WO2024054518A1 - Systèmes et procédés d'amélioration des populations immunitaires réactives aux tumeurs à l'aide d'organoïdes - Google Patents

Systèmes et procédés d'amélioration des populations immunitaires réactives aux tumeurs à l'aide d'organoïdes Download PDF

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WO2024054518A1
WO2024054518A1 PCT/US2023/032111 US2023032111W WO2024054518A1 WO 2024054518 A1 WO2024054518 A1 WO 2024054518A1 US 2023032111 W US2023032111 W US 2023032111W WO 2024054518 A1 WO2024054518 A1 WO 2024054518A1
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cells
tumor
days
tils
time period
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Marc LEUSHACKE
Jihang JU
Lisiena HYSENAJ
Adam MARGOLIN
Cecile Chartier-Courtaud
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Nextvivo, Inc.
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
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    • C12N2513/00—3D culture

Definitions

  • Adoptive cellular therapy utilizes cells from immune systems, such as tumor-reactive immune ceils, for cancer or tumor treatment.
  • This form of immunotherapy obtains cells, e.g., tumor-reactive immune cells, from patients. These cells are then expanded or, in some instances, engineered or reprogrammed to enhance their ability to target and eliminate cancer cells. These cells are then given back to the patients for cancer treatment. The process of expanding tumor- reactive immune cells and the quality of tumor-reactive immune cells are still limited.
  • the present disclosure provides a method of generating subject derived immune ceils, the method comprising: (a) obtaining one or more tumor samples from a subject, wherein the one or more tumor samples comprise immune cells; (b) incubating the one or more tumor samples using an in vitro culture process, wherein the one or more tumor samples are not submerged in culture medium, wherein one or more agents are added to the culture medium; and (c) collecting the immune ceils from the one or more tumor samples, thereby generating the subject derived immune cells.
  • the incubating of (b) comprises a first time period and a second time period.
  • the first time period is at least about 1 day. Ln some embodiments, the first time period is about 7 to about 14 days.
  • the second time period is at least about I day. In some embodiments, the second time period is about 11 days.
  • the one or more agents are added to the culture medium at a first concentration during the first time period. In some embodiments, the one or more agents are added to the culture medium at a second concentration during the second time period. In some embodiments, the first concentration is lower than the second concentration.
  • the in vitro culture process comprises an air-liquid interface setup.
  • the method further comprises, cry opreserving the immune cells from (c). In some embodiments, the method further comprises (d) expanding the immune cells obtained from (c) using one or more agents for a time period. In some embodiments, the method further comprises (e) collecting the immune cells from (d). In some embodiments, the method further comprises after (e), (1) cry opreserving the immune cells.
  • the immune cells comprise tumor infiltrating lymphocytes.
  • the tumor infiltrating lymphocytes comprise T cells.
  • the T cells comprise aetivated T cells, naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4+ I cells, helper T cells, e.g. T H l , Tu2, T H 9, T H 17, Tu22, L m memory T cells, e.g. central memory T cells, T stem cell memory cells (TSCM), effector memory T cells, NKT cells, or yd T cells.
  • the one or more agents in (b) comprises a cytokine.
  • the cytokine comprises IL-2, an IL-2 variant, II.. -7, an IL-7 variant, IL- 15, an IL- 15 variant, IL-18, an IL- 18 variant, IL-21, an IL-21 variant, or combination thereof.
  • the concentration of the cytokine added during the first time period in (b) i s at least about 10 IL ml... In some embodiments, the concentration of the cytokine added during the first time period in (b) is about 50 IL ml...
  • the concentration of the cytokine added during the second time period in (b) is at least about 4000 IU/mL. In some embodiments, the concentration of the cytokine added during the second time period in (b) is about 6000 IU/mL.
  • the expanding of the immune cells in (d) does not comprise an air-liquid interface setup.
  • the time period in (d) is at least 1 day. In some embodiments, the time period in (d) is about 14 days.
