodiments of the methods of the present disclosure, the subject may be a mammal. In some preferred embodiments, the subject may be a human of any gender or race. In some embodiments, the human is an adult human. In some embodiments of the methods of the present disclosure, the subject may be a healthy subject, e.g., a healthy adult human subject. In this context, a healthy subject is a subject that is not suffering from a disease. Preferably, the subject is a human or a mammal with a humanized immune system, e.g., a human immune system (HIS) mouse. Most preferably, the subject is a human.

[0191] In some embodiments of the methods of the present disclosure, the subject may have, may be suspected of having, or may have been diagnosed with a disease selected from the group consisting of cancer, an autoimmune disease, or an infectious disease (also called a microbial disease).

[0192] As used herein, "cancer" refers to a disease characterized by uncontrolled growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0193] In some embodiments of the present disclosure, the cancer is a malignant neoplasm of the lips, malignant neoplasm of the tonsils, malignant neoplasm of the tongue, malignant neoplasm of the gums, malignant neoplasm of the oral cavity, malignant neoplasm of the parotid gland, malignant neoplasm of the salivary gland, malignant neoplasm of the pharynx, malignant neoplasm of the esophagus, malignant neoplasm of the stomach, malignant neoplasm of the small intestine, malignant neoplasm of the colon, malignant neoplasm of the rectosigmoid junction, malignant neoplasm of the rectum, malignant neoplasm of the anus, malignant neoplasm of the liver, malignant neoplasm of the gallbladder, malignant neoplasm of the biliary tract, malignant neoplasm of the pancreas, malignant neoplasm of the intestinal tract, malignant neoplasm of the spleen, malignant neoplasm of the nasal cavity and middle ear. malignant neoplasms of the nasal sinuses, larynx, trachea, bronchi and lungs, thymus, heart, mediastinum and pleura, respiratory system and intrathoracic organs, bones and articular cartilage of the limbs, skull and facial bones, spine, ribs, sternum and clavicle, pelvis, sacrum and coccyx, skin, lips, eyelids including canthus, ear and external auditory canal auricular canal), malignant melanoma of the face, malignant melanoma of the skin of the anus, malignant melanoma of the skin of the breast, malignant melanoma of the extremities including shoulder, Merkel cell carcinoma, basal cell carcinoma of the skin of the lips, squamous cell carcinoma of the skin of the lips, other and unspecified malignant neoplasms of the skin / eyelid including canthus, malignant neoplasms of the skin / ear and external auric canal, other and unspecified malignant neoplasms of the skin / and unspecified parts of the face, basal cell carcinoma of the skin of other and unspecified parts of the face, squamous cell carcinoma of the skin of the face and unspecified parts of the face, basal cell carcinoma of the skin of the scalp and neck, squamous cell carcinoma of the skin of the scalp and neck, basal cell carcinoma of the skin of the trunk, basal cell carcinoma of the skin of the anus, basal cell carcinoma of the skin of the breast, squamous cell carcinoma of the skin of the trunk, squamous cell carcinoma of the skin of the anus, squamous cell carcinoma of the skin of the breast Cancer, squamous cell carcinoma of the skin of other parts of the trunk, other and unspecified malignant neoplasms of skin / extras including shoulder, basal cell carcinoma of skin / extras including shoulder, squamous cell carcinoma of skin / extras including shoulder, basal cell carcinoma of skin of extremities including buttocks, squamous cell carcinoma of skin of extremities including buttocks, mesothelioma, Kaposi's sarcoma, malignant neoplasms of the peripheral and autonomic nervous system, malignant neoplasms of the retroperitoneum and peritoneum, malignant neoplasms of other connective and soft tissues, malignant neoplasms of connective and soft tissues of the breast,Malignant neoplasms of connective tissue and soft tissue of the abdomen, malignant neoplasms of connective tissue and soft tissue of the pelvis, malignant neoplasms of connective tissue and soft tissue of the trunk, unspecified, malignant neoplasms of overlapping areas of connective tissue and soft tissue, malignant neoplasms of connective tissue and soft tissue, unspecified, gastrointestinal stromal tumors, malignant neoplasms of the breast, malignant neoplasms of the vulva, malignant neoplasms of the vagina, malignant neoplasms of the cervix, malignant neoplasms of the corpus of the uterus, malignant neoplasms of unspecified parts of the uterus, malignant neoplasms of the ovaries, malignant neoplasms of other and unspecified female genital organs, malignant neoplasms of the placenta malignant neoplasms of the penis, malignant neoplasms of the prostate, malignant neoplasms of the testes, malignant neoplasms of the male genital organs other and unspecified, malignant neoplasms of the kidneys, malignant neoplasms of the renal pelvis, malignant neoplasms of the ureters, malignant neoplasms of the urinary bladder, malignant neoplasms of the urinary tract other and unspecified, malignant neoplasms of the eyes and adnexa, malignant neoplasms of the meninges, malignant neoplasms of the brain, spinal cord, cranial nerves, malignant neoplasms of the optic nerves, malignant neoplasms of the cranial nerves other and unspecified, malignant neoplasms of the central nervous system, unspecified, malignant neoplasms of the thyroid gland, malignant neoplasms of the adrenal glands, endocrine glands malignant neoplasms of the bladder and other and unspecified urinary tract, secondary malignant neoplasms of the skin, secondary malignant neoplasms of the brain and cerebral meninges, secondary malignant neoplasms of the nervous system and of the nervous system secondary malignant neoplasms of the ovary, secondary malignant neoplasms of the adrenal gland, Hodgkin's lymphoma, follicular lymphoma, nonfollicular lymphoma, small B-cell lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic (diffuse) lymphoma, Burkitt's lymphoma, other nonfollicular lymphoma, nonfollicular (diffuse) lymphoma, unspecified, mature T / NK-cell lymphoma, Sézary's disease, peripheral T-cell lymphoma, unspecified, anaplastic large cell lymphoma, ALK-positive, anaplastic large cell lymphoma, ALK-negative, cutaneous T-cell lymphoma, unspecified, other mature T / NK-cell lymphoma, mature T / NK-cell lymphoma, unspecified,Other and unspecified types of non-Hodgkin's lymphoma, Malignant immunoproliferative disorders and certain other B-cell lymphomas, Multiple myeloma and malignant plasma cell neoplasms, Lymphocytic leukemia, Acute lymphocytic leukemia [ALL], Chronic lymphocytic leukemia of B-cell type, Prolymphocytic leukemia of B-cell type, Hairy cell leukemia, Adult T-cell lymphoma / leukemia (HTLV-1 associated), Prolymphocytic leukemia of T-cell type, Mature B-cell leukemia Burkitt type, Other lymphocytic leukemia, Lymphocytic leukemia, unspecified, Myeloid leukemia, Acute myeloblastic leukemia, Chronic myelogenous leukemia, BCR / ABL positive, Atypical chronic myelogenous leukemia, BCR / A BL negative, myeloid sarcoma, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute myelogenous leukemia with 11q23 abnormality, other myeloid leukemia, myeloid leukemia, unspecified, monocytic leukemia, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, other monocytic leukemia, monocytic leukemia, unspecified, other leukemia of specific cell type, acute erythroid leukemia, acute megakaryoblastic leukemia, mast cell leukemia, acute panmyelopathy with myelofibrosis, myelodysplastic disorders, unspecified, other specified leukemia, leukemia of unspecified cell type, chronic leukemia of unspecified cell type, leukemia, unspecified, leukemia other and unspecified malignant neoplasms of the lymphatic system, hematopoietic tissue, intraepithelial carcinoma of the oral cavity, esophagus and stomach, intraepithelial carcinoma of the colon, intraepithelial carcinoma of the rectosigmoid junction, intraepithelial carcinoma of the rectum, intraepithelial carcinoma of the anus and anal canal, intraepithelial carcinoma of other and unspecified parts of the intestine, intraepithelial carcinoma of unspecified parts of the intestine, intraepithelial carcinoma of other parts of the intestine, intraepithelial carcinoma of the liver, gallbladder and bile duct, intraepithelial carcinoma of other specified digestive organs, intraepithelial carcinoma of digestive organs, unspecified, intraepithelial carcinoma of the middle ear and respiratory system, intraepithelial carcinoma of the larynx, intraepithelial carcinoma of the trachea, intraepithelial carcinoma of the bronchi and lungs, intraepithelial carcinoma of other parts of the respiratory system, intraepithelial Melanoma, melanoma in situ of lips, melanoma in situ of eyelids including canthus, melanoma in situ of ear and ear canal, melanoma in situ of unspecified parts of face, melanoma in situ of scalp and neck, melanoma in situ of trunk, melanoma in situ of anal skin, melanoma in situ of breast (skin) (soft tissue), melanoma in situ of upper extremities including shoulders, melanoma in situ of lower extremities including buttocks, melanoma in situ of other sites, carcinoma in situ of skin, carcinoma in situ of skin of lips, carcinoma in situ of skin of eyelids including canthus, carcinoma in situ of skin of ear and ear canal, carcinoma in situ of skin of other and unspecified parts of face,Cutaneous intraepithelial carcinoma of the scalp and neck, Cutaneous intraepithelial carcinoma of the trunk, Cutaneous intraepithelial carcinoma of the upper extremities including shoulders, Cutaneous intraepithelial carcinoma of the lower extremities including buttocks, Cutaneous intraepithelial carcinoma of other sites, Cutaneous intraepithelial carcinoma of the breast, Lobular carcinoma in situ of the breast, Ductal carcinoma in situ of the breast, Other specified types of carcinoma in situ of the breast, Unspecified types of carcinoma in situ of the breast, Carcinoma in situ of the cervix, Carcinoma in situ of other parts of the cervix, Carcinoma in situ of the cervix, Unspecified, other and unspecified carcinoma of the genital organs, Carcinoma in situ of the endometrium, Carcinoma in situ of the vulva, Carcinoma in situ of the vagina, Other and unspecified carcinoma of the female genital organs, Carcinoma in situ of the penis, Prostate carcinoma in situ of glands, carcinoma in situ of unspecified male genital organs, carcinoma in situ of the scrotum, carcinoma in situ of other male genital organs, carcinoma in situ of the bladder, carcinoma in situ of other and unspecified urinary tract, carcinoma in situ of the eye, carcinoma in situ of the thyroid gland and other endocrine glands, benign neoplasms of the oral cavity and pharynx, benign neoplasms of the major salivary glands, benign neoplasms of the colon, rectum, anus and anal canal, benign neoplasms of the digestive system and obscure parts of the digestive system, benign neoplasms of the esophagus, benign neoplasms of the stomach, benign neoplasms of the duodenum, benign neoplasms of other and unspecified parts of the small intestine, benign neoplasms of the liver, benign neoplasms of the extrahepatic bile ducts, benign neoplasms of the pancreas, benign neoplasms of the endocrine pancreas organisms, benign neoplasms of obscure sites within the digestive system, benign neoplasms of the middle ear and respiratory system, benign neoplasms of the respiratory system, unspecified, benign neoplasms of other and unspecified intrathoracic organs, benign neoplasms of the thymus, benign neoplasms of the heart, benign neoplasms of the mediastinum, benign neoplasms of other specified intrathoracic organs, benign neoplasms of intrathoracic organs, unspecified, benign neoplasms of bone and articular cartilage, benign neoplasms of short bones of the upper limbs, benign neoplasms of long bones of the lower limbs, benign neoplasms of short bones of the lower limbs, benign neoplasms of bones of the skull and face, benign neoplasms of the mandible, benign neoplasms of the spine, benign neoplasms of the ribs, sternum and clavicle, benign neoplasms of the pelvis, sacrum and coccyx Organisms, benign neoplasms of bone and articular cartilage, unspecified, benign lipomatous neoplasms, benign (Ben) lipomatous neoplasms of the skin of the head, face and neck, benign lipomatous neoplasms of the intrathoracic organs, benign lipomatous neoplasms of the intraperitoneal organs, benign lipomatous neoplasms of the spermatic cord, benign lipomatous neoplasms of other sites, benign lipomatous neoplasms of the kidney, benign lipomatous neoplasms of the genitourinary system, hemangiomas and lymphangiomas, any site, hemangiomas, hemangiomas unspecified site, hemangiomas of the skin and subcutaneous tissue, hemangiomas of intracranial structures, hemangiomas of intraperitoneal structures, hemangiomas of other sites, lymphangiomas, any site, benign neoplasms of mesothelial tissue,Benign neoplasms of retroperitoneal and peritoneal soft tissues, other benign neoplasms of connective tissue and other soft tissues, Pigmented nevus, Pigmented nevus of lip, Pigmented nevus of eyelid including canthus unspecified, Pigmented nevus of ear and external auditory canal, Pigmented nevus of other and unspecified parts of face, Pigmented nevus of scalp and neck, Pigmented nevus of trunk, Pigmented nevus of upper limbs including shoulder, Pigmented nevus of lower limbs including buttocks, Pigmented nevus, unspecified, other benign neoplasms of skin of eyelid including canthus, other benign neoplasms of skin / ear and external auditory canal, other benign neoplasms of skin / left ear and external auditory canal (external auric canal), other benign neoplasms of the skin of other parts of the face and unspecified, other benign neoplasms of the skin of other parts of the face, other benign neoplasms of the skin of the scalp and neck, other benign neoplasms of the skin of the trunk, other benign neoplasms of the skin / upper extremities including shoulders, other benign neoplasms of the skin of the lower extremities including buttocks, other benign neoplasms of the skin, unspecified, benign neoplasms of the breast, benign neoplasms of the breast, leiomyomas of the uterus, other benign neoplasms of the uterus, benign neoplasms of the ovaries benign neoplasms of the female genital organs, other and unspecified, benign neoplasms of the male genital organs, benign neoplasms of the urinary tract, benign neoplasms of the kidney, benign neoplasms of the renal pelvis, benign neoplasms of the ureter, benign neoplasms of the bladder, benign neoplasms of the urethra, benign neoplasms of the urinary tract other specified, benign neoplasms of the urinary tract, unspecified, benign neoplasms of the eye and adnexa, benign neoplasms of the conjunctiva, benign neoplasms of the cornea, benign neoplasms of the retina, benign neoplasms of the choroid, benign neoplasms of the ciliary body, lacrimal glands and lacrimal ducts benign neoplasms of, benign neoplasms of unspecified parts of orbit, benign neoplasms of unspecified parts of eye, benign neoplasms of meninges, benign neoplasms of brain and central nervous system, benign neoplasms of thyroid, benign neoplasms of other and unspecified endocrine glands, benign neoplasms of other and unspecified parts, benign neoplasms of lymph nodes, benign neoplasms of peripheral and autonomic nervous system, benign neoplasms of other specified parts, benign neuroendocrine tumours, other benign neuroendocrine tumours, neoplasms of indeterminate behaviour of oral cavity and digestive organs, neoplasms of indeterminate behaviour of major salivary glands, neoplasms of indeterminate behaviour of pharynx, neoplasms of indeterminate behaviour of parts of oral cavity, neoplasms of indeterminate behaviour of stomach, neoplasms of indeterminate behaviour of small intestine, neoplasms of indeterminate behaviour of appendix, neoplasms of indeterminate behaviour of colon, neoplasms of indeterminate behaviour of rectum, neoplasms of indeterminate behaviour of liver, neoplasms of indeterminate behaviour of GB & bile duct, other digestive organs, neoplasms of indeterminate behaviour of digestive organs, neoplasms of middle ear and intrathoracic organs,Neoplasms of indeterminate behavior of the larynx, neoplasms of indeterminate behavior of the trachea, bronchi and lungs, neoplasms of indeterminate behavior of the pleura, neoplasms of indeterminate behavior of the mediastinum, neoplasms of indeterminate behavior of the thymus, neoplasms of indeterminate behavior of other respiratory system organs, neoplasms of indeterminate behavior of respiratory system organs, unspecified, neoplasms of indeterminate behavior of the female genital organs, neoplasms of indeterminate behavior of the uterus, neoplasms of indeterminate behavior of the ovaries, neoplasms of indeterminate behavior of the ovaries unspecified, neoplasms of indeterminate behavior of the placenta, neoplasms of indeterminate behavior of the male genital organs, neoplasms of indeterminate behavior of the urinary tract, neoplasms of indeterminate behavior of the kidneys, neoplasms of indeterminate behavior of the kidneys unspecified, neoplasms of the renal pelvis, neoplasms of uncertain behavior, neoplasms of uncertain behavior of the ureter, neoplasms of uncertain behavior of the bladder, neoplasms of uncertain behavior of other urinary tract, neoplasms of uncertain behavior of the urinary tract, unspecified neoplasms of uncertain behavior, neoplasms of uncertain behavior of the meninges, neoplasms of uncertain behavior of the cerebral meninges, neoplasms of uncertain behavior of the spinal meninges, neoplasms of uncertain behavior of the meninges, unspecified, neoplasms of uncertain behavior of the brain, neoplasms of uncertain behavior of the brain, neoplasms of uncertain behavior of the brain, infratentorial neoplasms of uncertain behavior of the brain, unspecified, neoplasms of uncertain behavior of the cranial nerves, neoplasms of uncertain behavior of the spinal cord, neoplasms of uncertain behavior of the central nervous system neoplasms of indeterminate behavior of endocrine glands, neoplasms of indeterminate behavior of the thyroid gland, neoplasms of indeterminate behavior of the adrenal gland, neoplasms of indeterminate behavior of unspecified adrenal glands, neoplasms of indeterminate behavior of the parathyroid gland, neoplasms of indeterminate behavior of the pituitary gland, neoplasms of indeterminate behavior of the craniopharyngeal duct, neoplasms of indeterminate behavior of the pineal gland, neoplasms of indeterminate behavior of the carotid body, neoplasms of indeterminate behavior of the aortic body and other paraganglia, neoplasms of indeterminate behavior of unspecified endocrine glands, polycythemia vera, myelodysplastic syndrome, refractory anemia without ringed sideroblasts, as described, with ringed sideroblasts Refractory anemia, refractory anemia with excess blasts [RAEB], myelodysplastic syndromes, other neoplasms of indeterminate behavior of lymphoid tissue, hematopoietic tissue, histiocytic and mast cell tumors of indeterminate behavior, chronic myeloproliferative disorders, monoclonal hypergammaglobulinemia, essential (hemorrhagic) thrombocythemia, myelofibrosis, other neoplasms of indeterminate behavior of lymphoid, hematopoietic & unspecified, neoplasms of indeterminate behavior of other and unspecified sites, neoplasms of indeterminate behavior of bone / articulate cartilage, neoplasms of indeterminate behavior of connective / soft tissue Neoplasms, neoplasms of unspecified behavior of the peripheral and autonomic nervous system, neoplasms of unspecified behavior of the retroperitoneum, neoplasms of unspecified behavior of the peritoneum, neoplasms of unspecified behavior of the skin, neoplasms of unspecified behavior of the breast, neoplasms of unspecified behavior of the digestive system, neoplasms of unspecified behavior of the respiratory system, neoplasms of unspecified behavior of bone, soft tissue and skin, neoplasms of unspecified behavior of the breast, neoplasms of unspecified behavior of the bladder, neoplasms of unspecified behavior of other genitourinary organs, neoplasms of unspecified behavior of the kidneys, neoplasms of unspecified behavior of other GU organs, neoplasms of unspecified behavior of the brain, endo glands and nervous systemIt may be a neoplasm of unspecified behavior in other parts of the retina (sys), a neoplasm of unspecified behavior in the retina and choroid, or a neoplasm of unspecified behavior in an unspecified site.

