Allogeneic invariant natural killer t cells adjuvant for treatment of disease and related methods

Abstract

The present disclosure relates to compositions comprising allogeneic invariant natural killer T (iNKT) cells, including allogeneic iNKT cell adjuvants, and methods of using the compositions comprising the iNKT cells for treatment of diseases and conditions, including cancer.

Description

ALLOGENEIC INVARIANT NATURAL KILLER T CELLS ADJUVANT FOR TREATMENT OF DISEASE AND RELATED METHODSRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application, U.S.S.N. 63 / 596,085, filed November 3, 2023, the entire contents of which are herein incorporated by reference.BACKGROUND

[0002] Invariant natural killer T (iNKT) cells are a unique subset of T cells that exhibit both direct and indirect anti-tumor activities. iNKT cells can recognize and kill tumor cells directly through the release of cytotoxic molecules, such as perforin and granzyme B, and by inducing apoptosis through the Fas / FasL pathway. Additionally, iNKT cells can act indirectly by stimulating other immune cells, such as dendritic cells, natural killer (NK) cells, and CD8+ T cells, to target and kill tumor cells. iNKT cells also play a role in regulating the immune response by producing cytokines, such as interferon-gamma and interleukin-4, which promote the activation and differentiation of other immune cells.

[0003] Prior clinical trials utilizing iNKT cells have focused on administering glycolipid a-galactosylceramide (a-GalCer) to patients to activate autologous iNKT cells or have focused on adoptive transfer of autologous iNKT cells. These approaches have been met with limited success. .SUMMARY

[0004] The present disclosure relates to adjuvant compositions comprising allogeneic invariant natural kill T (iNKT) cells (e.g., unmodified, iNKT cells from healthy donors), and methods of using the compositions for treatment of diseases and conditions such as cancer. The present disclosure, at least in part, is based on the observation that allogeneic iNKT cells prepared from healthy donors and stimulated and expanded ex vivo, modify the immunosuppressive tumor microenvironment and counteracting exhausted T cells to potentiate the anti-tumor effects of anti-cancer therapeutics, including PD-1 inhibitors. By administering an allogeneic iNKT cell adjuvant with anti-cancer therapeutics such as PD-1 inhibitors, the allogeneic iNKT cells increase the tumor killing activity of the anti-cancer therapeutic.

[0005] The present discloser is partly based on a surprising observation that unmodified allogeneic iNKT cells harvested from healthy donors and stimulated and expanded ex vivo prior to administration as a single dose to subjects were detectable in the subjects six months after the administration. Moreover, persistence of the allogeneic iNKT cells in the body long after administration correlates with the response of the subject to the anti-cancer therapeutic.

[0006] Unlike some anti-cancer therapeutics, the iNKT cell compositions (including adjuvants of the disclosure) of the disclosure do not require toxic lymphodepletion to modify the tumor microenvironment. The present disclosure established that even in the absence of lymphodepletion, it is unnecessary to HLA match the donor and recipient. HLA matching between donor and recipient is not required and does not affect the persistence of allogeneic iNKT cells in the subject.

[0007] In some embodiments, the disclosure includes methods of potentiating the effect of a therapeutic (e.g., immune checkpoint inhibitors) in a subject suffering from a disease or condition (e.g., cancer) by administering to the subject an allogeneic iNKT cell adjuvant with the therapeutic. As shown herein, allogeneic iNKT cells that have been harvested from a healthy donor, isolated from other donor cells, stimulated ex vivo, and expanded ex vivo, have the potential to modify the immune system to potentiate the effect of many types of therapeutics.

[0008] In some embodiments, the disclosure includes methods of modifying the tumor microenvironment in a subject with cancer to potentiate the tumor killing effects of an anti-cancer therapeutic. As shown herein, administration of allogeneic iNKT cell adjuvant potentiates the effects of an anti-cancer therapeutic. Moreover, since the allogeneic iNKT cell adjuvant persists in the subject for weeks after administration, a single dose of an allogeneic iNKT cell adjuvant of the disclosure administered to a subject can act as an adjuvant for multiple administrations of an anti-cancer therapeutic.

[0009] In some embodiments, administration of the iNKT cell adjuvant modifies the tumor microenvironment to make it more susceptible to an anti-cancer therapeutic. For instance, In some embodiments, the tumor microenvironment is modified by (i) an increase in dendritic cells; (ii) a decrease in M2 macrophages, (iii) an increase in Ml macrophages, (iv) a decrease in myeloid derived suppressor cells, and / or an increase in tumor infiltration of cytotoxic lymphocytes as compared to treatment with the anti-cancer therapeutic alone. Insome embodiments, administration of the allogeneic iNKT cell adjuvant with the anti-cancer therapeutic results in an increase of cytotoxic lymphocytes to the tumor as compared to treatment with the anti-cancer therapeutic alone. For instance, administration of the allogeneic iNKT cell adjuvant of the disclosure can result in an increase of CD8+ T cells infiltrating the tumor. The disclosure includes methods of increasing T cell tumor infiltration in a subject with cancer to potentiate the anti-tumor effects of an anti-cancer therapeutic by administering to the subject an allogeneic iNKT cell adjuvant with the anti-cancer drug. In some embodiments, a single dose of iNKT cell adjuvant is administered to increase T cell infiltration of the tumor.

[0010] Resistance to PD-1 blockage occurs in many subjects receiving treatment for cancer. In some embodiments, administration of the allogeneic iNKT cell adjuvant allows the subject’s body to overcome resistance to PD-1 blockade and positively respond to subsequent anti-cancer therapeutics, including immune checkpoint inhibitors (ICI) such as anti-PD-1 therapeutics.

[0011] In some embodiments of the disclosure, the allogeneic iNKT adjuvant is administered before the anti-cancer therapeutic, for instance, on a day prior to administration of the anti-cancer therapeutic. In some embodiments, the iNKT adjuvant is administered at the same time as administration of a dose of the anti-cancer therapeutic. Due to the long halflife of the allogeneic cell compositions of the disclosure in the body, it is possible to administer the allogeneic iNKT adjuvant once for the cells to be active during two or more rounds of administration of the anti-cancer therapeutic.

[0012] In some embodiments, the allogeneic iNKT cell adjuvant is administered to the subject by infusion. For instance, In some embodiments, the iNKT cell adjuvant is administered to the subject by infusion over a period of thirty minutes.

[0013] The allogeneic iNKT cell adjuvant of the disclosure is potentiates the effects of multiple types of therapeutics, including chemotherapeutic agents and immune checkpoint inhibitors. In some embodiments, one or more chemotherapeutic agents are administered with the allogeneic iNKT cell adjuvant. In some embodiments, one or more immune checkpoint inhibitors are administered with the allogeneic iNKT cell adjuvant.

[0014] In some embodiments, the iNKT cell adjuvant is administered to the subject at a dosage of about 4.3 x 106to about 1.4 x 107allogeneic iNKT cells / kg subject’s body weight. In some embodiments, the iNKT cell adjuvant is administered to a subject at about4.3 x 106iNKT cells / kg subject’s body weight. In some embodiments, the iNKT cell adjuvant is administered at about 1.4 x 107allogeneic iNKT cells / kg subject’s body weight. In some embodiments of the disclosure, the iNKT cell adjuvant comprises about 10 x 106to 1,000 x 106allogeneic iNKT cells.

[0015] The iNKT cell adjuvant of the disclosure is prepared using healthy donor iNKT cells. In some embodiments, the cells are from a single donor. In some embodiments, the healthy donor has CD8+ iNKT cells, CD4+ iNKT cells, and CD8- and CD4- (double negative) iNKT cells. In some embodiments, the iNKT cells are harvested from the peripheral blood, PBMCs, leukocytes, or bone marrow of the donor. The iNKT cells may obtained from a single donor mononuclear apheresis unit.

[0016] After iNKT cells are harvested from a healthy donor, it is necessary to isolate the donor’s iNKT cells from the donor’s non-iNKT cells. In some embodiments, the allogeneic iNKT cells are isolated from PBMCs (or other donor cells) by use of a microbeadbound monoclonal antibody to the iNKT’s invariant TCR.

[0017] The allogeneic iNKT cells of the compositions of the disclosure are stimulated and expanded ex vivo prior to administration to a subject. In some embodiments, the cells are stimulated with iNKT-specific ligand aGalCer-pulsed irradiated PBMCs. In some embodiments, the allogeneic iNKT cells are expanded by culturing the cells with IL-2, IL- 15 and / or IL-21. For instance, the disclosure includes culturing the cells in IL-2 over a period of time where IL-2 is added to the media one or more times. In some embodiments, the iNKT cells are expanded with IL- 15 alone or with IL-21. In some embodiments, the iNKT cells are expanded ex vivo by culturing the cells with IL-21.

[0018] The compositions (including allogeneic iNKT cell adjuvants) of the disclosure have a long half-life and persists in the subject’s body for weeks after a single administration. In some embodiments, administration of the allogeneic iNKT cell adjuvant results in allogeneic iNKT cells detectable in the subject for at least about 5 months, 6 months, 7 months, 8 months, or 9 months.