  • the one or more agents in (d) comprises a cytokine, an antibody, a modulator, or any combination thereof.
  • the one or more agents further comprises irradiated feeder cells.
  • the irradiated feeder cells are irradiated allogeneic PBMC-derived feeder cells.
  • a ratio of the immune cells to the irradiated feeder cells is about 1 : 100.
  • the cytokine comprises IL-2, an IL-2 variant, IL-7, an IL-7 variant, IL-15, an IL-15 variant, IL-18, an IL-18 variant, IL-2.1, an IL-21 variant, or any combination thereof.
  • the cytokine is added at a concentration of at least about 2000 ll ; ml... In. some embodiments, the cytokine is added at a concentration of about 3000 IU ml..
  • the antibody comprises an anti-CD3 antibody.
  • the concentration of the anti-CD3 antibody is at least about 10 ng/mL. Ln some embodiments, the concentration of the anti-CD3 antibody is about 10 ng/mL. In. some embodiments, the concentration of the anti-CD3 antibody is about 30 ng ml .
  • the modulator comprises a Notch, signaling pathway modulator, an interferon gamma. (IFNy) modulator, or a combination thereof.
  • the Notch signaling pathway modulator comprises a Notch activator.
  • the Notch activator comprises an antibody, a small molecule, or a combination thereof.
  • the IFNy modulator comprises an IFNy inhibitor.
  • the IFNy inhibitor comprises an antibody, a small molecule, or a. combination thereof.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise one or more of PD-1 , CD39, 4-lBB-positive T cells, or CXCR3-binding chemokines.
  • the CXCR3-binding chemokines comprise CXCL9 or CXCL 10.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise interferons.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise checkpoint inhibitors.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise TLR3, TLR.7, TLR9, or other TLR. agonists.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise modulators of RIG-I-like receptors, modulators of NOD-like receptors, modulators ofC-type lectin receptors, modulators of STING, or combination thereof
  • the method further comprises combining tumor organoids with immune cells obtained from, other sources.
  • the other sources comprise peripheral blood cells or organoids grown from lymphoid tissue.
  • the method further comprises stimulating antigen presentation.
  • the method further comprises depletion of i mmune inhibi tory cell types.
  • the immune inhibitory cell types comprise Tregs, myeloid derived suppressor cells, TAMs, vascular endothelial cells, or CAEs.
  • the method further comprises negative selection of bystander tumor reactive immune cells.
  • the method further comprises knock-down exhaustion regulator.
  • the knock-down exhaustion regulator is TOX.
  • the method further comprises reprogramming the immune cells.
  • the immune cells are reprogrammed by activation of Notch signaling pathway during (d), inhibition of interferon gamma (IFNy) signaling pathway during (d), or both.
  • the method further comprises identifying T cell receptor (TCR) to identify TCRs enriched in the air-liquid interface culture.
  • TCR T cell receptor
  • the identifying TCR is performed using sequencing technology.
  • the method comprises providing one or more tumor antigens during the first time period or the second time period.
  • the providing of one or more tumor antigens comprises providing cells expressing the tumor antigens.
  • FIG. 1 depicts an exemplary timeline for a pre-Rapid Expansion Protocol (prc-REP) step of a Standard Tumor Infiltrating Lymphocytes (STD TILs) protocol and of an air-liquid interface Tumor Infiltrating Lymphocytes (ALI TILs) protocol.
  • FIG. 2 shows a graph illustrating that co-culturing of air-liquid interface Tumor Infi ltrating Lymphocytes (ALI TILs) with autologous tumor epi thelial cells demonstrates a better tumor killing capacity compared to co-culturing of standard Tumor Infiltrating Lymphocytes (or STD TILs) with autologous tumor epithelial cells.
  • the Y-axis represents % of live tumor epithelial cells.
  • FIGS. 3A-3C show that ALI TILs are more reactive to tumor cells compared to STD TILs
  • FIG. 3A shows an experimental outline.