[0194] In some embodiments of the present disclosure, the cancer may not be one of the cancers listed above.

[0195] In some preferred embodiments of the present disclosure, the cancer may be selected from the group consisting of lymphoma, squamous cell carcinoma (such as epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma; peritoneal cancer; hepatocellular carcinoma; gastric or stomach cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; hepatocellular carcinoma; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; and head and neck cancer. In some particularly preferred embodiments of the present disclosure, the cancer may be lymphoma. In more particularly preferred embodiments of the present disclosure, the cancer may be B-cell lymphoma or T-cell lymphoma. In some particularly preferred embodiments of the present disclosure, the cancer may be non-Hodgkin's lymphoma. In some particularly preferred embodiments of the present disclosure, the cancer may be Burkitt's lymphoma, T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL) or diffuse large B-cell lymphoma (DLBCL). In other preferred embodiments, the cancer may be post-transplant lymphoproliferative disorder. In some other particularly preferred embodiments of the present disclosure, the cancer may be solid tumor cancer.

[0196] In embodiments in which the methods of the present disclosure are practiced on subjects who have, are suspected of having, or have been diagnosed with cancer, the NKT-like cells generated by these methods can treat the cancer. In this context, "treating" means exerting a beneficial therapeutic effect in the subject, which can be any overall clinical benefit resulting from the methods of the present disclosure. This overall clinical benefit may include, for example, extended survival, partial or complete disease remission (e.g., as assessed by % normal maturation of myeloblasts and / or cell lines), slowing or absence of disease progression (e.g., as assessed by change in c%), tumor shrinkage (e.g., a 5, 10, 20, 30, 40% or greater reduction in tumor volume), reduction in tumor burden (e.g., a 5, 10, 20, 30, 40% or greater reduction in tumor burden), slowing or absence of tumor growth, slowing or absence of tumor burden increase, improved quality of life (e.g., as assessed using a health-related quality of life questionnaire such as the Functional Assessment of Cancer Therapy (FACT) questionnaire), progression-free survival, overall survival, hematological improvement (e.g., increased blood hemoglobin, platelet count and / or neutrophil count), bone marrow response (e.g., a bone marrow with ≦5% myeloblasts, The outcome may be either a 30%, 40%, 50% or more reduction in myeloblasts; absence of circulating myeloblasts and myeloblasts with Auer rods; absence of extramedullary disease), hematological recovery (e.g.: ≧11 g / dL hemoglobin, ≧100×109 / L platelets and / or ≧1×109 / L neutrophils in peripheral blood), a negative response in genetic markers (e.g., CEBPA, NPM1 or FLT3) or any other positive patient outcome.

[0197] The overall clinical benefit may be an "anti-tumor effect". As used herein, "anti-tumor effect" refers to a biological effect that may be present as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an amelioration of various physiological symptoms associated with tumors. Anti-tumor effect may also refer to the prevention of tumor development, for example, a vaccine. Suitable methods for determining tumor volume / tumor burden are well known to those skilled in the art, for example, using computed tomography (CT) or magnetic resonance imaging (MRI) imaging techniques, X-ray imaging, for example, mammography, ultrasound imaging, nuclear imaging, for example, positron emission tomography (PET), PET / CT scan, bone scan, gallium scan or metaiodobenzylguanidine (MIBG) scan, bioluminescence imaging (BLI), fluorescence imaging (FLI), BD ToF (infrared-based 3D time-of-flight camera) imaging.

[0198] Thus, in some embodiments, the NKT-like cells of the present disclosure can treat cancer by tumor infiltration. In some embodiments, the NKT-like cells of the present disclosure can treat cancer through the release of immune-activating cytokines. In some embodiments, the NKT-like cells of the present disclosure can phagocytose and kill cancer cells in a subject. In some embodiments, the NKT-like cells of the present disclosure promote the infiltration of other immune cells into tumors. In some embodiments, the NKT-like cells of the present disclosure directly kill cancer cells through CD1d-directed apoptosis.

[0199] In some embodiments, the NKT-like cells of the present disclosure directly kill cancer cells by inducing apoptosis, for example, by expressing a ligand that binds to a death receptor on the target cell. In some embodiments, the NKT-like cells of the present disclosure can engulf or phagocytose cancer cells in a subject. In some embodiments, the NKT-like cells can secrete cytotoxic molecules that kill cancer cells. In some embodiments, the NKT-like cells can treat cancer through a bispecific attack by both TCR gamma / delta and invariant TCR (iTCR).

[0200] "Autoimmune disease" as used herein refers to autoimmune disorders and other diseases resulting from an abnormal immune condition in which the immune system abnormally attacks the subject's own constituents. (In healthy subjects, the immune system avoids damaging autoimmune reactions by establishing tolerance to the subject's own constituents). Examples of various autoimmune diseases are described herein and include, but are not limited to, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, transient osteoporosis, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0201] Autoreactive immune cells express high levels of phosphoantigens, which are produced by stressed cells and by microorganisms such as mycobacteria, E. coli, and malaria parasites that contain diphosphate-containing metabolites, in particular (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). Humans do not produce HMB-PP, but the majority of gram-negative bacteria, including Mycobacterium tuberculosis, Mycobacterium bovis, Clostridium difficile, Listeria monocytogenes, malaria parasites, and toxoplasma gondii and Schistosoma japonicum, do. Gamma delta T cells / receptors are highly responsive to HMB-PP, zoledronate and isopentyl pyrophosphate (IPP), mycolyl arabinogalactan peptidoglycan (mAGP) and iso-butylamine (IBA). Butyrophilin family members such as BTN2A1, BTN3A1, BTNL3, BTNL8, BTNL1, BTNL6, Skint1, and Skint2 play important roles in gamma delta T cell recognition of phosphoantigens. Aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMCs) can also activate gamma delta T cell receptors. IL-18 can enhance the response of gamma delta T cell receptors to phosphoantigens.

[0202] In some embodiments of the present disclosure, the autoimmune disease is selected from the group consisting of allergies, asthma, graft-versus-host disease (GvHD), steroid-resistant GvHD, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, alopecia, transient osteoporosis, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal nephropathy, and autoimmune urticaria. & Neuron neuropathy (AMAN), Baro disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica) optic), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogamma Hypogammalglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignoconjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease,Multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (P A), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, monkeys coidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, hematopoietic syndrome, It could be cytophagocytic lymphohistiocytosis, multiple myeloma, allergen specific immunotherapy, autosomal dominant haploinsufficiency, anterior interosseous nerve syndrome, Churg-Strauss syndrome, systemic vasculitis, chronic graft-versus-host disease, opsoclonus-myoclonus syndrome, necrotizing autoimmune myopathy (NAM), pulmonary sarcomatoid carcinoma, Waldenström's macroglobulinemia (WM), fertility, Behçet's disease, alopecia areata (AA), acute exacerbation of chronic hepatitis, melanoma, "organizing bronchiolitis syndrome" or encephalitis. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, uncerative protitis, severe combined immunodeficiency (SCID),DiGeorge syndrome, ataxia telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperglobulinemia E syndrome, Hashimoto's thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham chorea, myasthenia gravis, polyglandular syndrome, bullous pemphigoid, Henoch-Schönlein purpura, poststreptococcal nephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, or cancer.

[0203] In some embodiments of the present disclosure, the autoimmune disease may not be one of the autoimmune diseases listed above.

[0204] In some preferred embodiments of the present disclosure, the autoimmune disease may be selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes (T1D), scleroderma, pemphigus and lupus. In some other preferred embodiments of the present disclosure, the autoimmune disease may be selected from the group consisting of graft-versus-host disease (GvHD) and allergic disorders such as asthma. In some particularly preferred embodiments of the present disclosure, the autoimmune disease may be type 1 diabetes (T1D).

[0205] In embodiments in which the methods of the present disclosure are practiced on subjects who have, are suspected of having, or have been diagnosed with an autoimmune disease, the NKT-like cells generated by these methods can treat the autoimmune disease. In this context, "treating" means exerting a beneficial therapeutic effect in the subject, which can be any overall clinical benefit resulting from the methods of the present disclosure. This overall clinical benefit may be, for example, any of the following: reduced fatigue, reduced muscle pain, reduced swelling and redness, reduced low-grade fever, reduced ability to concentrate, reduced numbness and tingling in the hands and feet and arms or legs, reduced urination, reduced hair loss, reduced skin rash, restoration of normal blood sugar, increased C-peptide, improved wound healing, reduced diarrhea, reduced muscle spasms, improved muscle tone and control, reduced rash or scaly plaques or discoloration on the skin, improved weight maintenance, reduced muscle or joint pain, improved gastrointestinal comfort, normal heart rate, reduced anxiety, reduced Expanded Disability Status Scale (EDSS) score, reduced specific active lesions in the brain as measured by gadolinium-enhanced MRI.

[0206] In some embodiments, the NKT-like cells of the present disclosure can treat autoimmune diseases through directly killing autoreactive T and / or B lymphocytes, increasing the Treg:T lymphocyte ratio, inhibiting the activity of autoreactive T and / or B lymphocytes, reducing inflammation, or reducing trafficking of autoreactive lymphocytes.

[0207] "Infectious disease" (or "microbial disease"), as used herein, refers to a disease or illness resulting from infection of a subject's body with an infectious agent (pathogen), such as a virus, bacteria, or fungus. In some embodiments of the present disclosure, the infectious disease is selected from the group consisting of Acinetobacter infection (Acinetobacter baumannii), actinomycosis (Actinomyces israelii, Actinomyces gerencseriae, and Propionibacterium propionicus), African sleeping sickness or African trypanosomiasis (Trypanosoma brucei), AIDS (Acquired Immune Deficiency Syndrome) (Human Immunodeficiency Virus), amebiasis (Entamoeba histolytica), and the like. histolytica), anaplasmosis (Anaplasma spp.), angiostrongyliasis (Angiostrongylus spp.), anisakiasis (Anisakis spp.), anthrax (Bacillus arthracis), hemolytic arcanobacterial infections (Arcanobacterium haemolyticum), Argentine hemorrhagic fever (Junin virus), ascariasis (Ascaris lumbricoides), aspergillosis (Aspergillus spp.), astrovirus infections (Astroviridae), babesiosis (Babesia spp.), Bacillus cereus cereus infection, bacterial pneumonia (multiple bacteria), bacterial vaginosis (list of bacterial vaginosis microflora), Bacteroides infection, Balantidiosis (Balantidium coli), Bartonella infection, Raccoon roundworm infection (Baylisascaris species), BK virus infection, Black Piedrapiedra (Piedraia hortae), Blastocystosis (Blastocystis species), Blastomycosis (Blastomyces dermatitidis), Bolivian hemorrhagic fever (Machupo virus), botulism (and infant botulism) (Clostridium botulinum botulinum; Note: botulism is not an infection with Clostridium botulinum, but is caused by the uptake of botulinum toxin), Brazilian hemorrhagic fever (Sabia virus), brucellosis (Brucella spp.), bubonic plague (bacterial family, Enterobacteriaceae), Burkholderia infection, usually Burkholderia cepacia and other Burkholderia spp., Buruli ulcer (Mycobacterium ulcerans), Calicivirus infection (Norovirus and Sapovirus) (Caliciviridae), Campylobacteriosis (Campylobacter spp.), Candidiasis (Moniliasis; oral candidiasis) (usually Candida albicans), albicans and other Candida species), capillariasis (intestinal disease due to Capillaria philippinensis, liver disease due to Capillaria hepatica and lung disease due to Capillaria aerophila), Carrion's disease (Bartonella bacilliformis), cat scratch disease (Bartonella henselae), cellulitis (usually group A streptococci and staphylococci), Chagas' disease (American trypanosomiasis) (Trypanosoma cruzi), chancroid (Haemophilus ducreyi),ducreyi), chickenpox (Varicella zoster virus (VZV)), chikungunya (Alphavirus), chlamydia (Chlamydia trachomatis), Chlamydia pneumoniae infection (Taiwan Acute Respiratory Agent or TWAR) (Clamydia pneumoniae), cholera (Vibrio cholerae), chromoblastomycosis (commonly Fonsecaea pedrosoi), chytridiomycosis (Batrachochytrium dendrabatidis), Clonorchiasis (Clonorchis sinensis), Clostridium difficile colitis (Clostridium difficile), coccidioidomycosis (Coccidioides immitis), immitis and Coccidioides posadasii), Colorado Tick Fever (CTF) (Colorado Tick Fever Virus (CTFV)), Common Cold (Acute Viral Rhinopharyngitis, Acute Colic) (Commonly Rhinoviruses and Coronaviruses), Coronaviruses, Creutzfeldt-Jakob Disease (CJD) (PRNP), Crimean-Congo Hemorrhagic Fever (CCHF) (Crimean-Congo Hemorrhagic Fever Virus), Cryptococcosis (Cryptococcus neoformans), Cryptosporidiosis (Cryptosporidium species), Cutaneous Larva Migrans (CLM) (Commonly Ancylostoma braziliense; several other parasites), Cyclosporiasis (Cyclospora cayetanensis), Cysticercosis (Taenia solium solium), Cytomegalovirus infection (Cytomegalovirus), Dengue fever (Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) - Flaviviruses), Desmodesmus infection (Desmodesmus armatus), Dientamoebiasis (Dientamoeba fragilis), Diphtheria (Corynebacterium diphtheriae)diphtheriae), Diphyllobothrium, Dracunculus medinensis, Ebola hemorrhagic fever (Ebola virus (EBOV)), Echinococcosis (Echinococcus species), Ehrlichiosis (Ehrlichia species), Intestinal pinworm infection (Enterobius vermicularis), Enterococcus infection (Enterococcus species), Enterovirus infection (Enterococcus species), Typhus (Rickettsia prowazekii), erythema infectiosum (fifth disease) (Parvovirus B19), exanthema subitum (sixth disease) (Human Herpesvirus 6 (HHV-6) and Human Herpesvirus 7 (HHV-7)), Fasciolasis (Fasciola hepatica and Fasciola gigantica), Fasciolopsis (Fasciolopsis buski), Fatal Familial Insomnia (FFI) (PRNP), Filariasis (Filarioidea superfamily), Clostridium perfringens perfringens food poisoning (Clostridium perfringens), free-living ameba infections (multiple), Fusobacterium infections (Fusobacterium spp.), gas gangrene (Clostridial myonecrosis) (usually Clostridium perfringens, other Clostridium spp.), geotrichum (Geotrichum candidum), Gerstmann-Sträussler-Scheinker syndrome (GSS) (PRNP), giardiasis (Lamblia giardiasis), glanders (Burkholderia mallei), gnathostomiasis (Gnathostoma spinigerum and Gnathostoma hispidum), gonorrhea (Neisseria gonorrhoeae), gonorrhoeae), granuloma venereum (lymphogranuloma venereum)(Klebsiella granulomatis), Group A Streptococcus infection (Streptococcus pyogenes), Group B Streptococcus infection (Streptococcus agalactiae), Haemophilus influenzae infection (Haemophilus influenzae), Hand, Foot and Mouth Disease (HFMD) (Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71)), Hantavirus Pulmonary Syndrome (HPS) (Sin Nombre virus), Heartland Virus Disease (Heartland Virus), Helicobacter pylori infection (Helicobacter pylori), pylori), Hemolytic uremic syndrome (HUS), Escherichia coli O157:H7, O111 and O104:H4, Hemorrhagic fever with renal syndrome (HFRS) (Family Bunyaviridae), Hepatitis A (Hepatitis A Virus), Hepatitis B (Hepatitis B Virus), Hepatitis C (Hepatitis C Virus), Hepatitis D (Hepatitis D Virus), Hepatitis E (Hepatitis E Virus), Herpes simplex (Herpes simplex virus 1 and 2 (HSV-1 and HSV-2)), Histoplasmosis (Histoplasma capsulatum), Hookworm infection (Ancylostoma duodenale and Necator americanus), human bocavirus infection (Human bocavirus (HBoV)), human ewingii-ehrlichiosis (Ehrlichia ewingii), human granulocytic anaplasmosis (HGA) (Anaplasma phagocytophilum), human metapneumovirus infection, human metapneumovirus (hMPV), human monocytic ehrlichiosis (Ehrlichia chaffeensis), human papillomavirus (HPV) infection (Human papillomavirus (HPV)), human parainfluenza virus infection (Human parainfluenza virus (HPIV)), hymenolepis tapeworm (Hymenolepis nana and Hymenolepisdiminuta), Epstein-Barr virus infectious mononucleosis (Mono) (Epstein-Barr virus (EBV)), influenza (flu) (Orthomyxoviridae), isosporosis (Isospora belli), Kawasaki disease (unknown; evidence supports that it is contagious), keratitis (multiple), Kingella kingae infection (Kingella kingae), kuru (PRNP), Lassa fever (Lassa fever virus), Legionnaires' disease (Legionella pneumophila), Legionnaires' disease (Pontiac fever) (Legionella pneumophila), leishmaniasis (Leishmania species), leprosy (Mycobacterium leprae and Mycobacterium lepromatosis, leptospirosis (Leptospira spp.), listeriosis (Listeria monocytogenes), Lyme disease (Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii), lymphatic filariasis (elephantiasis) (Wuchereria bancrofti and Brugia malayi), lymphocytic choriomeningitis (Lymphocytic choriomeningitis virus (LCMV)), malaria (Plasmodium species), Marburg hemorrhagic fever (MHF) (Marburg virus), measles (measles virus), Middle East respiratory syndrome (MERS) (Middle East respiratory syndrome coronavirus), melioidosis (Whitmore's disease) (Burkholderia pseudomallei), meningitis (multiple), meningococcal disease (Neisseria meningitidis), Metagonomiasis (commonly Metagonimusyokagawai), microsporidiasis (Microsporidia phylum), molluscum contagiosum (MC) (Molluscum contagiosum virus (MCV)), monkeypox (Monkeypox virus), mumps (Mumps virus), Murine typhus (Endemic typhus) (Rickettsia typhi), mycoplasma pneumoniae, mycetoma (disambiguation) (numerous species of bacteria (Actinomycetoma) and fungi (Mycomycetoma), myiasis (larvae of parasitic dipteran flies), neonatal conjunctivitis (ophthalmia neonatorum) (most commonly Chlamydia trachomatis and Neisseria gonorrhoeae), norovirus (Norovirus ... Viruses (children and infants) (variant Creutzfeldt-Jakob disease (vCJD, nvCJD), PRNP), Nocardial infections (commonly Nocardia asteroides and other Nocardia species), Onchocerciasis (river blindness) (Onchocerca volvulus), Opisthorchiasis (Opisthorchis viverrini and Opisthorchis felineus), Paracoccidioidomycosis (South American blastomycosis) (Paracoccidioides brasiliensis), Paragonimiasis (commonly Paragonimus westermani) westermani and other Paragonimus species), Pasteurellosis (Pasteurella species), Head Lice (Pediculus humanus capitis), Body Lice (Pediculus humanus corporis), Public Lice (Public Lice, Crab Lice) (Phthirus pubis), Pelvic Inflammatory Disease (PID) (multiple), Pertussis (Whooping cough) (Bordetella pertussis), Plague (Yersinia pestis) pestis), Pneumococcal infection (Streptococcus pneumoniae), Pneumocystis pneumonia (PCP) (Pneumocystis jirovecii), Pneumonia (multiple), Poliomyelitis (Poliovirus), Prevotella infection (Prevotella species), Primary amebic meningoencephalitis (PAM) (commonly Naegleria fowleri), Progressive multifocal leukoencephalopathy (JC virus), Psittacosis (Chlamydophila psittaci), Q fever (Coxiella brunettii),burnetii), rabies (Rabies virus), relapsing fever (Borrelia hermsii, Borrelia recurrentis and other Borrelia species), respiratory syncytial virus infection (Respiratory syncytial virus (RSV)), rhinosporidiosis (Rhinosporidium seeberi), rhinovirus infection (Rhinovirus), rickettsial infection (Rickettsia species), rickettsialpox (Rickettsia akari), Rift Valley fever (RVF) (Rift Valley fever virus), Rocky Mountain spotted fever (RMSF) (Rickettsia rickettsia rickettsii), Rotavirus infection (Rotavirus), Rubella (Rubella virus), Salmonellosis (Salmonella spp.), Severe Acute Respiratory Syndrome (SARS-CoV), Scabies (Sarcoptes scabiei), Schistosomiasis (Schistosoma spp.), Sepsis (multiple), Shigellosis (Bacillary dysentery) (Shigella spp.), Shingles (Herpes zoster) (Varella zoster virus (VZV)), Smallpox (Varia major or Variola minor), Sporotrichosis (Sporothrix schenckii) schenckii), Staphylococcal food poisoning (Staphylococcus species), Staphylococcal infection (Staphylococcus species), Strongyloides stercoralis), Subacute sclerosing panencephalitis (Measles virus), Syphilis (Treponema pallidum), Cestoderma (Taenia species), Tetanus (Clostridium tetani)tetani), tinea barbae (barber's rash) (commonly Trichophyton species), tinea capitis (ringworm of the scalp) (commonly Trichophyton tonsurans), tinea corporis (ringworm of the body) (commonly Trichophyton species), tinea cruris (ringworm of the groin) (commonly Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes), tinea manubrium (ringworm of the hand) (Trichophyton rubrum), tinea nigricans (commonly Hortaea wernekii) werneckii), Tinea pedis (athlete's foot) (commonly Trichophyton species), Tinea unguium (onychomycosis) (commonly Trichophyton species), Tinea versicolor (pityriasis versicolor) (Malassezia species), Toxocariasis (ocular larva migrans (OLM)) (Toxocara canis or Toxocara cati), Toxocariasis (visceral larva migrans (VLM)) (Toxocara canis or Toxocara cati), Trachoma (Chlamydia trachomatis), Toxoplasmosis (Toxoplasma gondii), Trichinosis (Trichinella spiralis), Trichomoniasis (Trichomonas vaginalis) vaginalis), Trichuriasis (whipworm infection) (Trichuris trichiura), Tuberculosis (commonly known as Mycobacterium tuberculosis), Tularemia (Francisella tularensis), Typhoid fever (Salmonella enterica subspecies enterica, serovar typhi), Typhoid fever (Rickettsia spp.), Ureaplasma urealyticum infection (Ureaplasma urealyticum), Valley fever (Coccidioides immitis or Coccidioides posadaciposadasii), Venezuelan equine encephalitis (Venezuelan equine encephalitis virus), Venezuelan hemorrhagic fever (Guanarito virus), Vibrio vulnificus infection (Vibrio vulnificus), Vibrio parahaemolyticus enteritis (Vibrio parahaemolyticus), viral pneumonia (multiple viruses), West Nile fever (West Nile virus), White piedra (Tinea blanca) (Trichosporon beigelii), Yersinia pseudotuberculosis infection (Yersinia pseudotuberculosis), Yersinia enterica (Yersinia enterocolitica), Yellow Fever (Yellow Fever Virus), Zygomycosis (Mucorales (mucormycosis) and Entomophthorales (entomophthorosis))Human immunodeficiency virus [HIV] disease, HIV disease with infectious and parasitic diseases, HIV disease with mycobacterial infections, HIV disease with cytomegalovirus disease, HIV disease with other viral infections, HIV disease with candidiasis, HIV disease with other fungal diseases, Pneumocysticcarinii) HIV disease with pneumonia, HIV disease with malignant neoplasms, HIV disease with Kaposi's sarcoma, HIV disease with Burkitt's lymphoma, HIV disease with other types of non-Hodgkin's lymphoma, HIV disease with other malignant neoplasms of lymphatic, hematopoietic and related tissues, HIV disease with multiple malignant neoplasms, HIV disease with other malignant neoplasms, HIV disease with unspecified malignant neoplasms, HIV disease with encephalopathy, HIV disease with lymphoid interstitial pneumonia, HIV disease with wasting syndrome, HIV disease with multiple diseases classified elsewhere, HIV disease with other conditions, HIV disease acute HIV infection syndrome, HIV disease with (persistent) generalized lymphadenopathy, HIV disease with hematologic and immunologic abnormalities, HIV disease with other specified conditions or unspecified HIV disease. In some embodiments of the present disclosure, the infectious disease is caused by a virus, e.g., a virus from one of the following families of viruses: a) Adenoviridae, e.g., Adenovirus species; b) Herpesviridae, e.g., Herpes simplex type 1, Herpes simplex type 2, Varicella zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpes virus type 8 species; c) Papillomaviridae, e.g., Human papillomavirus species; d) Polyomaviridae, e.g., Polyomaviridae species;dae), for example, BK virus, JC virus species; e) Poxviridae, for example, Variola species; f) Hepadnaviridae, for example, Hepatitis B virus species; g) Parvoviridae, for example, Human Bocavirus, Parvovirus B19 species; h) Astroviridae, for example, Human Astrovirus species; i) Caliciviridae, for example, Norwovirus species; j) Flaviviridae, e.g., Hepatitis C virus (HCV), Yellow fever virus, Dengue virus, West Nile virus species; k) Tigaviridae, e.g., Rubella virus species; 1) Hepeviridae, e.g., Hepatitis E virus species; m) Retroviridae, e.g., Human Immunodeficiency Virus (HIV) species; n) Orthomyxoviridae, e.g., daw) family, such as the influenza virus species; o) Arenaviridae, such as the Guanarito virus, Junin virus, Lassa fever virus, Machupo virus and / or Sabia virus species; p) Bunyaviridae, such as the Crimean-Congo hemorrhagic fever virus species; q) Filoviridae, such as the Ebola virus and / or Marburg virus species; Paramyxoviridae, such as the measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and / or Nipah virus species; r) Rhabdoviridae genera, such as the Rabies virus species; s) Reoviridae, such as the Rotavirus, Orbivirus, Cortivirus and / or Bannavirus species.