[0019] The disclosure includes methods of administering the iNKT cell adjuvant and iNKT cell compositions to a subject in need. In some embodiments, the subject in need has cancer or a blood malignancy. In some embodiments, the subject has a solid tumor cancer. In some embodiments, the cancer is relapsed / refractory cancer. In some embodiments, the cancer is a solid tumor cancer selected from the group consisting of gastric cancer,esophagogastric cancer, lung cancer, testicular cancer, ampullary cancer, appendiceal cancer, biliary duct cancer, cholangiocarcinoma, colorectal cancer (CRC), microsatellite stable (MSS) colorectal cancer, duodenal cancer, non-small cell lung carcinoma (NSCLC), ocular melanoma, pancreatic cancer, prostate cancer, sarcoma, and bladder cancer. In some embodiments, the relapsed / refractory cancer is selected from the group consisting of gastric cancer, esophagogastric cancer, lung cancer, testicular cancer, ampullary cancer, appendiceal cancer, biliary duct cancer, cholangiocarcinoma, colorectal cancer (CRC), micro satellite stable (MSS) colorectal cancer, duodenal cancer, non-small cell lung carcinoma (NSCLC), ocular melanoma, pancreatic cancer, prostate cancer, sarcoma, and bladder cancer. For instance, In some embodiments, the cancer is gastric cancer, lung cancer, or testicular cancer. In some embodiments, the gastric cancer is high micro satellite instability (MSI-High) gastric cancer. In some embodiments, biliary duct cancer or cholangiocarcinoma.

[0020] In some embodiments, the disclosure includes methods for treating a relapsed / refractory solid tumor cancer by administering to a subject in need thereof (i) a composition comprising an allogeneic iNKT cell adjuvant, wherein said adjuvant comprises at least 95% allogeneic invariant natural killer cells (iNKTs) isolated from a single donor; and (ii) an immune checkpoint inhibitor (ICI ). In some embodiments, the ICI comprises an anti-PDl antibody, including, for instance, pembrolizumab, nivolumab, cemiplimab, or balstilimab. In some embodiments, the ICI comprises an anti-PDLl antibody, including, for instance, atezolizumab, avelumab, or durvalumab. In some embodiments, the ICI comprises an anti-CTLA4 antibody. For instance, In some embodiments the ICI comprises ipilimumab, tremelimumab, or botensilimab. In some embodiments, the ICI comprises an anti-LAG3 antibody, for instance, relatlimab.

[0021] The disclosure includes administering the compositions of the disclosure (including iNKT cell adjuvant) to a subject suffering with cancer that has previously been treated with one or more anti-cancer therapeutics. The allogeneic iNKT cell compositions of the disclosure (including allogeneic iNKT cell adjuvant) can be administered to heavily pretreated subjects who have had limited to no response to prior anti-cancer therapeutics. In some embodiments, the subject has been previously treated with one or more of the following prior to the administration of an allogeneic iNKT cell composition of the disclosure: nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilumumab, tremelimumab, botensilimab, relatlimab, FOLFOX, CAPOX, FOLFIRI, oxaliplatin, docetaxel, irinotecan, trifluridine and / or tipiracil, leucovorin, fluorouracil, capecitabine,trastuzumab, bleomycin, etopidide, cisplatin, vinblastine, ramucirumab, carboplatin, doxorubicin, vincristine, paclitaxel, ifosfamide, vincristine, methotrexate, cyclophosphamide, etoposide, Bacillus Calmette Guerin (“BCG”), CD ID RM 684, focal radiation, an antibody drug conjugate, a bispecific antibody (e.g., a bispecific antibody targeting CD3 and a tumor antigen), an oncolytic virus, or an autologous stem cell transplant. In some embodiments of the disclosure, the subject suffering from cancer has been previously administered an agent capable of causing T cell exhaustion in the tumor microenvironment or is suffering from T cell exhaustion.

[0022] In some embodiments, the subject has not previously been administered an anti-CDld monoclonal antibody.

[0023] In some embodiments, the subject has liver metastases. For instance, the disclosure includes methods of treating a subject with relapsed / refractory cancer with liver metastases with allogeneic iNKT cells and compositions of the disclosure.

[0024] In some embodiments, after administration of the allogeneic iNKT cell adjuvant to the subject, a level of one or more immune-modulatory cytokines or chemoattractant chemokines is increased relative to the level of the one or more immune- modulatory cytokines or chemoattractant chemokines prior to the administration. For instance, In some embodiments, the level of one or more immune-modulatory cytokines or chemoattractant chemokines is measured in the blood or in biopsied tumor samples. In some embodiments, the immune-modulatory cytokine is IFNy or TNFa.

[0025] In some embodiments of the disclosure, after administration of the allogeneic iNKT cell composition (including adjuvant) to the subject, T cell infiltration of the tumor microenvironment in the subject is increased relative to the level of immune infiltration of the tumor microenvironment prior to the administration.

[0026] In some embodiments of the disclosure, after administration of the allogeneic iNKT cell composition (including adjuvant) to the subject, an expression level of one or more of the following genes is increased relative to the expression level of the one or more genes prior to the administration: GZMB, GZMH, GZMA, GZMK, GZMM, GNLY, PRF1, LEF1, TRAIL, FASLG, LAMP1, NKG7, CD25, CD69, 4-1BB, HLA-DRA, CD44, TCF1, TBET, EOMES, CCL3, CCL4, IFNG, TNF, DNAM1, 2B4, SLAMF6, NKG2D, NKG2C, NKG2A, or CD56.

[0027] In some embodiments, administration of the allogeneic iNKT cell composition of the disclosure (including adjuvant) to the subject results in inhibition of growth or shrinkage of a tumor in the subject.

[0028] In some embodiments of the disclosure, the subject suffering from a disease or condition, including cancer, is a human.

[0029] The disclosure includes pharmaceutical compositions comprising an allogeneic iNKT cell adjuvant. For instance, In some embodiments, the disclosure includes a pharmaceutical composition comprising an allogeneic iNKT cell adjuvant and a pharmaceutically acceptable carrier or excipient.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

[0031] FIG. 1 is a schematic depicting iNKT cell anti-cancer mechanisms.

[0032] FIG. 2 is the study design for agenT-797 in solid tumors.

[0033] FIGs. 3A-3B show pro-inflammatory cytokine signature. FIG. 3A shows pre- and post-infusion serum levels of select pro-inflammatory and anti-inflammatory cytokines over the first month following infusion (n=30). Data grouped into three timepoints: day 1 preinfusion (Pre; single timepoint), early post-infusion window (0.5 hours post infusion to day 8; D1-D8), and a late post-infusion time window (days 15-29; D15-29). Upper limit of cytokines in healthy people as published in the literature: IFNy: 25 pg / ml; TNFa: 30 pg / ml; IL-ip: 12 pg / ml; IL-2: 20 pg / ml; IL-4: 1 pg / ml; IL-6: 25 pg / ml. Upper limit indicated as dotted line on graphs where possible. Significant changes post dosing are indicated with asterisks (** p<0.01; *** p<0.001; Analysis using non-parametric Friedman test). The most significant early post-dosing change was observed for IFNy. FIG. 2B shows IFNy data for each timepoint. IFNy release is a hallmark of iNKT activation, and increased detection in serum may indicate iNKT activation at tumor site. No corresponding increase in IFNy or TNFa levels has been observed in our trial with agenT-797 in COVID-19 / ARDS, where an anti-inflammatory signature predominates.

[0034] FIGs. 4A-4B show persistence of agenT-797 in the periphery. Quantification of agenT-797 in patient PBMC by digital droplet PCR (ddPCR) based on single nucleotidepolymorphisms (SNPs) unique to donor material. FIG. 4A shows average persistence measurements for DL1, DL2 and total dataset highlighting peak of agenT-797 levels in the periphery on D2 post-infusion, which scales with dose level. Low level persistence in blood detected up to week 8 post-infusion. FIG. 4B shows the observed decline of agenT-797 levels in blood post D2 is consistent with the dynamics of blood- to-tis sue distribution of agenT-797 observed in vivo in a previously described murine xenograft. Analysis of tissue persistence of agenT-797 in patients is currently in-process using cell-free DNA (cfDNA) analysis (readout Q2 2023).

[0035] FIGs. 5A-5B show the change in baseline scans in patients who received agenT-797 and have baseline as well as at least one post-baseline measure of target lesions. FIG. 5A shows the percent target lesion change form baseline. FIG. 5B shows percent target lesions change from baseline over time.

[0036] FIGs. 6A-6B show responses in patients treated with agenT-797 + nivolumab. FIG. 6A are scans from MSLhigh gastric cancer patient with partial response. FIG. 5B show characteristics of patients with response to combination therapy.

[0037] FIG. 7 shows multiplex immunofluorescence (mIF) on FFPE tumor sections from a responding gastric cancer patient. On-treatment biopsy demonstrates dramatic infiltration of tumor by T cells. Infiltrate consists predominantly of CD8+ cytotoxic T lymphocytes (CTL), with Ki-67 biomarker expression indicative of recent CTL activation and cell division, (images analyzed by automated image analysis as shown in FIG. 8)

[0038] FIG. 8 shows profiling tumor and immune cells in gastric cancer patient with partial response. The lines representative of Gastric cancer patient is annotated with an arrow, and the lines representative of all other patients are not annotated with arrow . (Left to right, top to bottom) High mutational burden correlated with a high neoantigen score. Tumor infiltrating T cells demonstrated a high degree of clonality, which increased following treatment with agenT-797. TCR CDR3 sequence analysis indicates a post-treatment switch in the predominant TCR clone, with infusion of agenT-797 followed by an expansion of a minority pre-existent T cell clone (open circles, dotted line). This increase of a specific T cell clone corresponds with markers of T cell activation and proliferation as measured by mIF and automated image analysis, indicating intra-tumoral expansion of mainly CTL following treatment with agenT-797. Taken together, high tumor mutational burden leads to increased neoantigen presentation which is only partially effective at driving clonal expansion of antitumor T cells, even in the presence of anti PD-1 checkpoint inhibitors. Following administration of agenT-797 systemic and local TH-1 signatures are detected, which lead toenhanced immune infiltration of tumor and relieves the blockage of neoantigen driven expansion of anti-tumor CTL. WES / RNAseq data (top 4 graphs) from 10 patients and mIF data (bottom 3 graphs) from 16 patients.