  • FIG* 3B and FIG. 3C show graphs illustrating that ALI Tumor Infiltrating Lymphocytes (ALI TILs) are more reactive to tumor cells compared to standard TILs (STD TILs) based on the percentage of CD3-r cells secreting IFNy as shown in FIG. 3B, and percentage of CD3+ cells expressing GDI 07a. as shown in FIG. 3C.
  • FIGS. 4A-4D show that ALI TILs express higher levels of HLA-DR compared to STD TILs.
  • FIG. 4A shows an experimental outline.
  • FIG. 4B, FIG. 4C, and FIG. 4D show graphs illustrating the expression levels ofPDI, HLA-DR, and CD 137, respectively.
  • FIG. 4C shows that ALI Tumor Infiltrating Lymphocytes (ALI TILs) express higher levels of HLA-DR compared to standard TILs (STD TILs).
  • ALI TILs ALI Tumor Infiltrating Lymphocytes
  • STD TILs standard TILs
  • FIGS. 6A-6C illustrate an example of the establishment of ALI tumor organoids.
  • FIG. 6A shows an example of ALI tumor organoids generated from tissue obtained from kidney, lung, esophagus, and uterus
  • FIG. 6B shows an example of ALI tumor organoids generated from tissue obtained from colon and glioblastoma (GBM).
  • Top figures show samples grown as ALI organoids.
  • Bottom figures show submerged organoids.
  • FIG. 6C shows sequencing results for three colorectal cancer (CRC) organoid lines, which are shown as CNV plots and mutated genes (ARC, TP53, KRAS).
  • CRC colorectal cancer
  • FIGS. 7A-7C demonstrate the generation and phenotypic characterization of ALI TILs.
  • FIG. 7 A depi cts a schematic representation of the 2-step ALI TIL process and cryopreservation of the product.
  • FIG. 7B shows cell number of ALI TILs obtained from 14 preps (left) and the fold-expansion di stribution of the 14 products (right).
  • FIG. 7C shows results of flow cytometry analysis using CD3, ySTCR, CD4, CDS, CD45RA, and CD62L to detect T cells, lineage, and memory subsets. Results are plotted as percent parent for each individual sample with average and SEM, [0038] FIGS.
  • FIG. 8A and 8B show ALI TIL tumor reactivity and cytotoxicity detection.
  • FIG. 8 A shows flow cytometry analysis of ALI TILs from CRC co-cultured with autologous tumor organoids. Increased JFNy, CD 1.07a, and 4- IBB (or GDI 37) were observed.
  • FIG. 8B shows results obtained from confocal imaging at 0 hour (Oh) and 24 hours (24h) of co-culturing experiment of ALI TILs and autologous tumor organoids. Tumor cell killing was observed over time.
  • FIGS. 9 A and 9B display the results of single-cell RNA seq analysis of CRC and melanoma ALI-TILs.
  • FIG. 9 A shows numbers of unique clonotypes relative to number of sequenced cells (upper row) and distribution of clonotype frequencies (lower row).
  • FIG. 9B shows expression levels of 397 immune genes which identify 10 clusters using SeqGeq.
  • Adoptive cellular immunotherapy utilizes a patient’s own immune cells for cancer treatment.
  • the immune cells are isolated from a. patient’s own blood or tumor tissue, grown and expanded in the laboratory, and then given back to the patient to treat the patient’s cancer.
  • the immune ceils are engineered to enhance their ability to target cancer cells, e.g., as in chimeric antigen receptor (CAR) T ceil therapies.
  • CAR chimeric antigen receptor
  • Tumor Infiltrating Lymphocytes are another type of adoptive cellular immunotherapy that has been developed for treating solid tumors.
  • Lymphocytes or white blood cells e.g., T cells or B cells
  • TILs or TIL are parts of the immune system that help the body fight infections and eliminate abnormal cells, e.g., cancer or tumor.
  • TILs Tumor Infiltrating Lymphocytes
  • TILs are known to kill cancer cells.