[0208] In some embodiments of the present disclosure, the infectious disease may not be one of the infectious diseases listed above.

[0209] In some embodiments, the infectious disease can be a disease caused by infection with an influenza A (Flu A) virus. In some embodiments, the influenza virus can be an avian or swine origin pandemic influenza virus, such as H5N1, H7N3, H7N7, H7N9 and H9N2 (avian subtypes) or H1N1, H1N2, H2N1, H3N1, H3N2 or H2N3 (swine subtypes).

[0210] In some preferred embodiments of the present disclosure, the infectious disease may be HIV, e.g., residual HIV disease, herpes, hepatitis, or human papilloma virus. In other preferred embodiments, the infectious disease may be a disease resulting from infection with a coronavirus, e.g., COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, a disease caused by SARS-CoV-2).

[0211] In embodiments in which the methods of the present disclosure are carried out on subjects who have, are suspected of having, or have been diagnosed with an infectious disease, the NKT-like cells generated by these methods can treat the infectious disease. In this context, "treat" refers to exerting a beneficial therapeutic effect in the subject, which can be any overall clinical benefit resulting from the methods of the present disclosure. This overall clinical benefit can be, for example, reduced fever, reduced diarrhea, reduced cough, reduced muscle pain, reduced fatigue, reduced CRP, reduced time on a ventilator, reduced need for extra oxygen, or reduced organ damage after recovery.

[0212] In some embodiments, the NKT-like cells of the present disclosure can treat infectious diseases through phagocytosis and killing of infectious organisms, activating other innate and adaptive immune cells, recruiting other immune cells to the site of infection (e.g., organs infected by the virus), and depleting immune cells infected by the virus (e.g., monocytes activated by COVID-19).

[0213] In some embodiments, the NKT-like cells of the present disclosure can treat infectious diseases through the release of immune-activating cytokines. In some embodiments, the NKT-like cells of the present disclosure can treat infectious diseases through the release of cytokines (e.g., TNF-alpha, IFN-gamma) that have antibacterial or antiviral effects. In some embodiments, the NKT-like cells of the present disclosure can treat infectious diseases by inducing apoptosis, for example, by expressing a ligand that binds to a death receptor on a target cell. In some embodiments, the NKT-like cells can secrete cytotoxic molecules that kill infectious organisms. In some embodiments, the NKT-like cells of the present disclosure can engulf or phagocytose infectious organisms.

[0214] In embodiments where the infectious disease is a disease resulting from infection with a coronavirus, e.g., COVID-19, the NKT-like cells of the present disclosure can treat the disease through phagocytosis and killing of the coronavirus and / or activating other innate and adaptive immune cells.

[0215] Thus, the present disclosure also provides a method of treating a disease resulting from infection with a coronavirus in a subject, comprising administering to the subject a glucocorticoid receptor (GR) modulating agent at a dose equivalent to about at least 6 mg / kg of a human equivalent dose (HED) of dexamethasone base. In some embodiments, the glucocorticoid receptor (GR) modulating agent can be a glucocorticoid, preferably dexamethasone or betamethasone. In some embodiments, the glucocorticoid receptor (GR) modulating agent can be administered at a dose equivalent to about at least 15 mg / kg of a human equivalent dose (HED) of dexamethasone base. In some preferred embodiments, the glucocorticoid receptor (GR) modulating agent can be administered at a dose equivalent to between about 18 mg / kg and 30 mg / kg of a human equivalent dose (HED) of dexamethasone base. In some preferred embodiments, the disease is COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, a disease caused by SARS-CoV-2) or SARS-CoV or MERS. In some embodiments, the glucocorticoid receptor (GR) modulating agent induces / mobilizes a population of NKT-like cells as disclosed elsewhere herein.

[0216] In some preferred embodiments, the present disclosure provides a method of treating COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, a disease caused by SARS-CoV-2) in a subject, comprising administering to the subject dexamethasone or betamethasone at a dose equivalent to a human equivalent dose (HED) of about 15 mg / kg to 30 mg / kg of dexamethasone base.

[0217] In embodiments where the infectious disease is a disease resulting from infection with a coronavirus, e.g., COVID-19, the glucocorticoid receptor modulating agent may be administered in combination with a proton pump inhibitor (e.g., omeprazole) and / or hydrocortisone. In this context, "in combination with" can mean simultaneous administration or separate and / or sequential administration in any order.

[0218] In some embodiments of the method of the present disclosure, the method of generating / mobilizing a population of NKT-like cells can further comprise isolating the NKT-like cells of the present disclosure or the population of NKT-like cells of the present disclosure from a subject or from a sample derived from a subject.Thus, the present disclosure provides isolated NKT-like cells, as well as isolated populations of NKT-like cells.The isolated cells and populations of isolated cells can be characterized by the pattern of surface proteins they express as outlined above.

[0219] Suitable methods for isolating cells and cell populations from mixed samples are well known to those skilled in the art, such as flow sorting (e.g., fluorescence-activated cell sorting; FACS) and magnetic particle sorting (e.g., magnetically activated cell sorting; MACS), microfluidic cell sorting, density gradient centrifugation, immunodensity cell isolation, expansion in cell culture based on growth factors and other components in the medium. In some preferred embodiments of the present disclosure, the isolating step is performed by fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS).

[0220] In embodiments in which the NKT-like cells are isolated from a sample derived from a subject, the sample may be selected from the group consisting of blood, plasma, a tumor biopsy or surgically harvested tumor, bone marrow, liver, spleen biopsy and adipose or adipose tissue.

[0221] In some embodiments, the isolating step may be performed at least about 1, 3, 12, 24, 48, 72, 96, 120, 144, or 168 hours after administration of the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent. In some embodiments, the isolating step may be performed at least about 1, 3, 8, 9, 10, 11, 12, 13, 14, or 15 days after administration of the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent. In some preferred embodiments, the isolating step is performed at least about 48 hours after said administration. In some other preferred embodiments, the isolating step is performed about 1, 3, or 48 hours after said administration. In some embodiments, the isolating step may be performed between about 1, 3, or 48 hours and 13 days, between about 1, 3, or 48 hours and 168 hours, between about 1, 3, or 48 hours and 120 hours, between about 1, 3, or 48 hours and 96 hours, or between about 1, 3, or 48 hours and 72 hours after administration of the glucocorticoid receptor (GR) modulating agent or ICAM3 modulating agent. In some preferred embodiments, the isolating step is performed between about 1, 3, or 48 hours and 72 hours after said administration. In some embodiments, the isolating step may be performed within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after glucocorticoid administration. In some preferred embodiments, the isolating step may be performed within 3 hours after glucocorticoid administration. In some particularly preferred embodiments, the isolating step may be performed within 1 hour after glucocorticoid administration. In other particularly preferred embodiments, the isolating step may be performed between 30 and 60 minutes after administration of the glucocorticoid. In some preferred embodiments, where the subject has cancer, an infectious disease, or an autoimmune disease, the isolating NKT-like cells may be performed on a blood sample obtained from the subject within 3 hours after administration of the glucocorticoid, preferably within 1 hour after administration of the glucocorticoid, for example between 30 and 60 minutes after administration of the glucocorticoid.

[0222] In some preferred embodiments of the methods that include an isolating step, the subject may be a healthy subject, such as a healthy adult human subject. In this context, a healthy subject is one that is not afflicted by a disease.

[0223] The isolated NKT-like cells and isolated NKT-like cell populations of the present disclosure can be expanded in culture. Suitable methods and reagents for culturing and expanding cells are well known to those skilled in the art. For example, long-term culture with IL-2, soluble anti-CD28 antibody, anti-CD3 epsilon antibody, anti-TCR beta antibody, and glycosides such as KRN7000, PBS44, or PBS57 have been shown to generate robust expansion of NKT cells (Watarai et al 2008, incorporated herein by reference in its entirety). Thus, in some embodiments of the methods of the present disclosure, the method of generating a population of NKT-like cells may further comprise a step of expanding the NKT-like cells or the NKT-like cells isolated by the isolating step. In some embodiments of the methods of the present disclosure, the method may further comprise a step of activating the isolated cells (either before or after the expanding step) with an NKT cell activator, a T cell activator, and / or an NK cell activator, which may be as described in detail above.

[0224] In some embodiments, after isolating NKT-like cells or a population of NKT-like cells from a subject or from a sample derived from a subject, the method of the present disclosure may further comprise introducing a nucleic acid encoding a protein into the isolated cell(s). Suitable methods for introducing nucleic acid into cells are well known to those skilled in the art, for example, physical or chemical methods including electroporation, sonoporation, cell microinjection, microparticle delivery, calcium phosphate-mediated transfection and liposome-based transfection, or viral transduction. After introducing the nucleic acid encoding the protein, the cell(s) may be cultured under conditions that promote the expression of the encoded protein. Suitable methods, reagents and conditions for culturing cells are well known to those skilled in the art. The cell(s) into which the nucleic acid encoding the protein has been introduced may be referred to as transfected cell or transformed cell herein.

[0225] In some embodiments of the present disclosure, the nucleic acid encoding a protein is a nucleic acid encoding a protein selected from the group consisting of one or more of a T cell receptor (TCR), a chimeric antigen receptor (CAR), a split, and universal and programmable CAR (SUPRA-CAR).

[0226] After isolation, the NKT-like cells of the present disclosure can be genetically engineered to target specific targets. For example, the cells can be expanded with IL-2, activated with GalCer (galactosylceramide), pulsed with autologous irradiated PBMC, and then transduced to express CAR or recombinant TCR (rTCR). The CAR or rTCR can specifically bind to a target selected from GD2 (disialoganglioside) and CD19. For example, the CAR can be NCT03294954 (specifically binds to GD2) or NCT03774654 (specifically binds to CD19).

[0227] Additionally, isolated cells can undergo targeted activation. For example, the following procedures can be utilized: nanovectors for passive and active delivery; a-GalCer-loaded APCs for targeted activation of NKT-like cells to tumors; iv administration of α-GalCer; and / or bulk PBMC stimulation (2-3 times) by addition of α-GalCer to cultured cells (to generate an iNKT cell enriched population that can then be infused back into the patient).

[0228] Additionally, isolated cells can be directly linked to tumor targeting moieties (either on the tumor cells or on the TME). Chemical modification of stimulators for NKT cells, T cells and NK cells (polarization of immune responses with α-GalCer analogs) can also be used.

[0229] The term "chimeric antigen receptor" (CAR), as used herein, refers to, but is not exclusive to, constructs that contain an antigen-binding domain of an antibody fused to a potent T cell activator domain. T cells modified with CAR constructs can bind antigen and can be stimulated to attack the bound cell. Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, chimeric antigen receptors (CARs)) are engineered receptors that graft any specificity onto immune effector cells. The receptors are called chimeric because they are composed of parts from different sources. The receptors / ligands or antibodies expressed by chimeric antigen receptor T cells or cellular immunotherapy can be mono- or bispecific or multispecific.