[0039] FIGs. 9A-9D show RNAseq gene expression analysis of screening (left bar) and on-treatment (onTx) (right bar) tumor biopsies from gastric cancer patient. On-treatment tumor material (D15) exhibits signature of increased tumor immune infiltration and activity. On-treatment gene expression signatures indicate increased (FIG. 9A) cytotoxic activity, (FIG. 9B) immune cell activation, and (FIG. 9C) TH-1 polarization, and are consistent with the infiltration of tumor by cytotoxic lymphocytes including CTF, (FIG. 9D) NK cells or iNKT cells. Gene expression shown as transcripts per million (TPM).

[0040] FIG. 10 shows a schematic illustrating an assay setup to assess agenT-797 impact on immune checkpoint inhibitor (ICI) activity.

[0041] FIG. 11 shows that agenT-797 synergizes with anti-PD-1 / anti-CTFA-4 antibodies to potentiate T cell responses.

[0042] FIGs. 12A-12B show agenT-797 combination with anti-PD-1 / anti-CTFA-4 rescues exhausted T cells.

[0043] FIG. 13 shows agenT-797 boosts T cell function via DC maturation.DETAILED DESCRIPTION

[0044] The present disclosure, at least in part, relates to compositions comprising allogeneic invariant natural kill T (iNKT), and methods of using the compositions comprising the iNKT cells for treating cancer.

[0045] The foregoing and other aspects, implementations, acts, functionalities, features and embodiments of the present teachings can be more fully understood from the following description in conjunction with the accompanying drawings.I. Therapeutic Treatments Using Invariant Natural Killer T (iNKT) cells

[0046] A method of potentiating the effect of a therapeutic in a subject suffering from a disease or condition, the method comprising administering to the subject an allogeneic iNKT cell adjuvant with the therapeutic. In some embodiments, the disease or condition can be treating by methods provided herein include viral infection, inflammatory diseases, or cancers. As used herein, the terms “administering” or “administration” means to provide a therapeutic agent (e.g., iNKT cells) or a composition thereof (e.g., a composition comprisingiNKT cells) to a subject in a manner that is physiologically and / or pharmacologically useful (e.g., to treat a disease or a symptom or complication associated with the disease in the subject). As used herein, the term “subject” refers to a mammal. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has cancer. As used herein, the term “treating” or “treatment” refers to the application or administration of a composition including one or more active agents (e.g., unmodified, allogeneic iNKT cells) to a subject who has cancer, or a predisposition of cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the cancer. Alleviating cancer includes delaying or preventing the development or progression of the disease, reducing disease severity, reducing tumor volume, and / or promoting survival.

[0047] In some aspects, the present disclosure also provides a method for treating a subject having cancer.

[0048] As used herein “relapsed / refractory cancer” refers to a cancer has recurred or that did not respond to treatment or stopped responding during the course of treatment.

[0049] As used herein “cancer” refers to a group of diseases characterized by transformation of cells to a state where the cells divide uncontrollably and have the ability to infiltrate and destroy normal body tissue. In some embodiments, a cancer is a solid tumor cancer. Solid tumor cancers are characterized by development of tumors (e.g., masses of abnormal, cancerous cells) that generally do not contain cysts or liquid. Examples of solid tumors include but are not limited to sarcomas, carcinomas, and lymphomas.

[0050] Non-small cell Lung Cancer (NSCLC) is a solid tumor cancer and the most common type of lung cancer. NSCLC includes large cell lung adenocarcinoma and squamous cell carcinoma of the lung.

[0051] Gastric cancer, also referred to as stomach cancer, is a solid tumor cancer that develops in cells of the stomach lining.

[0052] Testicular cancer is a solid tumor cancer that begins in the cells of the testicles.

[0053] As used herein, an “anti-cancer therapeutic” is a therapeutic designed to treat a cancer. Examples of an anti-cancer therapeutic include, but are not limited to, nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilumumab, tremelimumab, botensilimab, relatlimab, EOLEOX, CAPOX, EOLEIRI, oxaliplatin,docetaxel, irinotecan, trifluridine and / or tipiracil, leucovorin, fluorouracil, capecitabine, trastuzumab, bleomycin, etopidide, cisplatin, vinblastine, ramucirumab, carboplatin, doxorubicin, vincristine, paclitaxel, ifosfamide, vincristine, methotrexate, cyclophosphamide, etoposide, Bacillus Calmette Guerin (“BCG”), CD ID RM 684, focal radiation, an antibody drug conjugate, a bispecific antibody (e.g., a bispecific antibody targeting CD3 and a tumor antigen), an oncolytic virus, or an autologous stem cell transplant. In some embodiments, the anti-cancer therapeutic is an immune checkpoint inhibitor (ICI).

[0054] In some embodiments, the present disclosure provides compositions (e.g., an allogeneic iNKT cell adjuvant with ICI and / or chemotherapeutic agent) for treating cancer. In some embodiments, the cancer is a blood malignancy. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a relapsed / refractory cancer.

[0055] In some embodiments, the relapsed / refractory cancer is a solid tumor cancer selected from the group consisting of gastric cancer, esophagogastric cancer, lung cancer, testicular cancer, ampullary cancer, appendiceal cancer, biliary duct cancer, cholangiocarcinoma, colorectal cancer (CRC), micro satellite stable (MSS) colorectal cancer, duodenal cancer, non-small cell lung carcinoma (NSCLC), ocular melanoma, pancreatic cancer, prostate cancer, sarcoma, and bladder cancer. In some embodiments, the relapsed / refractory cancer is gastric cancer, lung cancer, or testicular cancer. In some embodiments, the gastric cancer is high micro satellite instability (MSI-High) gastric cancer. In some embodiments, the gastric cancer is biliary duct cancer or cholangiocarcinoma. In some embodiments, the subject has liver metastasis from a primary tumor.

[0056] In some embodiments, the subject has been previously administered one or more of the following agents for treatment of cancer: nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilumumab, tremelimumab, botensilimab, relatlimab, FOLFOX, CAPOX, FOLFIRI, oxaliplatin, docetaxel, irinotecan, trifluridine and / or tipiracil, leucovorin, fluorouracil, capecitabine, trastuzumab, bleomycin, etopidide, cisplatin, vinblastine, ramucirumab, carboplatin, doxorubicin, vincristine, paclitaxel, ifosfamide, vincristine, methotrexate, cyclophosphamide, etoposide, Bacillus Calmette Guerin (“BCG”), CD ID RM 684, focal radiation, an antibody drug conjugate, a bispecific antibody (e.g., a bispecific antibody targeting CD3 and a tumor antigen), an oncolytic virus, or an autologous stem cell transplant.

[0057] The present disclosure, at least in part, relates to the discovery that administration of administration of an allogeneic iNKT cell adjuvant described herein compositions (e.g., agenT-797) to a subject having cancer (e.g., gastric cancer, lung cancer, or testicular cancer) that is resistant to ICI therapy can overcome resistance to ICI therapy. In some embodiments, a subject does not have an active autoimmune disorder. In some embodiments, subject has not previously received anti-CDld monoclonal antibody. In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein, and one or more ICI. In some embodiments, the one or more ICI comprises an anti-PDl antibody. In some embodiments, the anti-PDl antibody comprises pembrolizumab, nivolumab, cemiplimab, or balstilimab. In some embodiments, the one or more ICI comprises an anti-PDLl antibody. In some embodiments, the PDL1 antibody comprises atezolizumab, avelumab, or durvalumab. In some embodiments, the one or more ICI comprises an anti- CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody comprises ipilimumab, tremelimumab, or botensilimab. In some embodiments, the one or more ICI comprises an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody comprises relatlimab. In some embodiments, one or more of the previous treatment described herein causes T cell exhaustion in the tumor microenvironment.

[0058] In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein in with a chemotherapeutic agent reduces T cell exhaustion by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0059] In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein in with a chemotherapeutic agent. Any suitable chemotherapeutic agent can be used in methods described herein, for example, alkylating agents, plant alkaloids, antitumor antibiotics, antimetabolites, or topoisomerase inhibitors.

[0060] In some embodiments, a subject does not undergo lymphodepletion prior to, or concurrent with, treatment with administration of an allogeneic iNKT cell adjuvant described herein. Lymphodepletion is frequently performed prior to immunotherapies, such as adoptive cell therapy such as CAR T therapies. In some instances, subject receiving adoptive cell therapy receive a course of chemotherapy to deplete the T cells from the subject, for instance, to debulk a tumor, alter tumor phenotype, modify the tumor microenvironment, remove cytokine sinks (e.g., make IL-2, IL-7, and IL-15 more available) and suppress the hostimmune system. In some embodiments, lymphodepletion can effectively prolong the persistence of infused cells and increase the effectiveness of treatment. However, lymphodepletion has multiple negative effects including, neutropenia, anemia, thrombocytopenia, and immunosuppression, and toxicities associated with lymphodepletion agents such as fludarabine and cyclophosphamide. In some embodiments, a subject receiving administration of an allogeneic iNKT cell adjuvant described herein does not need lymphodepletion therapy prior to administration of the iNKT cells. In some embodiments, the subject does not receive treatment with, fludarabine, or cyclophosphamide prior to, or concurrent with, administration of an allogeneic iNKT cell adjuvant described herein. In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein without lymphodepletion are effective in reducing tumor burden and / or improve survival. In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein without lymphodepletion exhibit long term efficacy in killing cancer cells. In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein without lymphodepletion does not induce graft-versus-host disease (GVHD). In some embodiments, the tumor microenvironment is modified by (i) an increase in dendritic cells; (ii) a decrease in M2 macrophages, (iii) an increase in Ml macrophages, (iv) a decrease in myeloid derived suppressor cells, and / or an increase in tumor infiltration of cytotoxic lymphocytes as compared to treatment with the anti-cancer therapeutic alone.