  • TILs are obtained directly from the tumor, they recognize targets on the cancer cells. Further, it is possible to obtain a group of TILs from the tumor which can recognize multiple unique targets on the cancer cells in order to anticipate and prevent the tumor from adapting to the therapy.
  • TILs are first collected from the tumor during a biopsy. Expansion of TILs in vitro is next performed in order to obtain a large population of these immune cells suitable for administration to the patient.
  • the present disclosure provides systems and methods for enhancing tumor-reactive immune populations using patient derived organoids (PDOs).
  • PDOs patient derived organoids
  • pre-REP Rapid Expansion Protocol
  • STD standard
  • REP Rapid Expansion Protocol
  • STD standard
  • the method of pre-REP step discloses an additional time period (e.g,, an additional week) of low concentration of IL-2 treatment as compared to standard protocol.
  • TILs obtained from PDOs also called “air-liquid interface TIL” or “ALI TIL”
  • AL1 TILs obtained from this present disclosure may provide effective immune ceils for cancer immunotherapy, e.g., adoptive cellular therapy.
  • the present disclosure provides methods for reprogramming TILs to stem memory immune cells at the REP step by inhibiting IFNy signaling pathway and activating Notch signaling pathway.
  • the stem cell-like properties of the reprogrammed immune cells provide improved lifespan, self-renewal capacity, and effector differentiation potential, towards generating anti-tumor or anti-cancer TILs with augmented anti-tumor or anti-cancer activity.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifical ly disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute.
  • a “subject” can be a biological entity containing expressed, genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro,
  • the subject can be a. mammal
  • the mammal can be human, primate, a non-human primate, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, etc.
  • the subject may include, but is not limited to, human, cow, dog, mouse, rat, rabbit, guinea pig, chicken, fish, bird, reptile, camelid, bovine, chimpanzee, sheep, goat, and non-human primate.
  • the subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • in vivo is used to describe an event that takes place in a subject's body.
  • TThhee tteerrmm “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ⁇ ?x wvo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject . An example of an ex vivo assay performed on a sample is an “in vitro” assay.
  • in vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • in vitro assays can encompass cell-based assays in which living or dead cells arc employed.
  • In vitro assays can also encompass a cell -free assay in which no intact cells are employed.
  • the term “about” a number refers to that number plus or minus 10% of that number.
  • the term “about” a range refers to that range minus 10% of its lowest value and plus 1.0% of its greatest value.
  • the term “immune cell” refers to cells that are of hematopoietic origin and that play a role in the immune response.
  • the immune cells comprise lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • the immune cells comprise a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (Tll.si isolated from an individual in need of treatment.
  • the term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and/or T cell antigen receptor.
  • the T cells comprise naive CDS'" T cells, cytotoxic CDS' T cells, naive CD4" T cells, helper T cells, e.g. T H .l, T H 2, TH9, TH 17, TH.22, TFH; memory T cells, e.g. central memory T cells, T stem ceil memory ceils (TSCM), effector memory T cells, NKT cells, T cells.
  • TSCM T stem ceil memory ceils
  • adoptive cellular therapy refers to a type of immunotherapy that utilizes patient’s own immune cells, e.g., T cells or B cells, to help the body fight diseases, e.g., cancer or tumor.
  • adoptive cellular therapy is also known as adoptive cell transfer, cellular adoptive immunotherapy, or T-cell, transfer therapy.
  • cancer or “tumor” are used interchangeably to refer to cells which exhibit autonomous, unregulated growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation.
  • Cells or tissues of interest for detection, analysis, or treatment in the present disclosure comprise precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Cancers of virtually every tissue are known. Further, cancer is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation.
  • cancer cell refers to a cancer cell or is derived from a cancer cell e.g. clone of a cancer cell.
  • cancers are known to those of skill in the ail, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc.
  • cancer examples comprise ovarian cancer, breast cancer, coion cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, esophageal cancer, uterine cancer, and brain cancer.