[0230] In some embodiments, the TCR, CAR, and / or SUPRA-CAR is selected from the group consisting of proto-oncogene tyrosine protein kinase ABL1, citrullinated antigen, ErbB2 / HER2, CD16, WT-1, KRAS, glypican 3, CD3, CD20, CD226, CD155, CD123, HPV-16 E6, Melan-A / MART-1, TRAIL bound to the DR4 receptor, LMP, MTCR, ESO, NY-ESO-1, gp100, 4SCAR-GD2 / CD56, mesothelin (CAK1 antigen or prepro-megakaryocyte potentiating factor or MSLN); DNA synthesis inhibitors; histamine H1 receptor (HRH1) antagonists; prostaglandin G / H synthase 2 (cyclooxygenase 2 or COX2 or prostaglandin endoperoxide synthase 2 or PHS II or prostaglandin H2 synthase 2 or PTGS2 or EC 1.14.99.1) inhibitors, CD19 (B-lymphocyte surface antigen B4 or differentiation antigen CD19 or T-cell surface antigen Leu 12 or CD19), cell adhesion molecule 5 (carcinoembryonic antigen or CEA or meconium antigen 100 or CD66e or CEACAM5); interleukin 2 receptor (IL2R) agonists, epidermal growth factor receptor (proto-oncogene c ErbB 1 or receptor tyrosine protein kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1); DNA ligase (EC 6.5.1.) inhibitors; DNA ligase (EC 6.5.1.), DNA polymerase alpha (POLA or EC 2.7.7.7) inhibitors; DNA primase (EC 2.7.7.6) inhibitors; ribonucleoside diphosphate reductase (ribonucleotide reductase or RRM or EC 1.17.4.1) inhibitors;RNA polymerase II (RNAP II or Pol II or EC 2.7.7.6) inhibitors, DNA polymerase (EC 2.7.7.7) inhibitors;DNA topoisomerase II (EC 5.99.1.3) inhibitors;CD22, meso, DNA primase (EC 2.7.7.6);Programmed cell death 1 ligand 1 (PD L1 or B7 homolog 1 or CD274) inhibitors;RNA polymerase II (RNAP II or Pol II or EC 2.7.7.6), histone lysine N-methyltransferase EZH2 (ENX1 or enhancer of zeste homolog 2 or lysine N-methyltransferase 6 or EZH2 or EC 2.1.1.43) inhibitor; programmed cell death 1 ligand 1 (PD-L1 or B7 homolog 1 or CD274), CXC chemokine receptor type 4 (FB22 or fusin or HM89 or LCR1 or leukocyte-derived seven transmembrane domain receptor or lipopolysaccharide-related protein 3 or stromal cell-derived factor 1 receptor or NPYRL or CD184 or CXCR4) antagonist; granulocyte colony-stimulating factor receptor (CD114 or GCSFR or CSF3R) agonist, adenosine deaminase (adenosine aminohydrolase or ADA or EC 3.5.4.4) inhibitors; cytotoxic to cells expressing tumor necrosis factor receptor superfamily member 17 (B-cell maturation antigen or CD269 or TNFRSF17), inactive tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor related 1 or ROR1 or EC 2.7.10.1); T-cell surface glycoprotein CD3 epsilon chain (T-cell surface antigen T3 / Leu 4 epsilon chain or CD3E); dihydrofolate reductase (DHFR or EC 1.5.1.3) inhibitors; ephrin type A receptor 2 (epithelial cell kinase or tyrosine protein kinase receptor ECK or EPHA2 or EC 2.7.10.1) inhibitors; glucocorticoid receptor (GR or nuclear body subfamily group 3C member 1 or NR3C1) agonists; mast cell / stem cell growth factor receptor Kit (proto-oncogene c Kit or tyrosine protein kinase Kit or v Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog or spotted protein or p145 c Kit or CD117 or KIT or EC 2.7.10.1) inhibitor;Platelet-derived growth factor receptor beta (Beta-type platelet-derived growth factor receptor or CD140 antigen-like family member B or Platelet-derived growth factor receptor 1 or CD140b or PDGFRB or EC 2.7.10.1) inhibitors; tubulin inhibitors; tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Fyn (proto-oncogene Syn or proto-oncogene c Fyn or Src-like kinase or p59 Fyn or FYN or EC 2.7.10.2) inhibitors; tyrosine protein kinase Lck (leukocyte C-terminal Src kinase or protein YT16 or proto-oncogene Lck or T-cell specific protein tyrosine kinase or lymphocyte cell specific protein tyrosine kinase or p56 LCK or LCK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Yes (proto-oncogene c Yes or p61 Yes or YES1 or EC 2.7.10.2) inhibitors, tumor necrosis factor (cachectin or TNF alpha or tumor necrosis factor ligand superfamily member 2 or TNF a or TNF) inhibitors, signal transducer and activator of transcription 3 (acute phase response factor or DNA-binding protein APRF or STAT3) inhibitors, Bcr-Abl tyrosine kinase (EC 2.7.10.2) inhibitors; dihydrofolate reductase (DHFR or EC 1.5.1.3); ephrin type A receptor 2 (epithelial cell kinase or tyrosine protein kinase receptor ECK or EPHA2 or EC 2.7.10.1); obesity / stem cell growth factor receptor Kit (proto-oncogene c Kit or tyrosine protein kinase Kit or v Kit or Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog or speckled plasma protein or p145 c Kit or CD117 or KIT or EC 2.7.10.1); platelet-derived growth factor receptor beta (beta-type platelet-derived growth factor receptor or CD140 antigen-like family member B or platelet-derived growth factor receptor 1 or CD140b or PDGFRB or EC 2.7.10.1); tubulin; tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Fyn (proto-oncogene Syn or proto-oncogene c Fyn or Src-like kinase or p59 Fyn or FYN or EC 2.7.10.2) inhibitors; tyrosine protein kinase Lck (leukocyte C-terminal Src kinase or protein YT16 or proto-oncogene Lck or T-cell specific protein tyrosine kinase or lymphoid cell specific protein tyrosine kinase or p56 LCK or LCK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Yes (proto-oncogene c Yes or p61 Yes or YES1 or EC 2.7.10.2) inhibitors, caspase 9 (apoptotic protease Mch 6 or apoptotic protease activating factor 3 or ICE-like apoptotic protease 6 or CASP9 or EC 3.4.22.62) activators; prostate stem cell antigen (PSCA), melanoma antigen preferentially expressed in tumors (cancer / testis antigen 130 or Opa interacting protein 4 or OIP4 or preferentially expressed antigen in melanoma or PRAME), signal transducer and activator of transcription 3 (acute phase response factor or DNA binding protein APRF or STAT3) inhibitor, CD44 antigen (CDw44 or Epican or extracellular matrix receptor III or GP90 lymphocyte homing / adhesion receptor or HUTCH I or heparan sulfate proteoglycan or Hermes antigen or hyaluronan receptor or phagocytic glycoprotein 1 or CD44), AXL (anexelekto) receptor tyrosine kinase, GAS6, TAM receptor tyrosine kinase, TYRO-3 (also known as Brt, Dtk, Rse, Sky and Tif), AXL (also known as Ark, Tyro7 and Ufo) and MER (also known as Eyk, Nym and Tyro12), CTLA4, tumor necrosis factor receptor superfamily member 8 (CD30L receptor or Ki 1 antigen or lymphocyte activation antigen CD30 or CD30 or TNFRSF8), caspase 9 (apoptotic protease Mch 6 or apoptotic protease activating factor 3 or ICE-like apoptotic protease 6 or CASP9 or EC 3.4.22.62) activator; cytotoxic to cells expressing ganglioside GD2; prostaglandin G / H synthase 1 (cyclooxygenase 1 or COX1 or prostaglandin endoperoxide synthase 1 or prostaglandin H2 synthase 1 or PTGS1 or EC 1.14.99.1) inhibitor; cytokine, interleukin, claudin 6 (Skullin or CLDN6), NKG2D, MICA, MICB and ULBP 1-6, NKp30, B7H6 (NCR3LG1), Bag6, B7 family, CD40 ligand (T cell antigen Gp39 or TNF-related activating protein or tumor necrosis factor ligand superfamily member 5 or CD154 or CD40LG) activator; interleukin 12 (IL12) activator, interleukin 3 receptor subunit alpha (IL3RA), obesity / stem cell growth factor, receptor Kit (proto-oncogene c Kit or tyrosine protein kinase Kit or v Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog or spotted protein or p145 c Kit or CD117 or KIT or EC 2.7.10.1) antagonist; proto-oncogene tyrosine protein kinase receptor Ret (cadherin family member 12 or proto-oncogene c Ret or RET or EC 2.7.10.1) inhibitor; receptor tyrosine protein kinase FLT3 (FMS-like tyrosine kinase 3 or FL cytokine receptor or stem cell tyrosine kinase 1 or fetal liver kinase 2 or CD135 or FLT3 or EC 2.7.10.1) antagonist; vascular endothelial growth factor receptor 1 (Fms-like tyrosine kinase 1 or tyrosine protein kinase receptor FLT or tyrosine protein kinase FRT or vascular permeability factor receptor or VEGFR1 or FLT1 or EC 2.7.10.1) Antagonists; Vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insert domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1) antagonists; vascular endothelial growth factor receptor 3 (Fms-like tyrosine kinase 4 or tyrosine protein kinase receptor FLT4 or VEGFR3 or FLT4 or EC 2.7.10.1) antagonist, caspase 9 (apoptotic protease Mch 6 or apoptotic protease activating factor 3 or ICE-like apoptotic protease 6 or CASP9 or EC 3.4.22.62) activator, cytotoxic T lymphocyte protein 4 (cytotoxic T lymphocyte-associated antigen 4 or CD152 or CTLA4) antagonist, myeloid cell surface antigen CD33 (sialic acid-binding Ig-like lectin 3 or gp67 or CD33), hepatocyte growth factor receptor (proto-oncogene c Met or tyrosine protein kinase Met or HGF / SF receptor or scatter factor receptor or MET or EC 2.7.10.1), epithelial cell adhesion molecule (adenocarcinoma-associated antigen or cell surface glycoprotein Trop 1 or epithelial cell surface antigen or epithelial glycoprotein 314 or KS 1 / 4 antigen or KSA or tumor-associated calcium signaling factor 1 or CD326 or EPCAM), ganglioside GD2, Lewis Y antigen (CD174), latent membrane protein 1 (protein p63 or LMP1), mucin 1 (breast cancer associated antigen DF3 or episialin or H23AG or Krebs-von-den-Lungen 6 or PEMT or peanut-reactive urinary mucin or polymorphic epithelial mucin or tumor-associated epithelial membrane antigen or tumor-associated mucin or CD227 or MUC1), T cell receptor beta 1 chain C region (TRBC1), vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insert domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1), BCMA, PD-1, interleukin-6 Receptor, NKR2, CX-072, T-lymphocyte protein 4 (cytotoxic T-lymphocyte-associated antigen 4 or CD152 or CTLA4) antagonist; Serine / threonine protein kinase B Raf (p94 or proto-oncogene B Raf or v Raf murine sarcoma viral oncogene homolog B1 or BRAF or EC 2.7.11.1) inhibitors, mucin 16 (ovarian cancer associated tumor marker CA125 or ovarian cancer antigen CA125 or MUC16); Bcr-Abl tyrosine kinase (EC 2.7.10.2) inhibitors; tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Fyn (proto-oncogene Syn or proto-oncogene c Fyn or Src-like kinase or p59 Fyn or FYN or EC 2.7.10.2) inhibitors; tyrosine protein kinase Lck (leukocyte C-terminal Src kinase or protein YT16 or proto-oncogene Lck or T-cell specific protein tyrosine kinase or lymphocyte cell specific protein tyrosine kinase or p56 LCK or LCK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Yes (proto-oncogene c Yes or p61 Yes or YES1 or. or EC 2.7.10.2) inhibitors, cyclin-dependent kinase 1 (p34 protein kinase or cell division protein kinase 1 or cell division control protein 2 homolog or CDK1 or EC 2.7.11.22 or EC 2.7.11.23) inhibitors;cyclin-dependent kinase 2 (p33 protein kinase or cell division protein kinase 2 or CDK2 or EC 2.7.11.22) inhibitors;granulocyte-macrophage colony-stimulating factor receptor subunit alpha (CDw116 or CD116 or CSF2RA) agonists, EGF VIII, tyrosine protein kinase SYK (spleen tyrosine kinase or p72 Syk or SYK or EC 2.7.10.2) inhibitors, alpha fetoprotein (alpha 1 fetoprotein or alpha fetoglobulin or AFP), cancer / testis antigen 1 (autoimmunogenic cancer / testis antigen or cancer / testis antigen 6.1 or L antigen family member 2 or CTAG1A or CTAG1B); HBV antigen, EGFR family member, Herin, tyrosine protein kinase BTK (Bruton's tyrosine kinase or B-cell precursor kinase or agammaglobulinemia tyrosine kinase or BTK or EC 2.7.10.2) inhibitors, CD4, epithelial cell adhesion molecule (adenocarcinoma-associated antigen or cell surface glycoprotein Trop 1 or epithelial cell surface antigen or epithelial glycoprotein 314 or KS 1 / 4 antigen or KSA or tumor-associated calcium signaling factor 1 or CD326 or EPCAM), prolyl endopeptidase FAP (melanoma membrane-associated gelatinase of 170 kDa or dipeptidyl peptidase FAP or integral membrane serine protease or fibroblast activation protein alpha or gelatinolytic protease FAP or seprase or FAP or EC 3.4.21.26 or EC 3.4.14.5), neural cell adhesion molecule 1 (antigen recognized by monoclonal antibody 5.1H11 or CD56 or NCAM1); epidermal growth factor receptor (proto-oncogene c ErbB 1 or receptor tyrosine protein kinase erbB 1 or HER1 or ERBB1 or EGFR or EC2.7.10.1) antagonists, tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor related 1 or ROR1 or EC 2.7.10.1); Wilms tumor protein (WT33 or WT1); interleukin 13 receptor subunit alpha 2 (interleukin 13 binding protein or CD213a2 or IL13RA2), trophoblast glycoprotein (M6P1 or 5T4 oncofetal antigen or 5T4 oncofetal trophoblast glycoprotein or Wnt-activated inhibitory factor 1 or TPBG), SLAM family member 7 (CD319 or membrane protein FOAP 12 or CD2-like receptor-activated cytotoxic cells or novel Ly9 or protein 19A or CD2 subset 1 or CS1 or SLAMF7), B-cell lymphoma 2 (Bcl 2) Inhibitors; DNA (cytosine 5) methyltransferase 1 (CXXC-type zinc finger protein 9 or DNA methyltransferase HsaI or MCMT or DNMT1 or EC 2.1.1.37) inhibitors, ROR1, CD19&CD40L, avidin (EGFRiiiv), folate receptor, CD30, pmel CD*8 T, CD33, NKR2, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen, aberrant product of ras, p53, alpha fetoprotein (AFP), CA-125, CA15-3, CA27-29, CA19-9, calcitonin, calretinin, CD34, CD99MIC 2, CD117, chromogranin, cytokeratin (various types: TPA, TPS, Cyfra21-1), desmin, epithelial membrane antigen (EMA), factor VIII, CD31 FL1, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45, human chorionic gonadotropin (hCG), immunoglobulins, inhibin, keratin (various types), lymphocyte markers (various types), BCR-ABL, MyoD1, Muscle-specific actin (MSA), Neurofilament, Neuron-specific enolase (NSE), Placental alkaline phosphatase (PLAP), Prostate-specific antigen (PSA), PTPRC (CD45), S100 protein, Smooth muscle actin (SMA), Synaptophysin, Thymidine kinase, Thyroglobulin (Tg), Thyroid transcription factor-1 (TTF-1), Tumor M2-PK, Vimentin, SV40, Adenovirus E1b-58kd, IGF2B3, Ubiquitous (low levels), Kallikrein 4, KIF20A, Lengsin, Meloe, MUC5AC, Immature laminin receptor, Tag-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, LAGE-1, PRAME, SSX-2, pmel17, tyrosinase, TRP-1 / -2, P. polypeptide, MC1R, β-catenin, prostate-specific antigen, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, Ras, TGF-beta receptor II, T cell receptor (TCR), BLOC1S6, CD10 / neprilysin , CD24, CD248, CD5 / cluster of differentiation 5, CD63 / Tspan-30 / tetraspanin-30, CEACAM5 / CD66e, CT45A3, CTAG1A, CXORF61, DSE, GPA33, HPSE, KLK3, LCP1, LRIG3, LRRC15, megakaryocyte potentiating factor, MOK, MUC4, NDNL2, OCIAD1, PMPCB, PTOV1, RCAS1 / EBAG9, RNF43, ROPN1, RPLP1, SARNP, SBEM / MUCL1, TRP1 / TYRP1, CA19-9, inactive tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor-related 1 or ROR1 or EC 2.7.10.1), ALK tyrosine kinase receptor (anaplastic lymphoma kinase or CD246 or ALK or EC2.7.10.1), prostate stem cell antigen (PSCA), melanoma antigen preferentially expressed in tumors (cancer / testis antigen 130 or Opa interacting protein 4 or OIP4 or preferentially expressed antigen in melanoma or PRAME), signal transducer and activator of transcription 3 (acute phase response factor or DNA-binding protein APRF or STAT3) inhibitor, CD44 antigen (CDw44 or Epican or extracellular matrix receptor III or GP90 lymphocyte homing / adhesion receptor or HUTCH I or heparan sulfate proteoglycan or Hermes antigen or hyaluronan receptor or phagocytic glycoprotein 1 or CD44), CD40 ligand (T cell antigen Gp39 or TNF-related activator protein or tumor necrosis factor ligand superfamily member 5 or CD154 or CD40LG) activator; tumor necrosis factor receptor superfamily member 13B (transmembrane activator and CAML interactor or CD267 or TACI or TNFRSF13B); tumor necrosis factor receptor superfamily member 17 (B cell maturation antigen or CD269 or TNFRSF17), CD276 antigen (B7 homolog 3 or 4Ig B7 H3 or costimulatory molecule or CD276), myeloid cell surface antigen CD33 (sialic acid-binding Ig-like lectin 3 or gp67 or CD33), ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1 (cyclic ADP-ribose hydrolase 1 or T10 or 2'phospho ADP-ribosyl cyclase / 2'phosphocyclic ADP-ribose transferase or ADP-ribosyl cyclase 1 or CD38 or EC 3.2.2.6 or EC 2.4.99.20), C-type lectin domain family 14 member A (epidermal growth factor receptor 5 or EGFR5 or CLEC14A), hepatocyte growth factor receptor (proto-oncogene c Met or tyrosine protein kinase Met or HGF / SF receptor or scatter factor receptor or MET or EC 2.7.10.1), epithelial cell adhesion molecule (adenocarcinoma-associated antigen or cell surface glycoprotein Trop or EC 2.7.10.2), 1 or epithelial cell surface antigen or epithelial glycoprotein 314 or KS1 / 4 antigen or KSA or tumor-associated calcium signaling factor 1 or CD326 or EPCAM), ganglioside GD3, interleukin 13 receptor subunit alpha 2 (interleukin 13 binding protein or CD213a2 or IL13RA2); kappa myeloma antigen (KMA), lambda myeloma antigen (LMA), latent membrane protein 1 (protein p63 or LMP1), melanoma-associated antigen, T lymphocyte activation antigen CD80 (activating B7-1 antigen or CTLA 4 counterreceptor B7.1 or CD80); cytotoxic to cells expressing T lymphocyte activation antigen CD86 (activating B7-2 antigen or CTLA 4 counterreceptor B7.2 or CD86), inactive tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor-related 1 or ROR1 or EC 2.7.10.1), Fas apoptosis inhibitory molecule 3 (IgM Fc fragment receptor or regulator of Fas-induced apoptosis Toso or TOSO or FAIM3 or FCMR), T cell receptor beta 1 chain C region (TRBC1), vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insert domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1), alpha fetoprotein (alpha 1 fetoprotein or alpha fetoglobulin or AFP), cancer / testis antigen 1 (autoimmunogenic cancer / testis antigen NY ESO 1 or cancer / testis antigen 6.1 or L antigen family member 2 or CTAG1A or CTAG1B), T cell surface glycoprotein CD5 (lymphocyte antigen T1 / Leu 1 or CD5), prolyl endopeptidase FAP (melanoma membrane-associated gelatinase of 170 kDa or dipeptidyl peptidase FAP or integral membrane serine protease or fibroblast activation protein alpha or gelatinolytic protease FAP or seprase or FAP or EC 3.4.21.26 or EC3.4.14.5), neural cell adhesion molecule 1 (antigen recognized by monoclonal antibody 5.1H11 or CD56 or NCAM1), C-type lectin domain family 12 member A (myeloid inhibitory C-type lectin-like receptor or dendritic cell-associated lectin 2 or C-type lectin-like molecule 1 or CLEC12A), integrin alpha V (vitronectin receptor subunit alpha or CD51 or ITGAV); cytotoxic to cells expressing integrin beta 6 (ITGB6), interleukin-13 receptor subunit alpha 2 (interleukin-13 binding protein or CD213a2 or IL13RA2), trophoblast glycoprotein (M6P1 or 5T4 carcinoembryonic antigen or 5T4 oncofetal trophoblast glycoprotein or Wnt-activated inhibitor of factor 1 or TPBG), trophoblast glycoprotein (M6P1 or 5T4 oncofetal antigen or 5T4 oncofetal trophoblast glycoprotein or Wnt-activated inhibitor of factor 1 or TPBG), C-type lectin domain family 12 member A (myeloid inhibitory C-type lectin-like receptor or dendritic cell-associated lectin 2 or C-type lectin-like molecule 1 or CLEC12A), SLAM family member 7 (CD319 or membrane protein FOAP 12 or CD2-like receptor-activated cytotoxic cells or novel Ly9 or protein 19A or CD2 subset 1 or CS1 or SLAMF7), SLAM family member 7 (CD319 or membrane protein FOAP12 or CD2-like receptor-activated cytotoxic cell or novel Ly9 or protein 19A or CD2 subset 1 or CS1 or SLAMF7), immunoglobulin, multidrug resistance-associated protein 3 (MRP3), proto-oncogene tyrosine protein kinase ABL1, prostatic acid phosphatase, OY-TES-1, ACSM2A, alpha-actinin-4, perilipin-2, alpha-fetoprotein, lymphoid blast crisis oncogene (Lbc) oncoprotein, aldehyde dehydrogenase 1 family member A1 (ALDH1A1), AML, ANKRD17, NY-BR-1, annexin II, ARHGAP17, ARHGAP30, ARID1B, endoplasmic reticulum resident protein, 5'-aminoimidazole-4-carboxamide-1-beta-d-ribonucleotide transferrin nicase (AICRT / I), ATR, ATXN2, ATXN2L, BAGE1, BCL11A, Bcl-xL, breakpoint cluster region, survivin, Livin / ML-IAP, HM1.24, BTB domain containing 2 (BTBD2), C6ORF89, carbonic anhydrase IX, CLCA2, CRT2, CAMEL, CAN protein, caspase-5, caspase-8, KM-HN-1, CCDC88 B, cyclin B1, cyclin D1, CCNI, CDC2, CDC25A, CDC27, CDK12, intestinal carboxylesterase, CEP95, CHAF1A, coactosin-like 1, CPSF, CRYBA1, TRAG-3, macrophage colony-stimulating factor, CSNK1A1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), cathepsin H, Kitakyushu lung cancer antigen 1, P450 1B1 or CYP1B1, DDR1, DEK oncogene, DEK-CAN, Dickkopf-1 (DKK1), DNAJC8, DSCAML1, EEF2, elongation factor Tu GTP-binding domain-containing or SNRP116, EIF4EBP1, human Mena (Mena) protein, EP300, ETV5, TEL1 or ETV6, polycomb group protein enhancer of zeste homolog 2 (EZH2), F2R, F4.2, FAM53C, Fibroblast growth factor 5 or FGF5, formin-related protein in leukocytes 1 (FMNL1), fibromodulin (FMOD), FNDC3B, FKHR, GDP-L-fucose, GAS7, GFI1, GIGYF2, GPNMB, O, A1, GPSM3, GRK2, GRM5, H3F3A, HAUS3, HERC1, HERV-K-MEL, HIVEP2, HMGN, HMHA1, heme oxygenase-1 (HO-1), HNRPL, heparanase, HMSD-v-encoded mHA, HSPA1A, Hsp70, HSPB1, immediate early response gene X-1 (IEX-1), insulin-like growth factor (IGF)-II mRNA-binding protein 3 (IMP-3), IP6K1, IRS2, ITGB8, JUP, RU2AS, KANSL3, KLF10, KLF2, KLK4, KMT2A, K-ras, low-density lipid receptor (LDLR), LDLR-FUT, Mac-2-binding protein, KIAA0205, LPP, LRP1, LRRC41, LSP1, LUZP1, lymphocyte antigen 6 complex locus K (LY6K), MACF1, MAP1A, MAP3K11, MAP7D1, maltrin-2, Mcl-1, MDM2, malic enzyme, MEF2D, MEFV, milk fat globule membrane protein BA46 (lactadherin), melanotransferrin, GNT-V or N-acetylglucosaminyltransferase nsferase V, MIIP, MMP14, matrix metalloproteinase-2, MORC2, melanoma antigen p15, MUC2, MUM, MYC, MYL9, non-conventional myosin class I gene, N4BP2, NCBP3, NCOA1, NCOR2, NFATC2, NFYC, NIFK, ninein, NPM, NPM1-ALK1, N-ras, OAS3, P polypeptide, OGT, OS-9, ErbB3-binding protein 1, PAGE-4, P21-activated serine kinase 2 (PAK2), neo-PAP, PARP12, PAX3, PAX3-FKHR, PCBP2, phosphoglycerate kinase 1 (PKG1), PLEKHM2, promyelocytic leukemia or PML, PML-RARA, POLR2A, cyclophilin B, PPP1CA,PPP1R3B, Peroxiredoxin 5, Proteinase 3, Parathyroid Hormone-Related Protein (PTHrP), Receptor-Like Protein Tyrosine Phosphatase Kappa, MG50, NY-MEL-1 or RAB38, RAGE, RALGAPB, RAR alpha, RBM, RCSD1, Recoverin, RERE, RGS5, RHAMM / CD168, RPA1, Ribosomal Protein L10a, Ribosomal Protein S2, RREB1, RSRP1, RTCB, SART, SCAP, Mammaglobin A, Secernin 1, SDCBP, SETD2, SF3B1, renal ubiquitous protein 1, SIK1, SIRT2, SKI, hairpin binding protein, SLC35A4, prostein, SLC46A1, SNRPD1, SOGA1, SON, SOX10, SOX11, SOX2, SOX-4, sperm protein 17, SPEN, SRRM2, SRSF7, SRSF8, SSX1, SSX2 or HOM-MEL-40, SSX4, STAT1, STEAP, STRAP, ART-1, SVIL, HOM-TES-14 / SCP1, CD138, SYNM, SYN PO, SYT, SYT15, SYT-SSX1, SYT-SSX2, SZT2, TAPBP, TBC1D10C, TBC1D9B, hTERT, THNSL2, THOC6, TLK1, TNS3, TOP2A, TOP2B, ATP-dependent interferon responsive (ADIR), TP53, triosephosphate isomerase or TPI1, tropomyosin-4, TPX2, TRG, T-cell receptor gamma alternative reading frame protein (TARP), TRIM68, prostate-specific protein transient receptor potential-p8 (trp-p8), The antigen-binding domain may comprise an antigen that binds to an antigen selected from the group of receptors / ligands / targets consisting of TSC22D4, TTK protein kinase (TTK), thymidylate synthase (TYMS), UBE2A, ubiquitin conjugating enzyme variant Kua, COA-1, USB1, NA88-A, VPS13D, BING4, WHSC1L1, WHSC2, WNK2, WT1, XBP1, XPO1, ZC3H14, ZNF106, ZNF219, papillomavirus binding factor (PBF), E3 ubiquitin protein ligase UBR4.