[0061] In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant composition comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% iNKT cells (e.g., isolated from a single donor or two or more donors). In some embodiments, a subject is administered at least IxlO6cells / kg, at least 2xl06cells / kg, at least 3xl06cells / kg, at least 4xl06cells / kg, at least 5xl06cells / kg, at least 6xl06cells / kg, at least 7xl06cells / kg, at least8xl06cells / kg, at least 9xl06cells / kg, at least IxlO7cells / kg, at least 2xl07cells / kg, at least3xl07cells / kg, at least 4xl07cells / kg, at least 5xl07cells / kg, or more. In some embodiments, a subject is administered iNKT cells in the range of IxlO6to 5xl07cells / kg, IxlO6to 4xl07cells / kg, IxlO6to 3xl07cells / kg, IxlO6to 2xl07cells / kg, IxlO6to IxlO7cells / kg, IxlO6to 9xl06cells / kg, IxlO6to 8xl06cells / kg, IxlO6to 7xl06cells / kg, IxlO6to 6xl06cells / kg, IxlO6to 5xl06cells / kg, IxlO6to 4xl06cells / kg, IxlO6to 3xl06cells / kg, IxlO6to 2xl06cells / kg, 2xl06to 5xl07cells / kg, 2xl06to 4xl07cells / kg, 2xl06to 3xl07cells / kg, 2xl06to 2xl07cells / kg, 2xl06to IxlO7cells / kg, 2xl06to 9xl06cells / kg, 2xl06to 8xl06cells / kg,2xl06to 7xl06cells / kg, 2xl06to 6xl06cells / kg, 2xl06to 5xl06cells / kg, 2xl06to 4xl06cells / kg, 2xl06to 3xl06cells / kg, 3xl06to 5xl07cells / kg, 3xl06to 4xl07cells / kg, 3xl06to 3xl07cells / kg, 3xl06to 2xl07cells / kg, 3xl06to IxlO7cells / kg, 3xl06to 9xl06cells / kg, 3xl06to 8xl06cells / kg, 3xl06to 7xl06cells / kg, 3xl06to 6xl06cells / kg, 3xl06to 5xl06cells / kg, 3xl06to 4xl06cells / kg, 4xl06to 5xl07cells / kg, 4xl06to 4xl07cells / kg, 4xl06to 3xl07cells / kg, 4xl06to 2xl07cells / kg, 4.3xl06to 1.7xl07cells / kg, 4xl06to IxlO7cells / kg, 4xl06to 9xl06cells / kg, 4xl06to 8xl06cells / kg, 4xl06to 7xl06cells / kg, 4xl06to 6xl06cells / kg, 4xl06to 5xl06cells / kg, 5xl06to 5xl07cells / kg, 5xl06to 4xl07cells / kg, 5xl06to 3xl07cells / kg, 5xl06to 2xl07cells / kg, 5xl06to IxlO7cells / kg, 5xl06to 9xl06cells / kg, 5xl06to 8xl06cells / kg, 5xl06to 7xl06cells / kg, 5xl06to 6xl06cells / kg, 6xl06to 5xl07cells / kg, 6xl06to 4xl07cells / kg, 6xl06to 3xl07cells / kg, 6xl06to 2xl07cells / kg, 6xl06to IxlO7cells / kg, 6xl06to 9xl06cells / kg, 6xl06to 8xl06cells / kg, 6xl06to 7xl06cells / kg, 7xl06to 5xl07cells / kg, 7xl06to 4xl07cells / kg, 7xl06to 3xl07cells / kg, 7xl06to 2xl07cells / kg, 7xl06to IxlO7cells / kg, 7xl06to 9xl06cells / kg, 7xl06to 8xl06cells / kg, 8xl06to 5xl07cells / kg, 8xl06to 4xl07cells / kg, 8xl06to 3xl07cells / kg, 8xl06to 2xl07cells / kg, 8xl06to IxlO7cells / kg, 8xl06to 9xl06cells / kg, 9xl06to 5xl07cells / kg, 9xl06to 4xl07cells / kg, 9xl06to 3xl07cells / kg, 9xl06to 2xl07cells / kg, 9xl06to IxlO7cells / kg, IxlO7to 5xl07cells / kg, IxlO7to 4xl07cells / kg, IxlO7to 3xl07cells / kg, IxlO7to 2xl07cells / kg, 2xl07to 5xl07cells / kg, 2xl07to 4xl07cells / kg, 2xl07to 3xl07cells / kg, 3xl07to 5xl07cells / kg, 3xl07to 4xl07cells / kg, or 4xl07to 5xl07cells / kg.

[0062] In some aspects, the present disclosure provides composition and methods for treating a relapsed / refractory solid tumor cancer (e.g., lung cancer, gastric cancer, testicular cancer). In some embodiments, the method comprising administering to a subject in need thereof: (i) a composition comprising at least 95% allogeneic invariant natural killer cells (iNKTs); and (ii) an immune checkpoint inhibitor (ICI) (e.g., nivolumab, pembrolizumab, botensilimab or any known ICI therapy).

[0063] In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein combination with an ICI therapy, initiates cytokine (e.g., cytotoxic cytokine) release. In some embodiments, administration of iNKT cells or a composition thereof (e.g., agenT-797) increases cytotoxic cytokines (e.g., one or more of GZMB, GZMH, GZMA, GZMM, GNLY, FASLG, NKG7) in the subject by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 100%, at least 2 folds, at least 5 folds, at least 10 folds, at least 20 folds, at least 25 folds, at least 50 folds, at least 75 folds, at least 100 folds, at least 200 folds, at least 300 folds, at least 500 folds, at least 750 folds, at least 1000 folds, or more relative to the cytotoxic cytokine levels prior to administration.

[0064] In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein (e.g., agenT-797) in combination with an ICI therapy increases cytotoxic immune cell (e.g., CD8+ T cell, NK cell and / or iNKT cell) infiltration to the tumor by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 2 folds, at least 5 folds, at least 10 folds, at least 20 folds, at least 25 folds, at least 50 folds, at least 75 folds, at least 100 folds, at least 200 folds, at least 300 folds, at least 500 folds, at least 750 folds, at least 1000 folds, or more relative to the infiltrated cytotoxic immune cell level prior to administration. In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein (e.g., agenT-797) in combination with an ICI therapy increases cytotoxic immune cell (e.g., CD8+ T cell, NK cell and / or iNKT cell) infiltration to the tumor by the range of l%-100%, l%-90%, 1 %-80%, l%-70%, l%-60%, l%-70%, l%-60%, l%-50%, l%-40%, l%-30%, l%-20%, l%-10%, l%-8%, l%-5%, 1%- 3%, l%-2%, 5%-100%, 5%-90%, 5%-80%, 5%-70%, 5%-60%, 55%-50%, 5%-40%, 5%- 30%, 5%-20%, 5%-10%, 5%-8%, 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, 1-1000 folds, 1-750 folds, 1-500 folds, 1-100 folds, 1-50 folds, 1-25 folds, 1-20 folds, 1-10 folds, 1-5 folds, 1-4 folds, 1-3 folds, 1-2 folds, 2-1000 folds, 2-750 folds, 2-500 folds, 2-100 folds, 2-50 folds, 2-25 folds, 2-20 folds, 2-10 folds, 2-5 folds, 5-1000 folds, 5-750 folds, 5- 500 folds, 5-100 folds, 5-50 folds, 5-25 folds, 5-20 folds, 5-15 folds, 5-10 folds, 5-8 folds, 5- 6 folds, 10-1000 folds, 10-750 folds, 10-500 folds, 10-100 folds, 10-50 folds, 10-25 folds, 10- 20 folds, 10-15 folds, 20-1000 folds, 20-750 folds, 20-500 folds, 20-100 folds, 20-50 folds, 20-25 folds, 50-1000 folds, 50-750 folds, 50-500 folds, 50-100 folds, 50-75 folds, 100-1000folds, 100-750 folds, 100-500 folds, or 100-250 folds relative to the infiltrated cytotoxic immune cell level prior to administration.

[0065] In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein (e.g., agenT-797) in combination with an ICI therapy restores exhausted T cell (e.g., endogenous cytotoxic T cell and / or CAR T cell targeting a tumor antigen) tumor killing capacity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 2 folds, at least 5 folds, at least 10 folds, at least 20 folds, at least 25 folds, at least 50 folds, at least 75 folds, at least 100 folds, at least 200 folds, at least 300 folds, at least 500 folds, at least 750 folds, at least 1000 folds, or more relative to tumor killing capacity of exhausted T cell prior to administration. In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein (e.g., agenT-797) in combination with an ICI therapy restores exhausted T cell (e.g., endogenous cytotoxic T cell and / or CAR T cell targeting a tumor antigen) tumor killing capacity by the range of 1%- 100%, l%-90%, l%-80%, l%-70%, l%-60%, l%-70%, l%-60%, l%-50%, l%-40%, 1%- 30%, l%-20%, l%-10%, l%-8%, l%-5%, l%-3%, l%-2%, 5%-100%, 5%-90%, 5%-80%, 5%-70%, 5%-60%, 55%-50%, 5%-40%, 5%-30%, 5%-20%, 5%-10%, 5%-8%, 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, 1-1000 folds, 1-750 folds, 1-500 folds, 1-100 folds, 1-50 folds, 1-25 folds, 1-20 folds, 1-10 folds, 1-5 folds, 1-4 folds, 1-3 folds, 1-2 folds, 2-1000 folds, 2-750 folds, 2-500 folds, 2-100 folds, 2-50 folds, 2-25 folds, 2-20 folds, 2-10 folds, 2-5 folds, 5-1000 folds, 5-750 folds, 5-500 folds, 5-100 folds, 5-50 folds, 5-25 folds, 5- 20 folds, 5-15 folds, 5-10 folds, 5-8 folds, 5-6 folds, 10-1000 folds, 10-750 folds, 10-500 folds, 10-100 folds, 10-50 folds, 10-25 folds, 10-20 folds, 10-15 folds, 20-1000 folds, 20-750 folds, 20-500 folds, 20-100 folds, 20-50 folds, 20-25 folds, 50-1000 folds, 50-750 folds, SO- SOO folds, 50-100 folds, 50-75 folds, 100-1000 folds, 100-750 folds, 100-500 folds, or 100- 250 folds tumor killing capacity of exhausted T cell prior to administration.