  • TILs tumor infiltrating lymphocytes
  • TILs tumor infiltrating lymphocytes
  • I'll ,.s can recognize and destroy cancer cells.
  • TILs are obtained from patient’s tumor.
  • TILs are obtained from patient-derived tumor sample.
  • TILs are obtained from patient-derived organoids. Examples of TILs include, but are not limited to, T lymphocytes, e.g., CD4+ or CDSr T-cells, B lymphocytes, macrophages, or NK cells.
  • the term s "cell culture” or “culture'’ refer to the main tenance of cells in an artificial, in vitro environment.
  • the term “cell culture” is a generic term and may be used to encompass the cultivation not only of individual cells, but also of tissues or organs or samples that are parts of tissues or organs and are derived from a patient.
  • the term “culture system” is used herein to refer to the culture conditions in which the cells, tissues, organs, or samples that are derived from tissues or organs of a patient are grown. This culture system promotes prolonged tissue or cell expansion with proliferation, multilineage differentiation and recapitulation of cellular and tissue ultrastructure.
  • a culture system refers to an air-liquid interface (ALI) 3D culture system.
  • a culture system also refers to a non-ALI culture in which cells of interest can be expanded, e.g. on feeder layer cells.
  • the terms “air-liquid interface” or “ALI” refer to the interface to which the tumor cells, tissues, or samples derived from a patient’s tissue or organ are exposed to in the cultures described herein.
  • the primary tissue may be mixed with a gel solution which is then poured over a layer of gel formed in a container with a lower semi- permeable support, e.g., a membrane.
  • the container is placed in an outer container that contains the medium such that the gel containing the tissue in not submerged in the medium, In some instances, the gel solution containing the primary tissue is exposed to air from the top and to liquid medium from the bottom.
  • the term “container” refers to a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.
  • organoid refers to a 3 ⁇ dimensional growth of tumor tissue in culture that retains characteristics of die tumor in vivo. In some instances, organoid recapitulates cel lular and tissue ultrastructure, immune cell interactions, etc. In some instances, organoids for use in the methods described herein are generally cultured from a tumor biopsy section, In some instances, the organoids may be generated using any method known in the art, depending on the application. Methods of organoid culture as described in the present disclosure, comprise a submerged method, an air-liquid interface method, a droplet and bioreactor method, etc. [0063] As used herei n, the term “gel substrate” refers to conventional meaning of a semisolid extracellular matrix.
  • Gel described here in includes, but is not limited to, collagen gel, matrigel, extracellular matrix proteins, fibronectin, collagen in various combinations with one or more of laminin, entactin (nidogen), fibronectin, and heparin sulfate; or human placental extracellular matrix.
  • ultrastructure refers to the three-dimensional structure of a cell or tissue observed in vivo.
  • examples of ultrastructure include, but is not limited to, the ultrastructure of a ceil which can be its polarity or its morphology in vivo, or the ultrastructure of a tissue which can be the arrangement of different cell types relative to one another within a tissue.
  • biological sample refers to liquid samples of biological origin (e.g., blood, sputum, semen, mucus, urine, cerebrospinal fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof for example clinical samples or tissue obtained by surgical resection, tissue obtained by biopsy 7 , cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.
  • biological origin e.g., blood, sputum, semen, mucus, urine, cerebrospinal fluid
  • solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof for example clinical samples or tissue obtained by surgical resection, tissue obtained by biopsy 7 , cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.
  • a “biological sample” comprises a sample obtained from a patient’s cancer cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s cancer cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising cancer cells from a patient.
  • A. biological sample comprises a cancer cell from a patient and can also compose non-cancerous cells. In some instances, samples have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations.
  • the term sample also comprises sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.
  • air-liquid interface immune cells or “ALI immune cells’ are used interchangeably to refer to in vitro isolated immune cells obtained from systems and methods described herein. These immune cells are obtained from PDO culture derived from one or more tumor samples from a subject and are undergone activation and expansion using systems and methods described herein.