[0231] In some embodiments of the present disclosure, the TCR, CAR and / or SUPRA-CAR may not comprise an antigen-binding domain that binds to an antigen selected from the above-listed group of receptors / ligands / targets.

[0232] In some preferred embodiments, the TCR, CAR and / or SUPRA-CAR may comprise an antigen binding domain that binds to an antigen selected from the group consisting of CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, Her2.

[0233] After introduction of the nucleic acid encoding the protein, the NKT-like cell(s) can be expanded in culture. Suitable methods and reagents for culturing and expanding cells are well known to those skilled in the art. After expansion, the method of the present disclosure may further comprise activating the cells with an NKT cell activator, a T cell activator and / or an NK cell activator. The NKT cell activator, the T cell activator and the NK cell activator may be as described in detail above.

[0234] In some embodiments, the cells or targeted cells of the present disclosure as described above are used to deliver a payload, e.g., nucleic acids, dsRNA, siRNA, microRNA, dsDNA, ssDNA, cDNA, rRNA, mRNA, tRNA, siRNA, dsRNAi, RNAi, organic compounds, cytotoxic drugs, antibodies, vedotin, ozogamicin, emtansine, deruxtecan, mertansine, mafodotin, tubulin inhibitors, monomethylauristatin-E (MMAE) and monomethylauristatin-F (MMAF), which are peptide analogs of dolastatin-10, maytansinoids, vinca alkaloids, calicheamicin, Diocarmycin, pyrrolobenzodiazepine dimers, talirine, tesirine, indolinobenzodiazepine pseudodimers, soravtansine, DM1, DM4, neurotransmitters, DNA intercalators, antimetabolites, endostatin, neurotrophins, chemotherapeutic or growth factors or antibodies, toxins, radioactivity, antibiotics, antifungals, antivirals, receptors, viruses, cytokines, lipids, chemokines, peptides and proteins, antiparasitic drugs, hormones, antigens, neuroactive agents, receptor agonists or antagonists, small molecules or any type of biological or biologically active payload can be delivered.

[0235] In some embodiments of the present disclosure, the cells of the present disclosure may be used to deliver a payload that is not one or more of the payloads listed above.

[0236] Also provided by the present disclosure are methods of treating cancer, autoimmune disease, or infectious disease (also called microbial disease) in a subject. In some embodiments, the method of treatment is a method of generating a population of NKT-like cells in a subject as described elsewhere herein. In some embodiments, the method of treatment is a method of mobilizing a population of NKT-like cells in a subject as described elsewhere herein. In these embodiments, the NKT-like cells can treat cancer, autoimmune disease, or infectious disease by one of the mechanisms described elsewhere herein. In other embodiments, the method of treatment is a method comprising administering to a subject a therapeutically effective dose of isolated NKT-like cells of the present disclosure. These may be any of the isolated NKT-like cells or populations of NKT-like cells outlined above, including expanded and non-expanded, and / or activated or non-activated, and / or transfected or non-transfected cells. In these embodiments, the subject, cancer, autoimmune disease, infectious disease, and / or mechanism of therapeutic efficacy may be as detailed above.

[0237] In embodiments where the method of treatment comprises administering to a subject a therapeutically effective dose of isolated NKT-like cells of the present disclosure, the subject to which the isolated cells are administered can be the same subject from which the cells were isolated. In such embodiments, the treatment may be referred to as autologous cell treatment. The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced, whether the individual is a human or other animal. In other embodiments where the method of treatment comprises administering to a subject a therapeutically effective dose of isolated NKT-like cells of the present disclosure, the subject to which the isolated cells are administered can be different from the subject from which the cells were isolated. In such embodiments, the treatment may be referred to as allogeneic cell treatment. The term "allogeneic" refers to any material derived from one individual that is subsequently introduced into another individual of the same species, whether the individual is a human or other animal. That is, in embodiments where the method of treatment comprises administering to a subject a therapeutically effective dose of isolated NKT-like cells of the present disclosure, the cells can be from either an autologous or allogeneic source. The therapeutic efficacy of a method where isolated NKT-like cells of the present disclosure are administered to a subject is described in Example 16.

[0238] The method of treating cancer, autoimmune disease or infectious disease in a subject according to the present disclosure may further comprise administering to the subject an NKT cell activator, a T cell activator and / or an NK cell activator. These may be as detailed above. The method of treating cancer, autoimmune disease or infectious disease in a subject according to the present disclosure may comprise administering to the subject a glucocorticoid or cell of the present disclosure in combination with one or more additional agents, such as an NKT cell activator, a T cell activator and / or a dendritic NK cell activator as outlined above, or a chemotherapeutic agent, such as an immune checkpoint inhibitor. In this context, "in combination with" may mean simultaneous administration or separate and / or sequential administration in any order.

[0239] As used herein, the term "administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of agents disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, agents disclosed herein may be administered by a route other than parenteral, for example, orally. Other non-parenteral routes include topical, epithelial or mucosal routes of administration, for example, intranasally, vaginally, rectally, sublingually or topically.

[0240] The phrase "systemic injection" as used herein relates to, but is not exclusive to, intravenous, intraperitoneal, subcutaneous, via nasal submucosa, lingual, via bronchoscopy, intravenous, intraarterial, intramuscular, intraocular, intrastriatal, subcutaneous, intradermal, via a dermal patch, via a skin patch, via a patch, into the cerebrospinal fluid, into the portal vein, into the brain, into the lymphatic system, intrathoracic, retroorbital, intradermal, into the spleen, into the lymph, among others.

[0241] The term "site of injection" as used herein relates to, but is not exclusive to, intratumor or intraorgans such as the kidney or liver or pancreas or heart or lung or brain or spleen or eye, intramuscular, intraocular, intrastriatal, intradermal, by skin patch, by patch, in the cerebrospinal fluid, in the brain, among others.

[0242] In some preferred embodiments of the present disclosure, the glucocorticoid receptor modulating agent may be administered orally. In embodiments in which the method of treatment of the present disclosure comprises administering to a subject a therapeutically effective dose of the isolated NKT-like cells of the present disclosure, the cells may be applied directly to the organ or tumor via, among others, collagen matrix, extracellular matrix composition, biopolymer microthreads made from fibrin or other extracellular matrix materials, patches containing extracellular matrix and biodegradable materials, fibrin patches, alginate or agarose-based patches, scaffolds composed of biodegradable physiologically inert materials that may non-exclusively involve components such as extracellular matrix materials and dextran that coat stem cells with organ-specific antigens or binding molecules, residual extracellular matrix also known as scaffolds or decellularized organs from ex vivo digested organ donors or cadaveric organs, and contact lenses. Preferably, the cells are administered to the subject by a method selected from the group consisting of intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, injection into the cerebrospinal fluid (CSF), direct injection into a tumor, or as a gel placed on or near a solid tumor.

[0243] In some embodiments of the present disclosure, the route of administration of the agents and cells disclosed herein may not be one or more of the routes listed above.

[0244] The present disclosure also provides a glucocorticoid receptor (GR) modulating agent and an ICAM3 modulating agent for use in a method of generating / mobilizing a population of NKT-like cells as detailed above. The present disclosure also provides a glucocorticoid receptor (GR) modulating agent and an ICAM3 modulating agent for use in a method of treating cancer, an autoimmune disease or an infectious disease (also called microbial disease) in a subject, the method of treatment being a method of generating / activating / mobilizing a population of NKT-like cells as detailed above. Preferred embodiments include a glucocorticoid for use in a method of generating and / or mobilizing a population of NKT-like cells as detailed above and a glucocorticoid for use in a method of treating cancer, an autoimmune disease or an infectious disease in a subject, the method of treatment being a method of generating and / or mobilizing a population of NKT-like cells in a subject as detailed above. Other preferred embodiments include a glucocorticoid for use in a method of mobilizing a population of NKT cells as detailed above. In some particularly preferred embodiments, the glucocorticoid is dexamethasone.

[0245] Also provided by the present disclosure is the use of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent in the manufacture of a medicament for use in a method of generating / mobilizing a population of NKT-like cells as detailed above.The present disclosure also provides the use of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent in the manufacture of a medicament for use in a method of treating cancer, an autoimmune disease or an infectious disease (also called microbial disease) in a subject, the method of treatment being a method of generating and / or mobilizing a population of NKT-like cells in a subject as detailed above.

[0246] The present disclosure also provides the use of a glucocorticoid receptor (GR) modulating agent or an ICAM3 modulating agent to induce and / or mobilize a population of NKT-like cells, the population of NKT-like cells being induced by a method of generating and / or mobilizing a population of cells in a subject as detailed above.

[0247] The present disclosure also provides a method for generating induced pluripotent stem cells (iPSCs), the method includes reprogramming the NKT-like cells of the present disclosure to generate iPSCs.The NKT-like cells of the present disclosure to be used in the method for generating iPSCs can be the NKT-like cells generated and isolated by the method as described above.

[0248] In some embodiments of the disclosed methods of generating iPSCs, reprogramming comprises introducing into the cells of the present disclosure one or more expression cassettes encoding Oct3 / 4, Klf4, Sox2 and c-myc. In some embodiments, reprogramming comprises introducing into the cells mRNA encoding Oct3 / 4, KLF4, Sox2 and c-myc. In some other embodiments of the disclosed methods of generating iPSCs, reprogramming may further comprise introducing into the cells one or more expression cassettes encoding one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog and / or LIN28. In other embodiments, reprogramming may further comprise introducing into the cells one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog and / or LIN28 encoding mRNA. Suitable methods for introducing expression cassette or encoding mRNA into cells are well known to those skilled in the art, for example, by electroporation, cell microinjection or liposome-based transfection methods.The use of retroviral systems, including lentivirus and adenovirus systems, to reprogram non-pluripotent cells into iPSCs has been described (Stadtfeld et al., 2008, incorporated herein by reference in its entirety).Reprogramming of adult cells into iPSCs can also be achieved by plasmids without using viral transfection systems (Okita et al., 2008, incorporated herein by reference in its entirety).

[0249] [Oct-3 / 4] Oct-3 / 4 (Pou5f1; cDNA available from Bioclone, San Diego, CA) is a member of the family of octamer ("Oct") transcription factors that play a critical role in maintaining pluripotency. Absence of Oct-3 / 4 in Oct-3 / 4+ cells, e.g., blastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation, whereas presence of Oct-3 / 4 thus gives rise to pluripotency and differentiation potential of embryonic stem cells. Various other genes in the "Oct" family, including the close relatives of Oct-3 / 4, Oct1 and Oct6, fail to induce induction, thus demonstrating that Oct-3 / 4 is exclusive to the induction process.

[0250] [Klf Family] Klf4, a member of the Klf family of genes, is a factor for the generation of mouse iPS cells. Klf2 (cDNA available from Bioclone, Inc., San Diego, CA) and Klf4 (cDNA available from Bioclone, Inc., San Diego, CA) are factors capable of generating iPS cells, as are the related genes Klf1 (cDNA available from Bioclone, Inc., San Diego, CA) and Klf5 (cDNA available from Bioclone, Inc., San Diego, CA), but with reduced efficiency.

[0251] [Sox Family] The Sox family of genes is associated with maintaining pluripotency similar to Oct-3 / 4, but with multipotent and unipotent stem cells, in contrast to Oct-3 / 4, which is expressed exclusively in pluripotent stem cells (Bowles et al, 2000, incorporated herein by reference in its entirety). Sox2 (cDNA available from Bioclone, Inc., San Diego, CA) was the first gene used for induction, and other genes in the Sox family have been found to act similarly in the induction process. Sox1 (cDNA available from Bioclone, Inc., San Diego, CA) results in iPS cells with similar efficiency as Sox2, and the genes Sox3 (human cDNA available from Bioclone, Inc., San Diego, CA), Sox15 and Sox18 also generate iPS cells, but with reduced efficiency.