[0066] In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein restores T cell (e.g., endogenous cytotoxic T cell and / or CAR T cell targeting a tumor antigen) tumor killing capacity by inducing dendritic cell maturation. In some embodiments, administration of an allogeneic iNKT cell adjuvant described herein induces dendritic cell (DC) maturation. In some embodiments, administration of an allogeneic iNKT cell adjuvant (i.e., contacting immature DCs with an allogeneic iNKT cell adjuvant) described herein increases dendritic cell (DC) maturation by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 2 folds, at least 5 folds, at least 10 folds, at least 20 folds, at least 25 folds, at least 50 folds, at least 75 folds, at least 100 folds, at least 200 folds, at least 300 folds, at least 500 folds, at least 750 folds, at least 1000 folds, or more relative to immature DCs not in contact with iNKT cells. In some embodiments, increasing DC maturation restores recovery of T cell tumor killing capacity. In some embodiments, administration of an allogeneic iNKT cell adjuvant restores T cell (e.g., endogenous cytotoxic T cell and / or CAR T cell targeting a tumor antigen) tumor killing capacity to at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% relative to T cell tumor killing capacity prior to exhaustion. In some embodiments, administration of an allogeneic iNKT cell adjuvant restores T cell (e.g., endogenous cytotoxic T cell and / or CAR T cell targeting a tumor antigen) tumor killing capacity without the combination of an ICI treatment. In some embodiments, administration of an allogeneic iNKT cell adjuvant restores T cell (e.g., endogenous cytotoxic T cell and / or CAR T cell targeting a tumor antigen) tumor killing capacity with the combination of an ICI treatment.

[0067] In some embodiments, an allogeneic iNKT cell adjuvant described herein have extended serum half-life (e.g., relative to iNKT cells not prepared by methods described herein) by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 2 folds, at least 5 folds, at least 10 folds, at least 20 folds, at least 25 folds, at least 50 folds, at least 75 folds, at least 100 folds, at least 200 folds, at least 300folds, at least 500 folds, at least 750 folds, at least 1000 folds, or more. In some embodiments, iNKT cells from an allogeneic iNKT cells adjuvant are detectable for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months or longer. In some embodiments, iNKT cells from an allogeneic iNKT cells adjuvant are functionally equivalent relative to freshly administered iNKT cells for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months or longer.

[0068] In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein and a therapeutic (e.g., an anti-cancer therapeutic) at the same time. In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein and a therapeutic (e.g., an anti-cancer therapeutic) in one composition. In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein and a therapeutic (e.g., an anti-cancer therapeutic) in separate compositions at the same time. In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein and a therapeutic (e.g., an anti-cancer therapeutic) concurrently. In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein and a therapeutic (e.g., an anti-cancer therapeutic) sequentially. In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein prior to a therapeutic (e.g., an anti-cancer therapeutic). In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein after a therapeutic (e.g., an anti-cancer therapeutic). In some embodiments, a subject is administered an allogeneic iNKT cell adjuvant described herein and a therapeutic (e.g., an anti-cancer therapeutic) at different frequencies. In some embodiments, since the allogeneic iNKT cell adjuvant has extended serum half-life as described herein, a subject is administered an allogeneic iNKT cell adjuvant described herein once, and a therapeutic (e.g., an anti-cancer therapeutic) more than once (e.g., once, twice, three times, four times, five times, six times, seven times, eight times, nice times, or more.II. Unmodified, Allogeneic Invariant Natural Killer T (iNKT) Cells

[0069] In some aspects, the present disclosure provides a composition comprising invariant natural killer T (iNKT) cells. The term “invariant Natural Killer T cells”, or “invariant NKT cells”, “iNKT cells”, or “Type I NKT cell), as used herein, refer to a population of T lymphocytes expressing a conserved semi-invariant TCR specific for lipid antigens restricted for the monomorphic MHC class I-related molecule CDld. Natural killer T cells (NKT cells) were originally characterized in mice as T cells that express both a TCR and NK1.1 (NKR-Pla-c or CD161), a C-type lectin NK receptor. Invariant NKT (iNKT) cells express a semi-invariant aP TCR (e.g., formed by an invariant TRAV11-TRAJ18 (4) rearrangement in mice, or the homologous invariant TRAV10-TRAJ18 chain in humans), paired with a limited set of diverse VP chains, predominantly TRBV1, TRBV29, or TRBV13 in mice (6) and TRBV25 in humans (see e.g., Dellabona et al., An invariant V alpha 24-J alpha Q / V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med. (1994) 180:1171-6. 10.1084). The semi-invariant TCR recognizes exogenous and endogenous lipid antigens presented by the monomorphic MHC class I- related molecule CDld (see e.g., Brennan et al., Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol. (2013) 13:101-17. 10.1038). Exogenous lipid antigens include the prototypical a-Galactosylceramide (a- GalCer) (Kawano et al., CD Id-restricted and TCR-mediated activation of valphal4 NKT cells by glycosylceramides. Science. (1997) 278:1626-9. 10.1126) and a number of bacterial- derived Ags, which can activate iNKT cells.

[0070] iNKT cells undergo a distinct developmental pathway compared to T cells, leading to the acquisition of innate effector functions already in the thymus. Thymic iNKT cells indeed express markers usually upregulated by peripheral effector / memory T cells, such as CD44 and CD69, together with distinctive NK differentiation markers, such as NK1.1 (in some mouse genetic backgrounds, CD161 in humans), CD122 (the IL-2R / IL-15R P-chain), CD94 / NKG2 and Ly49(A-J), and a broad spectrum of TH1 / 2 / 17 effector cytokines. Once migrated in the periphery, iNKT cells form a tissue resident population that survey the cellular integrity and rapidly respond to local damage and inflammation, jump starting the reaction by cells of the innate and adaptive immune response.

[0071] Because iNKT cells can rapidly produce IFNy, IL-4, or both, they have been found to play a role in various diseases by establishing a context-dependent Thl- or Th2- based immune response. In bacterial and viral infections, iNKT cells typically help in early control of the pathogen by establishing a productive Thl response. In both mouse and human studies, roles for iNKT cells have been described in diseases associated with excessive Thlresponses like type 1 diabetes and chronic obstructive pulmonary disease. Roles have also been described for iNKT cells helping to suppress Thl responses and drive tolerogenic responses to grafts. As an example, following hematopoietic stem cell transfer, the presence of iNKT cells is predictive for survival with a reduction in graft versus host disease (GvHD) in patients and preclinical models.

[0072] Accordingly, the present disclosure provides a composition comprising iNKT cells. iNKT cell therapy can be autologous, allogeneic or xenogeneic. In some embodiments, the subject and the donor are allogeneic. The term “allogeneic”, as used herein, is a word denoting tissue and / or cells taken from different individuals of the same species, and the tissue and / or cells are genetically dissimilar and immunologically incompatible. iNKT cells, being restricted for the monomorphic CD Id molecule, are substantially devoid of alloreactivity, allowing them to be used off-the-shelf in a donor-unrestricted manner (e.g., without causing graft- versus-host disease (GvHD)).

[0073] In some embodiments, the iNKT cells are isolated (e.g., purified or enriched) from peripheral blood mononuclear cells (PBMCs) from apheresis of the donor. In some embodiments, the isolated iNKT cells are stimulated ex vivo. In some embodiments, the isolated iNKT cells are expanded ex vivo. In some embodiments, an initial population of the iNKT cells are purified from PBMCs using a suitable method known in the art (e.g., FACS or MACS). In some embodiments, the initial population of the iNKT cells are stimulated by a - galactosylceramide (a-GalCer) or any modified glycolipid thereof, e.g., as described by Zhang et al., a-GalCer and iNKT Cell-Based Cancer Immunotherapy: Realizing the Therapeutic Potentials, Front Immunol. 2019 Jun 6; 10: 1126; Schafer et al., iNKT cell stimulation by glycolipid ligands modified from a-galactosylceramide results in differential interleukin-2 secretion profiles, J Immunol May 1, 2019, 202 (1 Supplement) 177.1) for activation and expansion. In some embodiments, the initial population of the iNKT cells are stimulated by a-GalCer while being co-cultured with PBMCs. In some embodiments, the PBMCs are irradiated prior to being co-cultured with iNKT cells. In some embodiments, the PBMCs are pulsed with a-GalCer. In some embodiments, the stimulation of iNKT cells with a-GalCer and PBMCs (e.g., irradiated PBMCs) In some embodiments, the iNKT cells can go through more than one round of stimulation as described herein. After expansion, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cells in the composition are iNKT cells. In some embodiments, the iNKT cells are unmodified (e.g., not genetically modified to express exogenous genes). The presentdisclosure also contemplates the use of iNKT cells isolated and / or expanded using any suitable known methods in the art.

[0074] In some embodiments, expanded, unmodified iNKT cells express both Thl type cytokines (e.g., IFNy, TNFoc, GM-CSF) and Th2 type cytokines (e.g., IL-4, IL-13). In some embodiments, after expansion, the iNKT cells retain their inherent cytotoxic capacity against CD Id-expressing cells. In some embodiments, the iNKT cell therapy composition is AgenT-797.