  • immune cell refers to cells that are of hematopoietic origin and that play a role in the immune response.
  • the immune cells comprise lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • the immune cells comprise a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment.
  • TILs tumor infiltrating lymphocytes
  • air-liquid interlace immune cells refers to “air-liquid interface tumor infiltrating lymphocytes”, “AIJ TILs”, or “ALI TIL”.
  • treatment or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include, but are not limited to, a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptom s associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect comprises delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a. particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • Immunotherapy utilizes immune cells to defend the body from infection and disease.
  • immune checkpoint therapy utilizes immune cells, e.g., TILs, along with immune checkpoint inhibitors that block a negative immune checkpoint protein, e.g., CTLA-4, PD-1 L, or PD-.1, to allow T cells to continue working and to eliminate cancer cells.
  • Cancer vaccines stimulate immune cells to recognize and destroy cancer cells.
  • Monoclonal antibodies can attach to specific proteins on the cancer cell surface or on immune cells and increase the ability of immune cells to fight cancer.
  • Cytokine therapy utilizes cytokines, e.g,, interferons and or interleukins to trigger an immune response to fight cancer.
  • Adoptive cellular therapy utilizes patient’s own immune cells to fight cancer after in vitro expansion or modification.
  • Adoptive cellular therapy focuses on increasing the number and/or improving the effectiveness of immune cells.
  • patient In adoptive cellular therapy, patient’s immune cells are isolated, expanded or modified in vitro, and then infused back into the patient to help the immune system fight cancer.
  • Chimeric Antigen Receptor (CAR) T cell therapy modifies T cells so that they are better at recognizing and attacking cancer cells.
  • Chimeric Antigen Receptor (CAR) natural killer (NK) cell therapy modifies NK cells instead of T cells to fight cancer.
  • Some T cell therapies obtain tumor reactive T cells from blood and selects only those that recognize specific signatures of the cancer cells. These cells are then expanded before being given back to the patient.
  • Tumor Infiltrating Lymphocyte (Til.,) therapy utilizes patient’s lymphocytes that are isolated from a tumor, expanded m vitro, and then given back to the patient.
  • Adoptive cellular therapy involves obtaining immune cells, e.g., TILs, from a patient’s tumor.
  • the tumor can be obtained from the patient via. biopsy or surgical resection.
  • the patient’s tumor is derived and cultured as an organoid, e.g., patient derived organoids (PDOs) in an in vitro environment.
  • PDOs patient derived organoids
  • TILs immune cells
  • pre-REP pre-Rapid Expansion Protocol
  • TCR T cell receptor
  • the present disclosure pro vides systems and methods for enhancing tumor-reactive immune populations using patient derived organoids (PDOs).
  • PDOs patient derived organoids
  • the methods provided herein involve a pre-Rapid Expansion Protocol or pre-REP step comprising an additional time period (e.g., a week) of low concentration IL-2 treatment.
  • Immune cells e.g., TILs, obtained from PDOs (also called “air-liquid interface immune cells, e.g., TILs,” or “ALI immune cells, e.g., TILs”) utilized in this protocol are shown to have a better tumor killing capacity and are more reactive to tumor cells when compared to immune cells, e.g., TILs, obtained from the standard protocol.
  • ALI immune cells e.g., ALI TILs
  • obtained from this present disclosure provides are more effective for cancer immunotherapy than TILs obtained from a standard protocol (e.g., without the additional time period of low concentration II.. -2 treatment).
  • the present disclosure provides systems and methods for reprogramming TILs, e.g,, obtained from PDOs, to have stem cell-like properties. In some instances, the reprogramming of TILs can be performed at the REP step.
  • the systems and methods described herein are improved as compared to standard methods of generating or expanding immune cells, e.g., TILs.
  • the systems and methods described herein for generating and/or expanding immune cells, e.g., TILs provide an improvement for immunotherapy.
  • the immune cells, e.g,, TILs, obtained from the present disclosure provide an improvement of tumor killing capacity and reactivity.