[0252] [Myc family] The Myc family of genes are proto-oncogenes involved in cancer. C-myc (cDNA available from Bioclone, Inc., San Diego, CA) is a factor involved in the generation of mouse iPS cells. However, c-myc may be unnecessary for the generation of human iPS cells. The use of the "myc" family of genes in the derivation of iPS cells has raised questions about the fate of iPS cells as a clinical therapy, as 25% of mice transplanted with c-myc-induced iPS cells developed lethal teratomas. N-myc (cDNA available from Bioclone, Inc., San Diego, CA) and L-myc have been identified as inducing alternatives to c-myc with similar efficiency.

[0253] [Nanog] In embryonic stem cells, Nanog (cDNA available from Bioclone, Inc., San Diego, Calif.), along with Oct-3 / 4 and Sox2, is required in promoting pluripotency (Chambers et al., 2003, incorporated herein by reference in its entirety).

[0254] [LIN28] LIN28 (cDNA available from Bioclone, Inc., San Diego, CA) is an mRNA-binding protein expressed in embryonic stem cells and embryonic carcinoma cells associated with differentiation and proliferation (Moss & Tang, 2003, incorporated herein by reference in its entirety).

[0255] In some embodiments, the disclosed method of generating iPSCs further comprises inducing differentiation of the iPSCs of the present disclosure. In some preferred embodiments, the disclosed method may further comprise inducing differentiation of the iPSCs of the present disclosure into NKT cells. Thus, the present disclosure also provides a method of generating a population of NKT cells, the method comprising differentiating the iPSCs generated by the method of the present disclosure into an NKT cell lineage. In some embodiments, the disclosed method may further comprise inducing differentiation of the iPSCs of the present disclosure into T cells. Thus, the present disclosure also provides a method of generating a population of T cells, the method comprising differentiating the iPSCs generated by the method of the present disclosure into a T cell lineage. Such differentiated cells may be used in methods of treating cancer, autoimmune disease, or infectious disease (also called microbial disease) in a subject according to the present disclosure.

[0256] The present disclosure also provides an in vitro method of generating a population of natural killer T-cell-like cells (NKT-like cells), isolated NKT-like cells, or a population of isolated NKT-like cells, as disclosed elsewhere herein. The in vitro method of the present disclosure may include obtaining a CD3 high CD49b- cell or a population of cells, and contacting the CD3 high CD49b- cell(s) with one or more cytokines, where the contacting induces the cell(s) to become an NKT-like cell of the present disclosure. The NKT-like cell may be characterized by a pattern of expressed surface proteins, as described elsewhere herein. In some embodiments, the CD3 high CD49b- cell or a population of cells may be obtained from a subject, e.g., a human subject. The subject may be a subject as defined elsewhere herein. In some embodiments, the contacting may include contacting the cell(s) with an NKT cell activator, a T cell activator, and / or an NK cell activator, as described elsewhere herein. In some embodiments, the one or more cytokines may include one or more cytokines described elsewhere herein as NKT cell activators, T cell activators and / or NK cell activators. In some embodiments, the one or more cytokines may include one or more cytokines described elsewhere herein. In some embodiments, the one or more cytokines may include IL-2 and IFN gamma. In some embodiments, the one or more cytokines may include IL-2, IFN gamma and one or more additional cytokines. The in vitro method of the present disclosure may further include steps such as isolating, activating, expanding, introducing, genetically engineering to target, linking to tumor targeting moieties, etc. of nucleic acid as described elsewhere herein. The cell(s) generated by the in vitro method of the present disclosure may be used in methods of treatment in which NKT-like cells are administered to a subject, as well as in methods of generating induced pluripotent stem cells (iPSCs) as described elsewhere herein.

[0257] Also provided by the present disclosure are isolated NKT-like cells generated or mobilized by any of the methods disclosed herein, as well as populations of isolated NKT-like cells generated or mobilized by any of the methods disclosed herein.Also provided are NKT-like cells and isolated populations of NKT-like cells characterized by patterns of surface proteins as detailed elsewhere herein, as well as the use of such cells in the methods of treatment of the present disclosure. EXAMPLES

[0258] The following examples demonstrate that high-dose glucocorticoid receptor agonists can induce the generation and mobilization of novel NKT-like cell populations in subjects, including human immune system (HIS) mice and humans, in addition to causing near-complete lymphodepletion of peripheral blood lymphocytes (without affecting neutrophil, platelet, RBC and stem cell counts).

[0259] These examples also show that NKT-like cell populations induced by high-dose glucocorticoid receptor agonists, in addition to displaying known properties of NKT cells, have a novel expression pattern of surface proteins that enable these cells to directly phagocytose cancer cells and display enhanced cytotoxic efficacy against solid tumors.

[0260] High-dose glucocorticoid receptor agonists therefore represent a promising therapy for use in the treatment of cancer and diseases mediated by immune cells such as lymphocytes.

[0261] [Abbreviation] term: ·ab Alpha beta A20 Mouse B lymphoma AVM_NKT CD56+ gdTCR+ invTCR+ human cells BM Bone marrow ·CanMod Cancer Model ·CBC complete blood count ·CD Cluster of differentiation CD19 B lymphocyte marker CD3 T lymphocyte marker CD4 helper T lymphocyte marker CD45 white blood cell marker ·CD49b Mouse natural killer marker ·CD56 human natural killer marker CD8 Cytotoxic T lymphocyte marker ·CNS Central Nervous System CR Complete Response Cy / Flu Cyclophosphamide / Fludarabine ·DN Double Negative DP Dexamethasone Phosphate DOB Date of birth ·DOM date of manufacture ·DSP Dexamethasone Sodium Phosphate ·FDA U.S. Food and Drug Administration ·FSC Forward Scatter (cell size) ·gd Gamma Delta ·GMP Good Manufacturing Practice ·GP grandparents hCD45 Human CD45 HED Human Equivalent Dose Inv Invariant ·LyDep Lymphocyte Depletion Test mCD45 Mouse CD45 ·MCL Mantle cell lymphoma ·MFI Mean Fluorescence Intensity Neoadj Neoadjuvant NHL Non-Hodgkin's Lymphoma NK Natural Killer Cells ·NKT Natural Killer T Cells ·NKT new AVM NKT cells ·NOD non-obese diabetic mice ·PBS Phosphate buffered saline ·PFA Paraformaldehyde ·PR Partial Response R / R Relapsed / Refractory ·SBIR Small Business Innovation Research SD stable disease ·SSC Side Scatter (Cell Complexity) ·TCRα / β T cell receptor alpha beta ·TCRg / d T cell receptor gamma delta ·TCRinv T cell receptor invariant ·UC Umbilical cord ·WBC white blood cells

[0262] [material and method] Acute high dose dexamethasone may also be referred to herein as Dex, AugmenStem™, PlenaStem™, or AVM0703. The novel population of NKT-like cells induced following administration of acute high dose dexamethasone (AVM0703) may also be referred to herein as NKT cells or AVM-NKT cells.

[0263] For the initial lymphodepletion study, naïve C57BL / 6 mice were treated with DP at 18 mg / kg HED by oral gavage. Male C57BL / 6 mice were obtained from Taconic Bioscience (Germantown, NY) and acclimated to laboratory conditions for at least one week. Mice were orally dosed once with 18 mg / kg dexamethasone phosphate (DP) or placebo and maintained until the time points. Each dosing time point group was accompanied by a placebo group of the same age and condition according to Table 3. Time points 24 hours, 48 ​​hours, 72 hours, 5 days, 7 days, 11 days, and 13 days were dosed with GLP grade AVM0703 and placebo. Time points 6 hours, 21 days, 28 days, and 35 days were dosed with GMP grade AVM0703 and placebo. When mice reached the study time points, they were euthanized as follows: Mice were anesthetized with isoflurane gas. After anesthesia, blood was collected by cardiac puncture and immediately placed into a heparin-lined microtube. 10 mL of 5 U / mL heparin / PBS was used to inject by slow pushing for retrograde perfusion through the abdominal aorta to flush all residual blood from the vasculature. 250 uL of blood was then transferred to a lavender-lidded EDTA-lined microtube and shipped to Lynette Brown at Flow Contract Site Labs (Bothell, WA) for analysis by flow cytometry. The remaining blood was sent to Phoenix Labs (Mukilteo, WA) for complete blood count and clinical chemistry.

[0264] For characterization of induced / mobilized populations of NKT-like cells in humans (AVM-NKT), whole blood from human subjects was collected into K2 EDTA Vacutainer tubes (367862, BD Biosciences, NJ) and shipped to AVM Biotechnology at room temperature. 100 μl of whole blood was stained with the following antibodies: CD45 AF700 (2D1), CD16 APC (3G8), iNKT PECy7 (6B11), CD8 PE (SK1), CD14 FITC (M5E2), CD56 BV650 (5.1H11), γδTCR BV510 (B1), CD19 BV421 (HIB19), NKp44 APC (P44-8) (all from Biolegend, San Diego, CA), and CD3 APCVio770 (BW264 / 56), (Miltenyi Biotec, San Jose, CA), 7-AAD (Biolegend, San Diego, CA) were included to distinguish live and dead cells. Antibody staining was performed at room temperature for 15 min. Red blood cells present in the samples were lysed with BD FACS lysing solution (BD Biosciences, San Diego CA) for 10 min at room temperature, then washed and resuspended in 300 μl of 1X DPBS CMF. Healthy patient blood, unstained, and fluorescence minus one (FMO) from BloodWorks (Seattle, WA) were included as controls. 250 μl of the samples were analyzed on a MACSQuant 16 (serial number 40150, Miltenyi Biotec, San Jose, CA) flow cytometer. Data were analyzed for different immune populations using Kaluza 2.1 (Beckman Coulter Lifesciences, Indianapolis, IN) software.Combining data obtained from flow cytometry analyzed by Kaluza, the following distinct cell populations gated from live CD45 lymphocytes were determined: D3+ cells, CD8+ cytotoxic T cells, CD3+CD56+ (NKT cells) CD3-CD56+, CD3-CD56bright (NK cells) and CD3-CD19+ (B cells), CD3+γδTCR+ cells, CD3+γδTCR bright cells, CD3+iTCR+ cells, live WBC CD3-CD16bright granulocytes, live WBC CD3-CD14+ monocytes. Counts reported as %WBC and cells / μl.

[0265] A high-concentration, large-volume formulation of AVM0703 containing the active pharmaceutical ingredient, dexamethasone sodium phosphate, was used in the humanized mouse studies. AVM0703 contains 26.23 mg / mL dexamethasone sodium phosphate (equivalent to 24 mg / mL dexamethasone phosphate, DP), 10 mg / mL sodium citrate, 0.5 mg / mL edetate disodium, and 0.035 mg / mL sodium sulfite (anhydrous). The AVM0703 material used in these studies was GMP grade and manufactured by Hospira, Australia. All AVM0703 dosage information in this report is referred to dexamethasone phosphate. Female huNOG-EXL mice (n=6) were obtained from Taconic Bioscience (Germantown, NY). Female huCD34-NCG mice (n=8) were obtained from Charles River (Wilmington, MA). Mice from both facilities were allowed to acclimate to laboratory conditions for at least 5–6 days.

[0266] Mice were orally dosed three times with 32 mg / kg dexamethasone phosphate (DP) or placebo and maintained until time points. After the first dose (03 / 01 / 2021) and the second dose one week later (03 / 08 / 2021), blood was collected up to 70 uL per mouse by cheek puncture when mice reached the designated time points. Blood was analyzed by flow cytometry. After the third dose (04 / 05 / 2021), 28 days after the previous dose, mice were euthanized according to standard operating procedures summarized here when they reached the study time points. Mice were anesthetized with isoflurane gas. After anesthesia, blood was collected by cardiac puncture and at least 300 uL of blood was immediately placed into EDTA-lined microtubes and analyzed by flow cytometry, and 300-400 uL of blood was collected separately in standard microcentrifuge tubes and allowed to clot for serum collection.

[0267] 10 mL of 5 U / mL heparin / PBS was used for retrograde perfusion via the abdominal aorta with slow push injection to flush all remaining blood from the vasculature. Spleen, thymus, femur, and sternum were harvested for all mice. Spleen and thymus were processed into single cell suspensions for analysis by flow cytometry. Muscle was removed from bones using gauze soaked in 70% ethanol and processed for bone marrow for analysis by flow cytometry. Resection of pancreas and colon was performed following the video and instruction JoVE file. Feces were drained from the colon by administering ice-cold PBS through a 22-gauge stomach tube. Other organs such as small intestine, cecum, kidney, lung, and liver were also harvested. All organ weights were recorded after removing excess fluid from outside. Soft organs were first fixed in 4% PFA and transferred to 70% ethanol after 24 hours and stored at 4°C. Bones were directly stored in 70% ethanol at 4°C. Blood was processed for flow cytometry analysis and serum collection. No CBC was performed.

[0268] All flow cytometry was performed in-house. Blood, spleen, thymus and bone marrow were processed via standard staining protocols for flow cytometry. MACSQUANT 16 was used for flow cytometry. The antibody panel was slightly changed between the first, second and third doses. Markers in the flow panel after the first dose analysis included Live / Dead, hCD45, mCD45, hCD56, hTCRgd, hCD3, hCD8, hCD16, hCD19, hCD14. Markers in the flow panel after the second dose included all of the above plus hTCRab and hiTCR. Blood analysis after the second dose included 7AAD, CD14 and CD19 together in the dump channel. It was later discovered that this novel AVM-NKT may also have a unique CD19 and CD14 expression profile. In the third dose panel design, when full sac was performed, all the above markers were assigned to unique channels to avoid duplication, but TCRab was not included in the final panel due to open channel restrictions.

[0269] For cheek bleeds, 70ul of blood was collected into EDTA coated microtainers and processed for immunostaining. Antibodies used in the panel were mouse CD45 (clone:30F-11), human CD45 (clone:2D1); human CD3 (clone:HIT3a); human CD8 (clone:SK1); human CD16 (clone:3G8); human CD14 (clone:M5E2); human TCRgd (clone:B1); human CD56 (clone:5.1H11); human CD19 (clone:HIB19). Blood was stained for live / dead markers for 10 minutes at room temperature, then stained for the above surface antibodies for 20 minutes at room temperature in the dark. RBCs were lysed in BD FACS lysis buffer, washed in DPBS, and resuspended in 300ul DBPS. 250ul of sample was run on a MACSquant 16 flow cytometer.

[0270] Example 1 - Acute high-dose glucocorticoid receptor agonists nearly completely lymphodeplete peripheral blood lymphocytes but induce a unique population of NKT cells. In International Patent Application PCT / US2019 / 054395, the inventors present a series of experiments showing that high-dose glucocorticoid receptor agonists can almost completely lymphodeplete peripheral blood lymphocytes, reduce the number of germinal centers in lymphoid organs, and deplete thymic lymphocytes. These effects are achieved without substantially affecting the cell numbers of neutrophils, platelets, erythrocytes, and stem cells, both hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs).

[0271] Here, studies performed in naïve mice show that administration of high doses of glucocorticoid receptor agonists leads to almost complete lymphodepletion of peripheral blood lymphocytes without substantial effects on neutrophil, platelet, red blood cell (RBC) and stem cell (both HSC and MSC) cell numbers. Interestingly, high doses of glucocorticoid receptor agonists were also found to induce upregulation of NKT cells.

[0272] As shown in Figure 1, in naive mice, high-dose dexamethasone (18 mg / kg HED DP) significantly reduced absolute lymphocyte counts (ALCs minus NK and NKT cells) compared to placebo, an effect that persisted for up to 21 days after treatment. Near-complete lymphocyte depletion was observed 6 and 48 hours after treatment, an effect comparable to that achieved with standard Cy / Flu chemotherapy (13 mg / kg HED cyclophosphamide and 0.8 mg / kg HED fludarabine).

[0273] In naive mice, high-dose dexamethasone selectively ablates T and B lymphocytes (equivalent to standard Cy / Flu chemotherapy; Figure 2), monocytes (superior to Cy / Flu chemotherapy; Figure 3), and lymphodepletes neutrophils (Figure 4) at targeted clinical doses. Basophils (reduced only at 6 hours), eosinophils (reduced only at 24 and 48 hours), platelets (see Figure 5), and RBCs are all spared, while HSCs (Figure 6) and MSCs are either spared or increased (*p<0.05; #p<0.0001).

[0274] Surprisingly, high-dose dexamethasone was shown to also induce NKT upregulation (Figure 7) and the generation of a novel NKT cell population (AVM-NKT). When examined by flow cytometry, these novel AVM-NKT cells are CD49b+ and CD3 very bright (CD3highCD49b+). Previously reported NKT cells express CD3 with a MFI 1 log lower than AVM-NKT cells (CD3medCD49b+; Figure 8). AVM-NKT cells appear in the blood of naïve mice 48 hours after administration of high doses (HED 18.1 mg / kg) of the glucocorticoid receptor agonists dexamethasone and betamethasone, but are not induced by standard Cy / Flu chemotherapy.

[0275] Dose escalation studies have shown that a single dose of 6-12 mg / kg HED dexamethasone base can induce AVM-NKT cells. 15 mg / kg HED dexamethasone base induces particularly robust production of AVM-NKT cells, as does a 6+6 mg / kg HED dosing schedule of 6 mg / kg at time 0 and 6 mg / kg 24 hours later.

[0276] Example 2 - AVM-NKT cells are involved in T and B lymphocyte elimination in vivo. Mononuclear cells from peripheral blood or single-cell splenocytes from naive male C57Bl / 6 mice were incubated with AVM0703 at concentrations equivalent to the peak blood concentrations achieved by acute high-dose AVM0703 in vivo. No apoptosis was observed up to 72 hours after addition of AVM0703 to peripheral blood mononuclear cells or single-cell splenocytes in vitro. The lack of apoptosis in peripheral blood mononuclear cells or splenocytes in vitro indicates that lymphodepletion in vivo is primarily due to the induction of AVM-NKT cells.

[0277] Example 3 - AVM-NKT cells home to tumor sites. In a preliminary study, naïve C57Bl / 6 mice were treated with high-dose dexamethasone and peripheral blood was examined by flow cytometry at defined time intervals to characterize the different immune populations. After high-dose dexamethasone administration, two NKT populations were identified: NKT cells defined as CD3medCD49b+ and a novel population of AVM-NKT defined as CD3highCD49b+ (Figure 8).

[0278] AVM-NKT cells were found to appear in the blood of naive mice 48 hours after administration of suprapharmacologic doses (HED 18.1 mg / kg) of dexamethasone (AVM0703) or betamethasone. Conversely, these cells are not induced to any significant extent by standard Cy / Flu chemotherapy or methylprednisone.