[0075] In some embodiments, the allogeneic iNKT cells or the composition thereof of the disclosure can be used as an adjuvant to potentiate the effect of a therapeutic to treat a subject with a disease or condition, including cancer. As used herein, an “adjuvant” increases or modifies the activity of a therapeutic. As used herein “allogeneic iNKT cell adjuvant” describes a composition comprising allogeneic iNKT cells which have been harvested from a healthy donor, isolated from other cell types, stimulated ex vivo, and expanded ex vivo. An allogeneic iNKT cell adjuvant of the disclosure is potentiates one or more effects of a therapeutic, including an anti-cancer therapeutic. For instance, an allogeneic iNKT cell adjuvant of the disclosure modifies the tumor microenvironment so that the tumor is not resistant to treatment by an anti-cancer therapeutic. The allogeneic iNKT cell adjuvant of the disclosure amplifies and accelerates an immune response by its interactions with other immune cells, for instance, promotion of Ml macrophages, suppression of myeloid-derived suppressor cells (MDSCs), and recruitment of tumor-reactive natural killer (NK) cells and cytotoxic lymphocytes (CLT).

[0076] The allogeneic iNKT cells of the disclosure persist in the subject’s body for weeks after administration of a single transfusion of cells. As used herein, “persistence” refers to the existence of allogeneic iNKT cells in the body after administration. In some embodiments, the allogeneic iNKT cells of the disclosure persist in the body of the subject for about six months after administration by infusion. The persistence of allogeneic iNKT cells after administration can be observed by detection of iNKT cells by methods known in the art such as SNP analysis using duplex sequencing or digital droplet PCR.

[0077] As used herein, “extended half-life allogeneic iNKT cells” refers to allogeneic iNKT cells that persist in the subject’s body for several weeks after administration by infusion. Allogeneic iNKT cells that are prepared using the methods described herein (e.g., harvesting from healthy donors, isolating iNKT cells from other cell types, stimulating iNKT cells ex vivo, and expanding iNKT cells ex vivo) persist in the subjects body for an extendedperiod of time after administration by infusion (for instance, up to at least 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months).

[0078] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials. In some embodiments, compositions of the present disclosure are formulated for intravenous administration.

[0079] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. Particular embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.EXAMPLES

[0080] The following examples are provided for illustrative purposes and are not intended to limit the scope of the disclosure.Example 1: Invariant Natural Killer T (iNKT) Cell Anti-cancer Mechanisms

[0081] iNKT cells directly recognize and kill tumor cells through TCR-dependent recognition of CD Id ligands on tumor cells or through recognition of stress signals through NK receptors NKG2D and DNAM1. Further, iNKT cells promoter anti-tumor responses within the tumor microenvironment (TME) by: 1) promoting anti-inflammatory Ml macrophages and killing tolerogenic M2 macrophages, 2) Relieving immunosuppression in the TME by suppression or killing of MDSCs, or 3) Recruitment and activation of other tumor-targeting immune cells such as NK cells and cytotoxic T cells (CTL) through interferon-gamma (IFN-y) and interleukin-2 (IL-2) (FIG. 1).Example 2: Phase 1 / 2 trial evaluating safety and efficacy of allogeneic iNKT cells, agenT-797, in patients with advanced or metastatic solid tumors

[0082] Patients were selected based on certain inclusion criteria. Individuals had a confirmed diagnosis of advanced or metastatic solid tumor that was refractory to standard therapy or for which no standard therapy was available. Patients with prior anti-CDld monoclonal antibody treatment or active autoimmune disease requiring systemic treatment were excluded. Additionally, patients had measurable disease on imaging based on RECIST1.1. As endpoints, the safety, persistence of agenT-797, and anti-tumor activity were measured as duration of response, progression-free survival, and time to response. The treatment was a 3+3 dose escalation of agenT-797 administered by single intravenous infusion. An overview of the study design is shown in FIG. 2.Example 3: agenT-797 was well tolerated in patients across multiple tumor types

[0083] Following treatment, the most common Treatment Adverse Event (TRAE) was fatigue (n=5) and the most severe TRAE (grade 3) was anemia (n=l). No grade > 3 neurotoxicity or cytokine release syndrome (CRS) was observed. agenT-797was well tolerated at all dose levels tested and no MTD was determined. Table 2 shows a summary of AEs.Table 1.

[0084] During the course of treatment, no DLTs were reported by the subjects treated. TEAEs were observed in 32 / 34 subjects (94.1%). The most common TEAEs were fatigue (n=16), nausea (n=8) and constipation (n=4) as well as peripheral oedema, abdominal distension, and dyspnea (n=6 each). TEAEs of grade >3 were experienced by a total of 19 subjects, largely consistent with disease progression. One subject (DL2, combination therapy) experienced an immune-related TEAEs (irTEAE) of grade >3 (skin rash, grade 3), likely as a result of anti-PD treatment. One subject (DL1, monotherapy) experienced a TRAE of grade 3 (anemia).Example 4: Biomarker Response in Specific Cancer Types

[0085] The biomarker response in PD-1 refractory testicular cancer showed a 41% AFP reduction at 9 months; response ongoing. The patient for this study was a 49 year old male with five failed prior therapies: 1) Bleomycin + etoposide + cisplatin (CR), 2) Vinblastine + ifosfamise +cisplatin +pembrolizumab (PR), 3) Etoposide + carboplatin +autologous stem cell transplant (PR), 4) Etoposide + ipilimumab +nivolumab (PD), 5) CD1D RM 684 (PD). A single dose of agent-797 + nivolumab (200mg) was administered at a dose level of 1.4 x 107cells. The baseline AFP response was 5.18 ng / mL and at three months the AFP was 3.06ng / mL. The AFP response and stable disease are ongoing.Example 5: Monotherapy and Combination Therapy

[0086] The agenT-797 alone and in combination with anti-PD-1 without lymphodepletion was well tolerated across multiple doses 60% DCR (2SD, 1PR) observed in 5 evaluable patients treated in combination with checkpoint inhibitors. agenT-797 persisted in the periphery for up to 8 weeks. agenT-797 demonstrated systemic and local proinflammatory TH- 1 signatures and increased tumor infiltration by cytotoxic lymphocytes (NK cells and CTL). Early signals of clinical and biomarker activity were observed in heavily pre-treated patients who progressed on prior checkpoint inhibitors. agenT-797 may drive clonal T cell expansion in cancers with high neoantigen burden. Translational data were consistent with iNKT cells to enable T cell and NK cell trafficking to tumors, reinvigorate partially exhausted CD8+ T cells, and improve effector functions within the tumor microenvironment. Observations of a proinflammatory signature associated with agenT-797 in the treatment of solid tumors contrasts with the use of agenT-797 in severe COVID19 / ARDS (NCT04582201), where an anti-inflammatory signature dominates, suggesting a plasticity of the agenT-797 mediated response in relation to the specific disease context. The Phase 1 / 2 study will be expanded into PD-1 refractory NSCLC and advanced gastric cancer to further investigate the cells in combination with immune-checkpoint- inhibitors.Example 6: AgenT-797 impact on immune checkpoint inhibitor (ICI) activity.

[0087] An assay was performed to assess agenT-797 impact on immune checkpoint inhibitor (ICI) activity (FIG. 10). PMBCs from healthy donors were co-cultured with agenT- 797 and immune checkpoint inhibitors anti-PD-1 and anti-CTLA-4 in the presence of Staphylococcal Enterotoxin A (SEA). Supernatants were collected after 96h of culture and analyzed for IL-2 by ELISA as a measure of T cell functionality.Example 7: AgenT-797 synergy with anti-PD-1 / anti-CTLA-4 antibodies

[0088] To determine whether agenT-797 synergizes with anti-PD-1 / anti-CTLA-4 antibodies to potentiate T cell responses, PBMCs were treated with anti-PD-1 and anti- CTLA-4 and / or agenT-797. Supernatants were collected after 96h of culture and analyzed for IL-2 by ELISA. As shown in EIG. 11, while agenT-797 does not respond to SEA in the absence of T cells, it significantly boosts SEA-mediated T cell activation, whether used alone or in combination with anti-PD-1 and anti-CTLA-4. The effect was maintained when matched T and iNKT donors were used, indicating absence of an allo-reactive response.Example 8: AgenT-797 combination with anti-PD-1 / anti-CTLA-4 rescues exhausted T cells

[0089] To determine whether agenT-797 combination with anti-PD-1 / anti-CTLA-4 rescues exhausted T cells, cells from the SEA assay were analyzed by flow cytometry to assess the expression of exhaustion markers, TIM-3 and LAG-3 on T cells. As shown in EIG. 12, treatment with agenT-797 led to a reduction of the proportion of TIM-3+ / LAG-3+ T cells, similar to that observed with ICI. When agenT-797 was combined with anti-PD-1 and anti-CTLA-4, the proportion of TIM-3+ / LAG-3+ T cells was reduced by an additional 20%.

[0090] To investigate whether the effect of agenT-797 on exhaustion marker expression could translate into the restoration of exhausted T cell cytotoxic potential, exhausted T cells were generated by repeatedly exposing Tumor-Associated Antigen (TAA)- specific T cells to target tumor cells. As expected, exhausted T cells showed a significantly reduced killing capacity compared to non-exhausted T cells, which was partially restored by combining them with ICIs (EIG. 12B). Addition of agenT-797 and immature dendritic cells (iDCs) to the exhausted T cells led to a similar recovery of T cell cytotoxicity without the need for ICI (anti-PD-l / anti-CTLA-4). Notably, the combination of ICIs, agenT-797, and iDCs fully restored the killing capacity of the exhausted T cells.Example 9: AgenT-797 boosts T cell function via DC maturation

[0091] To explore further the interaction between iDCs and agenT-797, CD 14+ monocytes were isolated from healthy donor PBMCs and cultured with GM-CSF and IL-4 to generate iDCs. iDCs were co-cultured with agenT-797 at 1:1 ratio to assess their differentiation into mature DCs (mDCs). Expression of the DC markers CD86, CD83 and HLA-DR was assessed by flow cytometry (FIG. 13). LPS treatment was used as positive control for DC maturation.OTHER EMBODIMENTS

[0092] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0093] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.EQUIVALENTS

[0094] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments ofthe present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0095] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. Definition of terms are disclosed throughout the specification, including but not limited to the “Definition” section.