  • the immune cells e.g., TILs
  • TILs stem memory immune cell phenotypes
  • stem-cell like properties such as improved lifespan, self-renewal capacity, and effector differential potential, thereby augmenting anti-tumor activity.
  • tissues used in the present disclosure comprise adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, glioblastoma (GBM), peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood NonHodgkin’s lymphoma, colon and. rectum cancer, endometrial cancer, esophagus cancer, Ewing's fami ly of tumors (e.g.
  • Ewing’s sarcoma eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, Mon-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma
  • the systems and methods provide immune cells, e.g., TILs, for adoptive cellular therapy.
  • the immune cells, e.g., TILs, obtained from the systems and methods described herein are used for adoptive cellular therapy alone.
  • the immune cells, e.g., TILs, obtained from the systems and methods described herein are used in combination with other immunotherapies, e.g., immune checkpoint therapy, monoclonal antibodies therapy, cytokine therapy, etc.
  • the immune cells, e.g., TILs, obtained from the systems and methods described herein are used in combination with chemotherapy.
  • the immune cells, e.g., TILs, obtained from the systems and methods described herein are used in combination with other cancer therapies, e.g,, drug therapy.
  • PDOs Patient-derived organoids
  • PDOs are 3-dimentional in vitro cultures that recapitulate the cellular architecture and ultrastnicture of the tumor or tissue samples from which they arc derived from.
  • PDOs comprise immune cells, e.g., tumor infiltrating lymphocytes, parenchymal element, stromal element, epithelial cells, etc., associated with the tumor or tissue in vivo.
  • PDOs are cultured in an air-liquid interface (AU) system.
  • AU air-liquid interface
  • PDO can be used for drug screening assay, in vitro disease modeling, or generation and/or expansion of immune cells, e.g., TILs, for immunotherapies, e.g., adoptive cellular therapy.
  • the air- liquid interface (ALI) method provides a culture system that allows the propagation of organoids both wi th epi thelial and stromal components of tumors.
  • the ALI method utilizes Boyden chambers (cell culture inserts) popularly used for cell migration assays.
  • cells are embedded in extracellular matrix (ECM) gels in an upper surface of the cell culture inserts with a porous membrane underneath, which substantially increases the oxygen supply to the cells compared to an epithelial-only submerged organoid method.
  • ECM extracellular matrix
  • cells obtain nutrients and growth factors from the medium placed in the outer dish through diffusion across the porous membrane on the lower surface.
  • the AU method provides many benefits for organoid culture - ALI method not only maintains stromal and immune cells from the tissue samples, but this system also retains the tumor microenvironment for an extended period of time.
  • Example of ALI method to culture PDOs is described, for example, as in Neal et al. Cell. 2018 Dec 13; 175(7): 1972-1988.e 16 which is incorporated herein in its entirety by reference.
  • alternative organoid cultures can be used.
  • tissue is embedded into droplets of cell culture matrix, e.g., basement membrane extract (BM E) or ECM gel, and then transferred into spinning bioreactors.
  • BM E basement membrane extract
  • ECM gel e.g., ECM gel
  • the continuous agitation provides improved absorption of nutrients and oxygen.
  • the present disclosure provides culture systems and methods for the generation and expansion of tumor-specific immune cells by organoid culture of solid tumors, including stromal and immune cells associated with the tumors in vivo, to activate and expand TIL cells, e.g. T cells, specific for the tumor-associated antigens.
  • the PDO culture and method described herein comprises tumor cells, immune cells, etc., associated with the tumor or tissue in vivo.
  • the PDO culture and method described herein recapitulate the cellular architecture and ultrastructure of the tumor or tissue samples from which they are derived from.
  • a biological sample is obtained to generate a PDO culture
  • the biological sample comprises a tumor tissue sample.
  • the tumor tissue samples can be obtained by any convenient method, e.g., by biopsy, e.g. during endoscopy, during surgery, by needle, etc.
  • a septic technique is utilized to obtain the tumor tissue samples.