[0279] As shown in Figure 9 and Table 2, in normal mice, AVM-NKT cells are induced in the spleen within 48 hours after dexamethasone administration, are evident in the peripheral blood starting 48 hours after dexamethasone administration, and continue to be found in the bloodstream up to 13 days after dexamethasone administration. AVM-NKT cells are not detectable in the spleens of naive placebo-treated mice. Cyclophosphamide / Fludarabine administration does not induce this novel NKT population. [Table 2]

[0280] In contrast to the time course of AVM-NKT upregulation observed in normal, disease-free mice, quantification of AVM-NKT cells in A20 B cell lymphoma tumor-bearing mice revealed the absence of AVM-NKT cells in the peripheral blood. Instead, in these tumor-bearing mice, AVM-NKT cells appear to home to the tumor site, where increased necrosis is evident when examined 48 hours after dexamethasone administration (Figure 10A). Additional time course studies demonstrated that AVM-NKT cells maximally cleared A20 lymphoma implanted in the flanks of mice within 3 hours after administration of 18 mg / kg HED dexamethasone phosphate, A20 metastases to blood and thymus were maximally cleared 24 hours after administration, and A20 metastases to bone marrow were maximally cleared 48 hours after administration (Figure 10B).

[0281] Consistent with this, high-dose dexamethasone was shown to significantly delay tumor growth in the A20 model (Figure 11; Example 6). Since A20 cells undergo only about 30% apoptosis 72 hours after high-dose dexamethasone treatment in vitro, AVM-NKT cells are likely to play a role in controlling tumor growth.

[0282] Two million A20 B-lymphoma cells, with a cell density of 1.8e7 cells / mL at harvest, were mixed with an equal volume of Matrigel (100 μl each) and injected subcutaneously into the left flank of BALB / c mice (200 μl total volume) to generate a solid tumor model of B-cell lymphoma. After tumors were established (approximately 7 days or approximately 100–150 mm 3 Mice were treated with 100 mg / kg of 10 ... 3The time when the mouse reached the study endpoint or when the body weight loss exceeded 20% was considered the study endpoint. Once the mouse reached the study endpoint, it was euthanized as follows: Mice were anesthetized with isoflurane gas. After anesthesia, blood was collected by cardiac puncture and perfused with 10 mL of 5 U / mL heparin / PBS. Tumors were excised from the right flank by peeling the skin over the right hind flank of the mouse. The skin was stretched and pinned in place, and the tumor was separated from the skin by gentle scraping with a scalpel. Tumors were fixed for 48 hours, then transferred to 70% ethanol and stored in cassettes at 4°C. Tumors were shipped to HistotoxLabs (Bolder, CO) for sectioning and staining. NKT cells in the tumor were identified by NKp46 staining.

[0283] [Example 4 - Hematological cancer increases the concentration of AVM-NKT cells in peripheral blood] Mice are inoculated with T-cell or B-cell lymphoma by tail vein injection of 1-5M lymphoma cells in logarithmic growth phase. Six hours to 13 days later, blood is collected from the mice and the number of AVM-NKT in the blood is determined by flow cytometry gating on cells that are CD3 very high (MFI at least 0.5 log higher than T lymphocytes) and CD49b positive, or by gating on NKp46. Compared to naive mice or mice bearing solid tumors such as T- or B-lymphoma cells encased in Matrigel and implanted subcutaneously in the flank, mice bearing circulating T- or B-lymphoma cells have significantly increased numbers of AVM-NKT in the peripheral blood.

[0284] Example 5 - AVM-NKT is induced in bone marrow and adipose tissue 48 hours after AVM0703 doses of approximately 29 mg / kg or more (as DP) in naive Balb / c mice. Balb / c mice have the MHC haplotype "d", and H-2K is d (H-2K d ) H-2D is d (H-2D d) H2-L is d (H-2L d) Aαβ is d, d. Eαβ is d, d. Mls1 is b. Mls2 is a. IA is d (I- Ad ) IE is d (I- Ed ) Qa-1 is b (Qa-1 b ) Qa-2 is a (Qa-2 a ).

[0285] C57Bl / 6 mice have the "b" MHC haplotype, and H-2K is the b (H-2K b ) H-2D is b (H-2D b) H2-L is null. Aαβ is b, b. Eαβ is b, b. Mls1 is b. Mls 2 is b. IA is b (I- Ab ) IE is null. Qa-1 is b (Qa-1 b ) Qa-2 is a (Qa-2 a ).

[0286] AVM NKT induced in naive Balb / c mice have high CD3 MFI, similar to peripheral blood AVM-NKT induced in naive C57Bl / 6 mice, and AVM-NKT in naive Balb / c mice are TCR gamma / delta positive. Many cells are NKp46 negative, indicating that they are not activated. This example shows that MHC expression can determine the target organ.

[0287] MHC may control the trafficking of AVM_NKT cells. AVM_NKT cells are present in the blood of naive AVM0703-treated male C57Bl6 mice. AVM_NKT cells are present in the fat and bone marrow of naive AVM0703-treated male Balb / c mice. AVM_NKT cells are present in the tumors of AVM0703-treated male tumor-bearing Balb / c mice. The novel NKT in naive Balb / c mice is also tCRgd positive, B220-, NKp46+ / -, Ly6G-, CD4-, CD8-, CD3high, MFI 10492, and CD49b+.

[0288] Example 6 - Acute high-dose dexamethasone has tumor-killing effects in T-cell and B-cell lymphoma, prevents or delays hyperglycemia in spontaneously diabetic NOD mice, and reverses diabetes in NOD mice with early-onset established diabetes. High-dose dexamethasone was shown to significantly delay tumor growth in the A20 B-cell lymphoma tumor model (Figure 11). A subsequent series of experiments (described in PCT / US2021 / 019773, the contents of which are incorporated herein by reference in their entirety) confirmed the tumor-killing effect of acute high-dose dexamethasone in the A20 B-cell lymphoma tumor model and a xenograft model of T-cell lymphoma (CCRF-CEM), and demonstrated the ability of high-dose dexamethasone to prevent hyperglycemia and reverse diabetes in NOD mice with early-onset established diabetes. [Table 3]

[0289] Example 7 - Identification of AVM-NKT cells in human subjects treated with acute high-dose dexamethasone. Following the identification of AVM-NKT cells in mice, data on file from human subjects treated with high-dose dexamethasone were reanalyzed.

[0290] In patients with osteoarthritis, generic dexamethasone was administered at 3 to 6 mg / kg in four patients under the Physician Practice of Medicine guidelines (by Dr. Loniewski, Advanced Orthopedic Specialists, Brighton, MI).

[0291] Flow cytometry data from four patients 48 hours after treatment with a dexamethasone dose six-fold lower than the dose used to maximally induce AVM_NKT in mice was reviewed. The CD / CD56 scattergram from one of the four patients shows that a new population of cells corresponding to the AVM-NKT cells identified in the mice appeared approximately 48 hours after treatment (Figure 12).

[0292] A novel population of CD56 very bright cells was also observed in a prostate cancer patient 1 hour after the fourth AVM0703 treatment at 6 mg / kg injection, as shown in Figure 13. This prostate cancer patient had no options after multiple years of cancer treatment and received a total of four AVM0703 injections at least 28 days apart.

[0293] Compared to healthy blood donors, prostate cancer patients had evidence of a novel CD3 dim population that was no longer evident 1 hour after AVM0703, but then a new CD56 very bright cell population became evident in the blood that was no longer observed 3 hours after infusion.

[0294] Compared to healthy blood donors, prostate cancer patients had CD3dim and NKp46dim cell populations prior to infusion, and one hour after infusion of 6 mg / kg AVM0703, the patients had a new CD56 very bright CD3dim population that was CD45 dim / negative and CD4 / CD8 double negative.

[0295] Example 8 - Generation and mobilization of human AVM-NKT cells in humanized mice. BRGSF humanized mice on a Balb / c background from Genoway, generated by transplanting human umbilical cord blood CD34+ stem cells into irradiated mice lacking mouse B and T lymphocytes and NK cells but with a functional mouse complement system, are orally administered HED 18-45 mg / kg DSP and after 24-48 hours a novel population of cells corresponding to the AVM-NKT cells identified in the non-human mice can be observed. Human CD56+ cells can be observed in the blood from approximately 36 hours up to 13 days later.

[0296] [HuCD34-NCG mouse model] HuCD34-NCG mice from Charles River are research-ready mouse models with a human-like immune system generated by adoptive transfer of CD34+ stem cells. HuCD34-NCG mice are an ideal in vivo platform for evaluating the efficacy of compounds that modulate the human immune system. Humanized mice do not or are slow to develop graft-versus-host disease (GvHD), making them suitable for long-term studies.

[0297] Following bone marrow ablation, NCG mice were humanized by adoptive transfer with human umbilical cord blood-derived CD34+ stem cells. NCG mice from four donors (n=2 per donor) were orally administered DSP at HED 18-45 mg / kg, and 24-48 hours later, cells corresponding to AVM-NKT cells identified in non-human mice could be observed as approximately 0.2-3% of total splenocytes by flow cytometry. Human CD56+ cells could be observed in the blood from approximately 36 hours after administration until 13 days later.

[0298] [huNOG-EXL mouse model] huNOG EXL from Taconic has an average of 54% CD45 cells positive for human CD45. Six huNOG EXL humanized immune system mice from three donors (n=2 per donor) were orally administered HED 18-45 mg / kg DSP, and 24-48 hours later, approximately 0.2-3% of total splenocytes were observed by flow cytometry, which corresponds to AVM-NKT cells identified in non-human mice. Human CD56+ cells can be observed in the blood from approximately 36 hours to 13 days later.

[0299] Example 9 - Characterization of induced / mobilized AVM-NKT (NKT-like cells) in human subjects Preclinically, AVM0703-induced AVM-NKT cells have been characterized and have shown activity against mouse melanoma, mouse B lymphoma, human xenograft T lymphoma and diabetes.

[0300] Blood samples were collected from human cancer patients treated with AVM0703, and the cells were characterized by the human whole blood surface staining protocol described above. AVM0703 induces the production and recruitment of γδ natural killer T-like cells (CD56+γδTCR+). Interestingly, the recruited NKT-like cells were also found to express iTCR (Figure 14). This finding may explain why AVM0703-induced cells have activity against both cancer and type 1 diabetes, even though it has been said that iNKT and γδT cells are generally active against one disease but not the other. The recruited cells are also typically CD16+ and NKp44+ (Figure 14).

[0301] Following AVM0703 administration, CD56+γδTCR+ (1.64% of WBCs) cells were mobilized into whole blood within 30 minutes of infusion. These cells were gated from all live WBCs (white blood cells). In a representative subject, these cells were iNKT positive (~96% of new cells), NKp44 positive (~97% of new cells), CD8 dim / - (~98% of new cells), CD19+ (85% of new cells), CD16+ (86% of new cells), and CD14+ (67% of new cells). Numbers reported here are % CD56+γδTCR+. CD56+γδTCR+iTCR+ cells were also found to express CD3, CD45, and in some cases not CD4. Some CD56+γδTCR+iTCR+ cells were also found to express αβTCR.

[0302] Example 10 - AVM-NKT cells are isolated and expanded and then used to precondition patients prior to cell therapy. Autologous or allogeneic AVM-NKT cells are administered to the patient either IV or IP 6 to 96 hours before the cell therapy is administered, which may be for regenerative purposes, treatment of cancer, treatment of autoimmune disease, or treatment of infectious disease or other medical conditions where cell therapy is warranted.

[0303] Example 11 - Induction of tumor lysis syndrome by AVM-NKT AVM-NKT targets the tumor, forming bands of attacking cells that invade the tumor like an army from all sides. Tumor lysis syndrome occurs, which is untreatable and leads to death in mice. Clinical chemistry markers of tumor lysis syndrome (e.g. uric acid) are elevated. Macroscopic examination of the tumor shows a sludge-like oil encased in the tumor membrane.

[0304] Example 12 - Using AVM-NKT cells to prepare patients for cancer or other serious medical procedures. Autologous or allogeneic AVM-NKT cells are administered either IV or IP to patients whose performance status prevents them from receiving medical therapy, such as chemotherapy, cell therapy, organ or bone marrow transplantation, etc. The patient's performance status is improved so that they become eligible for medical therapy.

[0305] Example 13 - AVM-NKT cells induce tumor pseudoprogression. Although AVM-NKT cell-treated tumors appear to continue to grow, this growth is a pseudoprogression of the tumor due to other immune cells that the AVM-NKT cells attract to the tumor, either by release of cytokines and chemokines or by direct engagement of other immune cells. Eventually, the tumor becomes completely acellular and resorbed.

[0306] [Example 14 - AVM-NKT cells are used to treat any type of cancer, graft-versus-host disease, autoimmunity, or immune-related adverse events of immunotherapy] AVM-NKT cells home to and target both hematologic and solid cancers, fibroid tumors, benign tumors, and autoreactive T and B lymphocytes.

[0307] Example 15 - AVM-NKT cells are detected in human R / R NHL subjects and humanized mice treated with acute high doses of dexamethasone. [overview]

[0308] In human R / R NHL clinical trial patients, bispecific gamma delta TCR+ and invariant TCR+ cells are mobilized into the blood within 30-60 minutes after AVM0703 doses of 6 mg / kg to 18 mg / kg. These novel induced immune cells are not found in the blood of healthy mice in a pathogen-free environment, nor in the blood of healthy human donors (not in a pathogen-free environment). However, cancer patients expressed low levels of these novel bispecific cells at baseline that also expressed CD56 (a marker for natural killer). Thus, the cells are gamma delta TCR+ invariant TCR+ bispecific natural killer T-like cells.

[0309] We hypothesize that the cancer environment may lead to induction of these cells, but that these cells are not maximally mobilized into the blood until after administration of AVM0703, similar to what was seen in the murine A20 lymphoma model (Figure 11, Examples 3 and 6). In one compassionate use patient with CNS squamous cell carcinoma, immune infiltration characterized by skin redness restricted to the CNS tumor area on the left side of the brain was observed within 30 minutes of initiating an 18 mg / kg AVM0703 infusion (data not shown).

[0310] Interestingly, the presence of these CD56+γδTCR+iTCR+ cells has been associated with clinical responses in patients in R / R NHL clinical trials. The only patient (108-004) who showed no evidence of these cells at baseline or after AVM0703 administration was also the only patient who did not show an objective clinical beneficial response after AVM0703.

[0311] In mice, AVM0703, at a human equivalent dose (HED) of 18 mg / kg calculated as dexamethasone phosphate, induces production of CD3 high cells in the spleen, bone marrow and thymus, and mobilizes A20 cells from the spleen to the blood and into the A20 murine B cell lymphoma, whether the cells are in a solid tumor injected into the flank or in the bone marrow, spleen, thymus or blood. The most rapid and major mobilization is to the tumor, where the maximal effect of killing A20 cells is observed approximately 3 hours after administration.

[0312] Naive mice do not normally express these cells in any of the organs examined. The sensitivity to AVM0703 to induce and mobilize these cells varies between mouse strains, and although the tumor environment itself may induce the production of these cells in the spleen, optimal recruitment and tumor targeting of the cells may require AVM0703.

[0313] Humanized mice with human lymphoid compartments purchased from Charles River and Taconic also mobilize hCD45+CD56+γδTCR+invTCR+ cells following AVM0703 administration.

[0314] [Summary of human data from AVM0703 R / R NHL clinical trial patients]

[0315] 101-001 (6 mg / kg):CD56+γδTCR+ were present in the blood at low levels prior to infusion and increased from 1.6% (76 cells / uL blood) to 3.48% (165 cells / uL blood) of total CD45+ cells 1 hour after infusion of 6 mg / kg AVM0703. When CD56+γδTCR+ cells were gated on histograms of invTCR and αβTCR expression, these cells were also invTCR+ and in this patient αβTCR+. By size and complexity, these cells are primarily large granular lymphoid cells (visible as red dots on the FSC vs SSC plot; Figure 15).

[0316] Using an alternative gating approach, gating CD56+ WBCs on a scatter plot with γδTCR on the X-axis and invTCR on the Y-axis, CD56+γδTCR+iTCR+ triple positive cells were present in WBCs at 0.15% (7 cells / uL) pre-infusion and 0.24% (11.4 cells / uL) 1 hour post-infusion. These cells were not seen on day 3 post-infusion, suggesting homing of the cells to the tumor site, and were 1.74% (78.7 cells / uL) of total WBCs on day 14. This patient showed evidence of tumor flare and stable disease (SD) by day 28 PET / CT. These data are shown in Figure 16 and summarized in Table 4 below with the %GP gate (GP is WBC). [Table 4]

[0317] 103-002 (6 mg / kg): Blood CD56+γδTCR+ cells were high pre-infusion (9.3% of total CD45+ cells, 735 cells / μL blood) and decreased 1 hour post-infusion (6.54% of total CD45+ cells, 517 cells / μL blood). This patient had evidence of tumor flare and PR on PET / CT on day 28, suggesting AVM-NKT was targeted to the tumor after AVM0703 infusion. Not all of this patient's cells were bispecific, with only ~10% of CD56+γδTCR+ cells co-expressing invTCR. This patient also had high levels of CD8 expression, including CD8 MFI high cells.

[0318] Using a different gating approach, where CD56+WBCs were gated on a scattergram with γδTCR on the X-axis and invTCR on the Y-axis, CD56+γδTCR+iTCR+ triple positive cells were measured and characterized for marker expression. On day 3, no CD19 staining was observed in any cells except for 4% of CD56+γδTCR+iTCR+ cells, whereas lymphocyte populations were evident in FCS vs. SSC and only 60% were CD3 positive in flow. These data are shown in Figure 17 and summarized in Table 5 below. [Table 5]

[0319] 103-005 (9 mg / kg): Baseline CD56+γδTCR+ cells were 2.7% of CD45+ cells, and all were bispecific for invTCR (Figure 18, top left). αβTCR was not included in this flow panel. Most were CD8 negative, but 95% were CD14 positive and 60% were CD16 positive, indicating an activated state. 13% were CD19 positive.

[0320] One hour after administration of 9 mg / kg AVM0703 (Figure 18, top right), CD56+γδTCR+ cells had decreased to 0.21% of CD45+ cells, and the patient showed evidence of tumor flare and PR by day 28 PET / CT, suggesting that AVM-NKT cells were targeted to the tumor site.

[0321] Using a different gating approach, CD56+ WBCs were gated on a scattergram with γδTCR on the X-axis and invTCR on the Y-axis, CD56+γδTCR+iTCR+ triple positive cells were measured and expression markers were characterized. This patient, who had a clinical response with PI within 1 week and a PR by PET / CT on day 28, had high levels of cells that dropped dramatically in the blood 1 hour after 9 mg / kg AVM0703 infusion, suggesting tumor homing. These data are shown in Figure 18 and summarized in Table 7 below. [Table 7]

[0322] 103-006 (9 mg / kg): Baseline CD56+γδTCR+ cells were 3.84% of CD45+. αβTCR and invTCR were not included in this flow panel. CD56+γδTCR+ cells were also positive for CD16, CD34, and ICAM3 (MFI for ICAM3 was 294, MFI for healthy controls was 760), and 42% were positive for NKp44. Most were CD8 negative, but 95% were CD14 positive and 20% were CD16 positive, indicating an activated state. 13% were CD19 positive.