[0096] The section heads are not meant to be interpreted to limit the scope of the present disclosure.

[0097] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0098] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0099] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “onlyone of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0100] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0101] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0102] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

CLAIMSWhat is claimed is:

1. A method of potentiating the effect of a therapeutic in a subject suffering from a disease or condition, the method comprising administering to the subject an allogeneic iNKT cell adjuvant with the therapeutic.

2. A method of modifying the tumor microenvironment in a subject with cancer to potentiate the tumor killing effects of an anti-cancer therapeutic, the method comprising administering to the subject an allogeneic iNKT cell adjuvant with the anti-cancer therapeutic.

3. The method of claim 2, wherein the tumor microenvironment is modified by (i) an increase in dendritic cells; (ii) a decrease in M2 macrophages, (iii) an increase in Ml macrophages, (iv) a decrease in myeloid derived suppressor cells, and / or an increase in tumor infiltration of cytotoxic lymphocytes as compared to treatment with the anti-cancer therapeutic alone.

4. The method of claim 2 or 3, wherein administration of the allogeneic iNKT cell adjuvant with the anti-cancer therapeutic results in an increase of cytotoxic lymphocytes to the tumor as compared to treatment with the anti-cancer therapeutic alone.

5. A method of increasing T cell tumor infiltration in a subject with cancer to potentiate the anti-tumor effects of an anti-cancer therapeutic, the method comprising administering to the subject an allogeneic iNKT cell adjuvant with the cancer drug.

6. The method of claim 5, wherein the T cells are CD8+ T cells.

7. The method of claim 5, wherein the T cells are cytotoxic T cells.

8. A method of overcoming resistance to PD-1 blockade in a subject with cancer, the method comprising administering to the subject an allogeneic iNKT cell adjuvant, wherein the subject has previously been treated with an immune checkpoint inhibitor (ICI).

9. The method of claim 6, wherein the allogeneic iNKT cell adjuvant are administered with an anti-cancer therapeutic.

10. The method of any one of claims 1-9, wherein the anti-cancer therapeutic administered with the allogeneic iNKT cell adjuvant comprises one or more chemotherapeutic agents.

11. The method of any one of claims 1-9, wherein the anti-cancer therapeutic administered with the allogeneic iNKT cell adjuvant comprises one or more immune checkpoint inhibitors (ICI) to the subject.

12. A method for treating a relapsed / refractory solid tumor cancer, the method comprising administering to a subject in need thereof:(i) a composition comprising an allogeneic iNKT cell adjuvant, wherein the allogeneic iNKT cell adjuvant comprises at least 95% allogeneic invariant natural killer cells (iNKTs) isolated from a single donor; and(ii) an immune checkpoint inhibitor (ICI ).

13. The method of claim 11 or 12, wherein the one or more ICI comprises an anti-PDl antibody.

14. The method of claim 13, wherein the anti-PDl antibody comprises pembrolizumab, nivolumab, cemiplimab, or balstilimab.

15. The method of claim 11 or 12, wherein the one or more ICI comprises an anti-PDLl antibody.

16. The method of claim 15, wherein the PDL1 antibody comprises atezolizumab, avelumab, or durvalumab.

17. The method of claim 11 or 12, wherein the one or more ICI comprises an anti-CTLA4 antibody.

18. The method of claim 17, wherein the anti-CTLA4 antibody comprises ipilimumab, tremelimumab, or botensilimab.

19. The method of claim 11 or 12, wherein the one or more ICI comprises an anti-LAG3 antibody.

20. The method of claim 19, wherein the anti-LAG3 antibody comprises relatlimab.

21. The method of claim 11 or 12, wherein the one or more ICI comprises nivolumab, pembrolizumab, and / or botensilimab.

22. The method of claim 14 or 21, wherein the method comprises administering 200 mg of nivolumab to the subject.

23. The method of 1-22, wherein the subject is administered one or more immune checkpoint inhibitors and one or more chemotherapeutic agents.

24. The method of any one of claims 1-23, wherein said disease or condition is a cancer or blood malignancy.

25. The method of any one of claims 2-24, wherein the cancer is a blood malignancy.

26. The method of any one of claims 2-24 wherein the cancer is a solid tumor cancer.

27. The method of any one of claims 2-26, wherein the cancer is a relapsed / refractory cancer.

28. The method of claim 27, wherein the relapsed / refractory cancer is a solid tumor cancer selected from the group consisting of gastric cancer, esophagogastric cancer, lung cancer, testicular cancer, ampullary cancer, appendiceal cancer, biliary duct cancer, cholangiocarcinoma, colorectal cancer (CRC), micro satellite stable (MSS) colorectal cancer, duodenal cancer, non-small cell lung carcinoma (NSCLC), ocular melanoma, pancreatic cancer, prostate cancer, sarcoma, and bladder cancer.

29. The method of claim 27 or 28, wherein the relapsed / refractory cancer is gastric cancer, lung cancer, or testicular cancer.

30. The method of claim 29, wherein the gastric cancer is high microsatellite instability (MSLHigh) gastric cancer.

31. The method of claim 29, wherein the gastric cancer is biliary duct cancer or cholangiocarcinoma.

32. The method of any one of claims 1-31, wherein the subject has been previously administered one or more of the following agents for treatment of cancer: nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilumumab, tremelimumab, botensilimab, relatlimab, FOLFOX, CAPOX, FOLFIRI, oxaliplatin, docetaxel, irinotecan, trifluridine and / or tipiracil, leucovorin, fluorouracil, capecitabine, trastuzumab, bleomycin, etopidide, cisplatin, vinblastine, ramucirumab, carboplatin, doxorubicin, vincristine, paclitaxel, ifosfamide, vincristine, methotrexate, cyclophosphamide, etoposide, Bacillus Calmette Guerin (“BCG”), CD ID RM 684, focal radiation, an antibody drug conjugate, a bispecific antibody (e.g., a bispecific antibody targeting CD3 and a tumor antigen), an oncolytic virus, or an autologous stem cell transplant.

33. The method of any one of claims 1-32, wherein the subject has been previously administered an agent capable of causing T cell exhaustion in the tumor microenvironment or is suffering from T cell exhaustion.

34. The method of any one of claims 1-33, wherein the subject has not previously been administered an anti-CDld monoclonal antibody.

35. The method of any one of claims 1-34, wherein the subject has liver metastases.

36. The method of any one of claims 1-35, wherein the subject does not have an active autoimmune disorder.

37. The method of any one of claims 1-36, wherein the allogeneic iNKT cell adjuvant is administered at the same time as the therapeutic.

38. The method of any one of claims 1-36, wherein the allogeneic iNKT cell adjuvant is administered prior to the therapeutic.

39. The method of any one of claims 1-38, wherein the allogeneic iNKT cell adjuvant comprises allogeneic iNKT cells obtained from the peripheral blood, PBMCs, or bone marrow of one or more healthy donors.

40. The method of any one of claims 1-39, wherein the allogeneic iNKT cell adjuvant comprises allogeneic iNKT cells obtained from a single healthy donor mononuclear apheresis unit.

41. The method of any one of claims 1-40, wherein the allogeneic iNKT cell adjuvant is prepared comprising the steps of(i) harvesting iNKT cells from one or more healthy human donors;(ii) isolating the iNKT cells from other cell types;(iii) stimulating the iNKT cells;(iv) expanding the iNKT cells ex vivo; and(vi) formulating the iNKT cells with pharmaceutically acceptable carrier or excipient.

42. The method of any one of claims 1-41, wherein the allogeneic iNKT cell adjuvant comprises iNKT cells which have been isolated from PBMCs by microbead-bound monoclonal antibody to the iNKT TCR.

43. The method of any one of claims 1-42, wherein the allogeneic iNKT cell adjuvant comprises allogeneic iNKT cells stimulated ex vivo with iNKT-specific ligand aGalCer- pulsed irradiated PBMCs.

44. The method of any one of claims 1-43, wherein the allogeneic iNKT cell adjuvant comprises allogeneic iNKT cells expanded ex vivo by culturing with interleukin-2 (IL-2), interleukin- 15 (IL- 15), and / or interleukin-21 (IL-21).

45. The method of claim 44, wherein the allogeneic iNKT cells have been expanded ex vivo by culturing with IL-2.

46. The method of claim 44, wherein the allogeneic iNKT cells have been expanded ex vivo by culturing with (i) IL- 15 or (ii) IL- 15 and IL-21.

47. The method of claim 44, wherein the allogeneic iNKT cells have been expanded ex vivo by culturing with IL-21.

48. The method of any one of claims 1-47, wherein the allogeneic iNKT cell adjuvant comprises from about 4.3 x 106to about 1.4 x 107iNKT cells.

49. The method of any one of claims 1-48, wherein the subject has gastric cancer and is administered about 4.3 x 106iNKT cells / kg of body weight from the allogeneic iNKT cell adjuvant.

50. The method of any one of claims 1-48, wherein the subject has testicular cancer and is administered about 1.4 x 107iNKT cells / kg of body weight from the allogeneic iNKT cell adjuvant.

51. The method of any one of claims 1-50, wherein administration of the allogeneic iNKT cell adjuvant results in detectable allogeneic iNKT cells in the subject for at least about 5 months, 6 months, 7 months, 8 months, or 9 months.

52. The method of any one of claims 1-51, wherein the allogeneic iNKT cell adjuvant is not HLA matched to the subject.

53. The method of any one of claims 1-52, wherein the allogeneic iNKT cell adjuvant administered to the subject is at least about 80%, 85%, 90%, 95%, or 99% free of other cell types.