[0323] One hour after administration of 9 mg / kg AVM0703, there was no change in the number or expression of CD56+γδTCR+ cells in the blood, although only 25% were now NKp44+. There was no change in ICAM3 MFI. On day 3, CD56+γδTCR+ cells represented 2.2% of total WBCs, 92% expressed CD16, 19% expressed NKp44, and the ICAM3 MFI was 240. On day 14, CD56+γδTCR+ cells represented 2.2% of total WBCs, 92% expressed CD16, 19% expressed NKp44, and the ICAM3 MFI was 240. On day 14, CD56+γδTCR+ cells represented only 0.11% of total WBCs.

[0324] 108-001 (9 mg / kg): One hour after AVM0703 infusion, blood CD56+γδTCR+iTCR+ cells increased approximately 20-fold over baseline (5.8 to 112 cells / uL) and remained elevated on day 3 (36 cells / uL). This patient demonstrated a significant clinical response with recovery of vision on day 3 after AVM0703 infusion. Interestingly, this patient had very few CD19+ lymphocytes at baseline or at any time point. The CD56+γδTCR+iTCR+ flow cytometry profile after the first infusion is shown in Figure 19 and summarized in Table 8 below. αβTCR was not included in this flow panel. [Table 8]

[0325] AVM_NKT CD56+γδTCR+iTCR+ cells were present at baseline or 1 hour after the second 9 mg / kg AVM0703 infusion, but increased 10-fold by day 3. This patient continued to respond with no CNS solid tumors and a 40% reduction in CSF blasts after the second 9 mg / kg infusion. The data are summarized in Table 9 below. [Table 9]

[0326] AVM_NKT CD56+γδTCR+iTCR+ cells were not observed in the blood following the third 9 mg / kg AVM0703 infusion, consistent with the patient's loss of response to the third infusion.

[0327] 108-003 (12 mg / kg): Data regarding CD56+γδTCR+iTCR+ cells in patient 108-003 are shown in FIG. 20 and summarized in Table 10 below. [Table 10]

[0328] 108-004 (12 mg / kg): CD56+γδTCR+invTCR+ cells were 0.09% (4.6 cells / uL) of total cells before infusion. No increase was observed 1 hour, 3 days, or 14 days after AVM0703 infusion. Interestingly, patient 108-004 was the only patient who did not have an objective beneficial response as measured by either PET / CT, clinical chemistry, CBC, or clinical symptoms. As shown in Figure 21, 108-004 did not mobilize CD56+γδTCR+invTCR+ cells.

[0329] 108-002 (18 mg / kg): At baseline, 2.8% of total WBCs were CD56+γδTCR+iTCR+ (163.2 cells / uL) and this did not change at 1 hour after AVM0703 18 mg / kg infusion. By day 3, these new cells had decreased to 0.04% in the blood, suggesting tumor homing as observed in the mouse model, and returned to 2.12% in the blood by day 14. Patient 108-002 has had an ongoing PR with SD on PET / CT and is alive from the date of dosing on August 30, 2021. The data is shown in Figure 22 and summarized in Table 11 below. [Table 11]

[0330] [Summary of the association between dose-escalating AVM-NKT and clinical and PET / CT responses]

[0331] A summary of AVM0703 for R / R NHL, dose escalation phase is shown in Table 12 below. As of July 15, 2022; administered to 12 patients; mean 5.6 prior therapies, 7 of 12 were refractory to HSCT or CART. The only patient without evidence of de novo AVM-NKT by flow cytometry also did not respond to treatment (108-004). All patients with evidence of de novo AVM-NKT cells had a clinical and / or PET / CT SD / PR / CR response. [Table 12] TIFF2024533093000013.tif250147

[0332] [Summary of data from healthy human control blood donors] In healthy control blood donors: CD56+γδTCR+ cells are usually absent. The few cells present are typically invTCR co-expressing cells and are also positive for CD14 and CD16. A scatter plot of γδTCR+iTCR+ cells from total CD56+WBCs is shown in FIG. 23 for 12 healthy blood donors.

[0333] Table 13 shows CD56+γδTCR+iTCR+ marker expression for healthy donors with some low levels found in the blood. The markers expressed are consistent with those expressed by these cells in the blood of R / R NHL patients, and we hypothesize that these "healthy" blood donors may in fact have had an infection or other asymptomatic problem that induced the production of these cells that are never seen in the placebo mice maintained in a pathogen-free environment. The characteristics of these cells when present in healthy blood donors are similar to those of these cells in the AVM0703-001 study patients. %GP gates are the % listed in the table (GP is WBC). [Table 13]

[0334] [Summary of data from humanized mouse studies LYDEP 43 and 45]

[0335] In mice with partially human blood cells, created by irradiating newborn mice and transplanting umbilical cord (UC) CD34+ cells, new human immune cells similar to those observed in naive mice and human patients treated with AVM0703 were increased in the blood after AVM0703 treatment compared to placebo mice from the same human UC blood CD34+ donors. Mice rechallenged one week later had more hCD45+CD56+TCRγδ+ human immune cells compared to placebo-treated mice. These humanized mice lacked both mouse and human bone marrow cells and, interestingly, began to produce both human and mouse bone marrow cells after AVM0703 treatment. Furthermore, after the third AVM0703 treatment, the humanized mice had hCD45+mCD45+ double positive cells.

[0336] Female huNOG-EXL mice (n=6) were obtained from Taconic Bioscience (Germantown, NY). Female huCD34-NCG mice (n=8) were obtained from Charles River (Wilmington, MA). Mice from both facilities were acclimated to laboratory conditions for at least 5-6 days.

[0337] Mice were orally dosed with 32 mg / kg dexamethasone phosphate (DP) or placebo three times and maintained until time points. After the first dose (03 / 01 / 2021) and the second dose one week later (03 / 08 / 2021), up to 70 uL of blood / mouse was collected by cheek puncture when mice reached the designated time points. Blood was analyzed by flow cytometry.

[0338] Following the third dose (04 / 05 / 2021), 28 days after the previous dose, mice were euthanized according to standard operating procedures when they reached the 48- or 60-hour study timepoints.

[0339] Female humanized mice studied were obtained from a total of six different cord blood donors and two different vendors. Responses to AVM0703 were consistent across both vendors and all six donors and are summarized below. AVM0703 induced expression and mobilization of human CD56+γδTCR+invTCR+ immune cells and also induced production of myeloid cells in mice that largely lack a myeloid compartment. [Table 14]

[0340] Humanized mice purchased from Taconic mobilized more hCD45+CD56+γδTCR+ cells after the first dose of AVM0703 than mice purchased from Charles River. The flow panel for the second dose included markers for invTCR, but not for the first dose. However, upon rechallenge, mice purchased from Charles River mobilized more hCD45+CD56+γδTCR+invTCR+ cells than after the first dose, while mice purchased from Taconic showed the same mobilization compared to placebo upon rechallenge. Placebo-treated mice cannot be considered naïve mice, since all mice were lethally irradiated and then transplanted with human cord blood CD34+ cells. Therefore, it is not surprising that placebo-treated mice may have a baseline level of these new immune cells, since we have shown that if the mice have cancer or diabetes, these cells are present but not optimally mobilized until after AVM0703 treatment. [Table 15]

[0341] Similar to human patients, AVM0703 induces CD56+TCRγδ+invTCR+ bispecific immune cell mobilization in humanized mice. As shown in Figure 24, AVM0703 induced CD56+TCRγδ+ cells that were CD16+ (12% of hCD45+ cells), suggesting an activated state (mouse 10 Taconic NOG-EXL). As shown in Figures 25 and 26, >18 mg / kg AVM0703 HED induces bispecific immune cell mobilization of 2-12% of hCD45+ cells. As shown in Figures 27 and 28, AVM0703 induced γδTCR+invTCR+ bispecific activated CD56+ bone marrow cells in humanized mice, which correlates with data from human patients. Over 60% of human CD45+CD56+ cells were bispecific for TCRγδ and invariant TCR and were CD16 positive, indicating an activated state. Bone marrow was analyzed 48-60 hours after the third repeat dose of AVM0703 32 mg / kg HED.

[0342] Figures 29 and 30 show FSC vs. SSC for humanized mice after the first (Figure 29) and second (Figure 30) doses of AVM0703. Mice have been reported by Charles River and Taconic to have no bone marrow compartment, but after AVM0703 administration, the mice began to make both human and mouse bone marrow cells. Scatter plots are shown for two placebo mice (top plots of Figures 29 and 30; placebo mouse M12 top left, placebo mouse M90 ​​top right) and an AVM0703 treated mouse (bottom plots of Figures 29 and 30; mouse M88). Mouse 88 had the fastest increase in bone marrow cells out of all 12 AVM0703 treated humanized mice. On average, in placebo-treated mice, 12.7% of total mouse WBCs were lymphoid cells, whereas in AVM0703-treated mice, 10.62% of total mouse WBCs were lymphoid cells (range 2%-20.4%).

[0343] This observation that mice begin to produce bone marrow cells, including neutrophils, is consistent with a report from a compassionate use patient in Germany who began to produce healthy, active neutrophils after AVM0703 administration. This 18-year-old male had not produced neutrophils since his first chemotherapy cycle, 6 years before being treated with AVM0703. Similarly, all human patients in our AVM0703-001 study in R / R NHL have shown evidence of neutrophilia.

[0344] Figures 31-33 show that the humanized mice have predominantly human lymphoid cells. These figures provide another perspective on the origin of lymphoid and myeloid cells in placebo-treated mice, again showing that the majority of lymphoid cells are human in origin while a small number of myeloid cells are predominantly murine in origin. There is significant debris in these flow cytometry samples, which is why many points in the ungated hCD45 vs. mCD45 scatter plots are negative for both human and mouse CD45.

[0345] Figure 31 shows that in placebo-treated mice, lymphocytes are predominantly human CD45+ (Figure 31, top row) and a small number of myeloid cells are predominantly mCD45+ (Figure 31, bottom row). Figures 32-33 show that AVM0703 treatment induces myeloid cell production in humanized mice. Scatter plots of FSC vs. SSC gated on mCD45+ cells (top left) and hCD45+ cells (top right), and hCD45+ cells vs. mCD45+ cells (bottom) are shown. Figure 32 shows data from placebo mouse M12, where mouse lymphocytes are 13% of mouse total WBCs (Figure 32, top left), human lymphocytes are 60% of human total WBCs (Figure 32, top right), and total lymphocytes are 45% of total WBCs. FIG. 33 shows data from placebo mouse M90, where mouse lymphocytes were 12.5% ​​of mouse total WBCs (FIG. 33 top left), human lymphocytes were 31.5% of human total WBCs (FIG. 33 top right), and total lymphocytes were 30% of total WBCs.

[0346] Figures 34-39 show that AVM0703 treatment induces myeloid cell production in humanized mice. These are flow cytometry scatter plots of AVM0703-treated mice after the first dose of AVM0703. In the scatter plots, lymphocytes are circled, but myeloid cells have a higher SSC and are plotted above lymphocytes. These FSC vs. SSC scatter plots show significantly higher numbers of mouse-derived myeloid cells compared to placebo (top left), suggesting that AVM0703 treatment induces myeloid cell production as observed in human clinical trial and compassionate use patients. The lymphoid cell population remains largely of hCD45+ origin (top right). Compared to placebo-treated humanized mice, which have roughly twice as many human CD45+ cells as mCD45+ cells, AVM0703-treated humanized mice have roughly equal numbers of mouse CD45+ and human CD45+ cells (bottom).

[0347] Figure 34 shows data from AVM0703 treated mouse M88, where mouse lymphocytes are only 5.7% of total WBCs (Figure 34 top left). Human lymphocytes are 58% of total human WBCs (Figure 34 top right). Total lymphocytes are 32% of total WBCs. Figure 35 shows data from AVM0703 treated mouse M01, where mouse lymphocytes are only 6.7% of total WBCs (Figure 35 top left). Human lymphocytes are 67% of total human WBCs (Figure 35 top right). Total lymphocytes are 35% of total WBCs. Figure 36 shows data from AVM0703 treated mouse M03, where mouse lymphocytes are only 23.7% of total WBCs (Figure 36 top left). Human lymphocytes are 47% of total human WBCs (Figure 36 top right). Total lymphocytes are 40% of total WBCs. Figure 37 shows data from AVM0703 treated mouse M05, where mouse lymphocytes are only 2.0% of total WBCs (Figure 37 top left). Human lymphocytes are 50.1% of human total WBCs (Figure 37 top right). Total lymphocytes are 20.9% of total WBCs. Figure 38 shows data from AVM0703 treated mouse M07, where mouse lymphocytes are only 20.4% of total WBCs (Figure 38 top left). Human lymphocytes are 58.2% of human total WBCs (Figure 38 top right). Total lymphocytes are 41.9% of total WBCs. Figure 39 shows data from AVM0703 treated mouse M10, where mouse lymphocytes are only 5.2% of ...

Claims

1. 1. A composition comprising a glucocorticoid for use in a method of generating a population of natural killer T-cell-like cells (NKT-like cells), the method comprising administering the glucocorticoid to a human subject at a dose equivalent to at least about a 6 mg / kg human equivalent dose (HED) of dexamethasone base, and isolating a population of NKT cells from the subject or a sample derived from the subject; wherein the population of NKT cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD56, TCR gamma / delta, and iTCR, or at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express TCR gamma / delta and iTCR.

2. A composition comprising a glucocorticoid for use in a method for treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising administering to the subject a glucocorticoid at a dose equivalent to at least about a 6 mg / kg human equivalent dose (HED) of dexamethasone base; wherein the glucocorticoid induces a population of NKT-like cells characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing CD56, TCR gamma / delta, and iTCR, or at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells expressing TCR gamma / delta and iTCR.

3. The population of NKT-like cells may comprise at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells: i) expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34, and / or ICAM3; ii) expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta; and / or iii) Absence of CD4 expression The composition of claim 1 , characterized in that 4. The population of NKT-like cells, wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells are: i) expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34, and / or ICAM3; ii) expressing CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta; and / or iii) Absence of CD4 expression The composition according to claim 2, characterized in that

5. the NKT-like cells express CD56, TCR gamma / delta, and iTCR; and i) CD16 and NKp44; (ii) TCR alpha / beta; (iii) CD16, NKp44, and TCR alpha / beta; (iv) CD16, NKp44, CD8, CD14, and CD19; (v) CD16, NKp44, CD3, CD8, CD14, CD19, and CD45; or (vi) The composition of claim 3, which expresses CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and TCR alpha / beta.

6. The NKT-like cells express CD56, TCR gamma / delta, and iTCR, and i) CD16 and NKp44; (ii) TCR alpha / beta; (iii) CD16, NKp44, and TCR alpha / beta; (iv) CD16, NKp44, CD8, CD14, and CD19; (v) CD16, NKp44, CD3, CD8, CD14, CD19, and CD45; or (vi) the composition of claim 4, which expresses CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and TCR alpha / beta.

7. A composition described in any one of claims 1 to 6, wherein the glucocorticoid is selected from the group consisting of dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide, betamethasone, flumethasone and beclomethasone.

8. The composition described in claim 7, wherein the glucocorticoid is dexamethasone sodium phosphate.

9. The glucocorticoid in an amount of about: i) a human equivalent dose (HED) of at least 6-12 mg / kg of dexamethasone base; ii) a human equivalent dose (HED) of at least 6 mg / kg dexamethasone base; iii) a human equivalent dose (HED) of dexamethasone base of at least 12 mg / kg; iv) a human equivalent dose (HED) of at least 15 mg / kg dexamethasone base; v) a human equivalent dose (HED) of at least 21 mg / kg dexamethasone base; vi) a human equivalent dose (HED) of at least 24 mg / kg dexamethasone base; vii) Human equivalent dose (HED) of dexamethasone base of 15 mg / kg; viii) 24 mg / kg human equivalent dose (HED) of dexamethasone base; or ix) Human equivalent dose (HED) of dexamethasone base of 45 mg / kg The composition of any one of claims 1 to 6, administered in a dose equivalent to

10. The composition described in any one of claims 1 to 6, wherein the method further comprises administering to the subject an NKT cell activator, a T cell activator, and / or an NK cell activator.

11. (i) the subject has, is suspected of having, or has been diagnosed with cancer; (ii) the NKT-like cells treat cancer in the subject; (iii) the subject has, is suspected of having, or has been diagnosed with solid tumor cancer; or (iv) the subject has, is suspected of having, or has been diagnosed with cancer that is lymphoma, preferably B-cell lymphoma, T-cell lymphoma, or non-Hodgkin's lymphoma, or leukemia, preferably T-ALL or B-ALL; The composition according to any one of claims 1 to 6.

12. (i) The NKT-like cells treat cancer through tumor infiltration. (ii) the NKT-like cells promote the infiltration of other immune cells into tumors; (iii) the NKT-like cells directly kill cancer cells via CD1d-induced apoptosis; or (iv) the NKT-like cells treat cancer by causing tumor necrosis; The composition of claim 11.

13. (i) the subject has, is suspected of having, or has been diagnosed with an autoimmune disease; (ii) the subject has, is suspected of having, or has been diagnosed with an autoimmune disease selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes (T1D), scleroderma, pemphigus, and lupus; (iii) the subject has, is suspected of having, or has been diagnosed with an infectious disease; or (iv) the subject has, is suspected of having, or has been diagnosed with an infectious disease selected from HIV, or a disease resulting from infection with a coronavirus, such as COVID-19; The composition according to any one of claims 1 to 6.

14. The isolating step comprising: i) at least 48 hours after glucocorticoid administration; or ii) 48 hours to 13 days after glucocorticoid administration The composition of any one of claims 1, 3, and 5, wherein 15. The method comprising: (i) expanding the isolated NKT-like cells; (ii) activating the isolated NKT-like cells with an NKT cell activator, a T cell activator, and / or an NK cell activator; (iii) activating the isolated NKT-like cells with an NKT cell activator selected from alphaGalCer and sulfatide; (iv) activating the isolated NKT-like cells with a T cell activator selected from zoledronate and mevastatin; (v) activating the isolated NKT-like cells with an NK cell activator selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and IL-21; (vi) introducing into the isolated NKT-like cells a nucleic acid encoding a protein and culturing the cells under conditions that promote expression of the protein; or (vii) introducing a nucleic acid encoding a protein selected from the group consisting of one or more of a T cell receptor (TCR), a chimeric antigen receptor (CAR), and a split, universal and programmable CAR (SUPRA-CAR), and culturing the cells under conditions that promote expression of the protein.

15. The composition of claim 14, further comprising:

16. 10. A composition for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, comprising isolated NKT-like cells produced by the method of any one of claims 1, 3, and 5, wherein the method of treating comprises administering to the subject a therapeutically effective dose of the isolated NKT-like cells.

17. An isolated NKT-like cell or a population of NKT-like cells produced by the method of any one of claims 1 to 5.

18. an isolated NKT-like cell or population of NKT-like cells, characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cell or population of cells express CD56, TCR gamma / delta, and iTCR, or at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cell or population of cells express TCR gamma / delta and iTCR, and optionally i) expresses CD56, TCR gamma / delta, iTCR, CD16, NKp44, CD3, CD8, CD14, CD19, CD45, TCR alpha / beta, CD34 and / or ICAM3; ii) expresses CD16, NKp44, CD3, CD8, CD14, CD19, CD45, and / or TCR alpha / beta; and / or iii) do not express CD4; NKT-like cells or populations of NKT-like cells.

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