54. The method of any one of claims 1-53, wherein the allogeneic iNKT cell adjuvant comprises CD8+ iNKT cells.

55. The method of any one of claims 1-54, wherein the allogeneic iNKT cell adjuvant comprises CD4+ iNKT cells.

56. The method of any one of claims 1-55, wherein the allogeneic iNKT cell adjuvant comprises CD4- and CD8- iNKT cells (double negative iNKT cells).

57. The method of any one of claims 1-56, wherein after administration of the allogeneic iNKT cell adjuvant to the subject, a level of one or more immune-modulatory cytokines or chemoattractant chemokines is increased relative to the level of the one or more immune- modulatory cytokines or chemoattractant chemokines prior to the administration.

58. The method of claim 57, wherein the level of one or more immune-modulatory cytokines or chemoattract chemokines is measured in the blood or in biopsied tumor samples.

59. The method of claim 57 or 58 , wherein the one or more immune-modulatory cytokine is IFNy or TNFa.

60. The method of any one of claims 2-59, wherein after administration of the allogeneic iNKT cell adjuvant to the subject, T cell infiltration of the tumor microenvironment in the subject is increased relative to the level of immune infiltration of the tumor microenvironment prior to the administration.

61. The method of any one of claims 2-60, wherein after administration of the allogeneic iNKT cell adjuvant to the subject, an expression level of one or more of the following genes is increased relative to the expression level of the one or more genes prior to the administration: GZMB, GZMH, GZMA, GZMK, GZMM, GNLY, PRF1, LEF1, TRAIL, FASLG, LAMP1, NKG7, CD25, CD69, 4-1BB, HLA-DRA, CD44, TCF1, TBET, EOMES, CCL3, CCL4, IFNG, TNF, DNAM1, 2B4, SLAMF6, NKG2D, NKG2C, NKG2A, or CD56.

62. The method of any one of claims 2-61, wherein administration of the allogeneic iNKT cell adjuvant to the subject results in inhibition of growth or shrinkage of a tumor in the subject.

63. The method of any one of claims 1-62, wherein the subject is a human.

64. An adjuvant for potentiating one or more effects of a therapeutic comprising allogeneic iNKT cells, wherein said allogeneic iNKT cells are prepared in a method comprising:(i) harvesting allogeneic iNKT cells from the peripheral blood, PBMCs, or bone marrow from a healthy donor or a single healthy donor mononuclear apheresis unit;(ii) isolating the allogeneic iNKT cells from other donor cells;(iii) stimulating the allogeneic iNKT cells ex vivo; and(iv) expanding the allogeneic iNKT cells ex vivo.

65. The adjuvant of claim 64, wherein the adjuvant potentiates the efficacy of a chemotherapeutic agent, checkpoint inhibitor, or other anti-cancer therapeutic.

66. The adjuvant of claim 64 or 65, wherein the adjuvant modifies the tumor microenvironment.

67. The adjuvant of any one of claims 64-66, wherein the allogeneic iNKT cells are isolated from the donor’s PBMCs by microbead-bound monoclonal antibody to the iNKT TCR.

68. The adjuvant of any one of claims 64-67, wherein the allogeneic iNKT cells are stimulated with iNKT-specific ligand aGalCer-pulsed irradiated PBMCs.

69. The adjuvant of any one of claims 64-68, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the with interleukin-2 (IL-2).

70. The adjuvant of any one of claims 64-68, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with interleukin- 15 (IL- 15).

71. The adjuvant of any one of claims 64-68, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with interleukin-21 (IL-21).

72. The adjuvant of any one of claims 64-71, wherein the allogeneic iNKT cells compriseCD8+ iNKT cells.

73. The adjuvant of any one of claims 64-72, wherein the allogeneic iNKT cells comprise CD4+ iNKT cells.

74. The adjuvant of any one of the claims of 64-73, wherein the allogeneic iNKT cells comprises CD8- and CD4- iNKT cells (double negative iNKT cells).

75. The adjuvant of any one of claims 64-74, wherein the allogeneic iNKT cells are detectable in the subject for at least about 5 months, 6 months, 7 months, 8 months, or 9 months or more after administration.

76. The adjuvant of any one of claims 64-75, wherein the adjuvant comprises 10 x 106to 1,000 x 106allogeneic iNKT cells.

77. A pharmaceutical composition comprising the adjuvant of claims 64-76 and a pharmaceutically acceptable carrier or excipient.

78. The pharmaceutical composition of claim 77, further comprising a checkpoint inhibitor.

79. The pharmaceutical composition of claim 77 or 78, wherein the pharmaceutical composition is used for the treatment of a subject with cancer or blood malignancy.

80. The pharmaceutical composition of claim 79, wherein the cancer is a relapsed / refractory solid tumor cancer.

81. The pharmaceutical composition of claim 80, wherein the relapsed / refractory solid tumor cancer is gastric cancer, esophagogastric cancer, lung cancer, testicular cancer, ampullary cancer, appendiceal cancer, biliary duct cancer, cholangiocarcinoma, colorectal cancer (CRC), micro satellite stable (MSS) colorectal cancer, duodenal cancer, non-small cell lung carcinoma (NSCLC), ocular melanoma, pancreatic cancer, prostate cancer, sarcoma, or bladder cancer.

82. The pharmaceutical composition of claim 80 or 81, wherein the relapsed / refractory solid tumor cancer is gastric cancer, lung cancer, or testicular cancer.

83. The pharmaceutical composition of claim 82, wherein the gastric cancer is high micro satellite instability (MSI-High) gastric cancer.

84. The pharmaceutical composition of claim 82, wherein the gastric cancer is biliary duct cancer or cholangiocarcinoma.

85. A pharmaceutical composition for treatment of a subject suffering from a disease or a condition comprising extended half-life allogeneic iNKT cells and a pharmaceutically acceptable carrier or excipient, wherein said iNKT cells are isolated from a healthy donor and expanded ex vivo prior to use by culturing said cells in interleukin-2 (IL-2), interleukin- 15 (IL- 15), and / or interleukin-21 (IL-21).

86. A pharmaceutical composition for treatment of a subject suffering from a disease or condition comprising extended half-life allogeneic iNKT cells and a pharmaceutically acceptable carrier or excipient, wherein said extended half-life allogeneic iNKT cells are produced by a method comprising(i) harvesting cells from the peripheral blood, PBMCs, or bone marrow from a healthy donor or a single healthy donor mononuclear apheresis unit;(ii) isolating the allogeneic iNKT cells from other donor cells;(iii) stimulating the allogeneic iNKT cells; and(iv) expanding the allogeneic iNKT cells ex vivo.

87. The pharmaceutical composition of claim 85 or 86, wherein the allogeneic iNKT cells are isolated from the donor’ s PBMCs by microbead-bound monoclonal antibody to the iNKT TCR.

88. The pharmaceutical composition of any one of claims 85-87, wherein the allogeneic iNKT cells are stimulated with iNKT-specific ligand aGalCer-pulsed irradiated PBMCs.

89. The pharmaceutical composition of any one of claims 85-88, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with interleukin-2 (IL-2), interleukin- 15 (IL-15), and / or interleukin-21 (IL-21).

90. The pharmaceutical composition of claim 89, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with IL-2.

91. The pharmaceutical composition of claim 89, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with IL- 15.

92. The pharmaceutical composition of claim 89, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with IL-21.

93. The pharmaceutical composition of claim 89, wherein the allogeneic iNKT cells are expanded ex vivo by culturing the cells with IL- 15 and IL-21.

94. The pharmaceutical composition of any one of claims 85-93, wherein the allogeneic iNKT cells comprise CD8+ iNKT cells.

95. The pharmaceutical composition of any one of claims 85-94, wherein the allogeneic iNKT cells comprise CD4+ iNKT cells.

96. The pharmaceutical composition of any one of claims 85-95, wherein the allogeneic iNKT cells comprise CD8- and CD4- iNKT cells (double negative iNKT cells).

97. The pharmaceutical composition of any one of claims 85-96, wherein the allogeneic iNKT cells are detectable in the subject for at least about 5 months, 6 months, 7 months, 8 months, or 9 months.

98. A method for manufacturing extended half-life allogeneic iNKT cells for treatment of a disease or a condition comprising(i) harvesting cells from the peripheral blood, PBMCs, or bone marrow from a healthy donor or a single healthy donor mononuclear apheresis unit;(ii) isolating iNKT cells from other donor cells;(iii) stimulating iNKT cells; and(iv) expanding iNKT cells ex vivo.

99. The method claim 98, wherein step (ii) comprises isolating iNKT cells from the donor’ s PBMCs by microbead-bound monoclonal antibody to the iNKT TCR.

100. The method of claim 98 or 99, wherein step (iii) comprises stimulating the iNKT cells with iNKT-specific ligand aGalCer-pulsed irradiated PBMCs.

101. The method of any one of claims 98-100, wherein step (iv) comprises expanding the iNKT cells ex vivo by culturing the cells with interleukin-2 (IL-2), interleukin- 15 (IL-15), and / or interleukin-21 (IL-21).

102. The method claim 101, wherein step (iv) comprises expanding the iNKT cells ex vivo by culturing the cells with IL-2.

103. The method of claim 101, wherein step (iv) comprises expanding the iNKT cells ex vivo by culturing the cells with IL- 15.

104. The method of claim 101, wherein step (iv) comprises expanding the iNKT cells ex vivo by culturing the cells with IL-21.

105. The method of claim 101, wherein step (iv) comprises expanding the iNKT cells ex vivo by culturing the cells with IL- 15 and IL-21.

106. The method of claims 98-105, wherein the manufactured allogeneic iNKT cells are detectable in the subject for at least about 5 months, 6 months, 7 months, 8 months, or 9 months.

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