  • the tumor tissue samples comprise human tissue, particularly cancer and other lesions, e.g., solid tumor microbiopsy samples such as needle or fine needle aspirate.
  • tlie tumor tissue samples are taken at a single timepoint. In some embodiments, the tumor tissue samples are taken at multiple timepoints.
  • tlie tumor tissue samples can be as small as ells, KF cells, 10 5 cells, or less.
  • the tumor tissue samples can be a tumor biopsy section of from about 0. 1 mm 2 , about 1 mm 2 , about 10 mm 2 , etc,
  • tissue sample is removed, the tissue is immersed in ice-cold buffered solution, e.g., PBS, Ham’s F12, MEM, culture medium, etc.
  • pieces of tissue can be minced to a size less than about 1 mm A 3, and can be less than about 0.5 mm 3. or less than about 0.1 mm A 3.
  • the minced tissue is mixed with a gel substrate, e.g., a collagen gel. solution, e.g. Cellmatrix type I- A collagen (Nitta Gelatin Inc.); a mairigel solution, etc.
  • a gel substrate e.g., a collagen gel. solution, e.g. Cellmatrix type I- A collagen (Nitta Gelatin Inc.); a mairigel solution, etc.
  • the tissue-containing gel substrate is layered over a layer of gel (a “foundation layer”) in a contai ner with a lower semi-permeable support, e.g., a membrane, supporting the foundation gel layer, and the tissue-containing gel substrate is allowed to solidify.
  • This container is placed into an outer container containing a suitable medium, for example HAMs F-12 medium supplemented with fetal calf serum (FCS) at a concentration of from about 1 to about 25%, usually from about 5 to about 20%, etc.
  • FCS fetal calf serum
  • an alternative serum to FCS can be used in the methods described herein.
  • parts of tumor tissue samples are preserved for different applications or experiments, e.g, histology or sequencing.
  • parts of tumor tissue samples are frozen for different applications or experiments.
  • Various methods known in the art can be used to preserve tumor tissue samples.
  • the arrangement of the PDO culture as described above al lows nutrients to travel from the bottom, through the membrane and the foundation gel layer to the gel layer containing the tissue
  • the level of the medium is maintained such that the top part of the gel, e.g., the gel layer containing the tissue sample, is not submerged in liquid but is exposed to air.
  • the tissue is grown in a gel with an air-liquid interface environment.
  • Example of an air-liquid interface culture system is described, for example, as in Ootanl el al. in Nat Med.
  • the air-liquid interface organoid cultures can be moved into other formats such as multi-wells for screening or in submerged 2D or 3D geometries where the cells are placed underneath the tissue culture medium.
  • the PDO cultures can be maintained for up to 5 days, up to 7 days, up to 10 days, up to 15 days, up to 21 days, up to 28 days, or more.
  • tissue e.g., primary tissue
  • tissue is obtained from a solid tumor.
  • one or more tumor samples are obtained from a subject to generate PDO culture.
  • the tumor tissue may be from any mammalian species, e.g., human, non-human primates, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, etc.
  • the subject includes, but is not limited to human, cow, dog, mouse, rat, rabbit, guinea pig, chicken, fish, bird, reptile, camelid, bovine, chimpanzee, sheep, goat, and non-human primate.
  • the subject is human.
  • the PDO cultures can be passaged for maintenance.
  • the PDO cultures can be cryopreserved.
  • the PDO cultures can be cryopreserved and then cryorecovered for downstream applications.
  • the PDO cultures described herein comprise cognate immune cells, e.g., endogenous immune cells present in the biopsy sample; allogeneic T cells; etc.
  • Immune cells that can be cultured with PDOs include, but are not limited to, T cells, macrophages, B cells, natural killer cells (NK cells), etc., including any of the T cell subsets.
  • the PDO cultures can comprise exogenous agents that are added to activate T cells that are present in the culture.
  • agents that activate T cells can be added to the culture.
  • the agents that activate T cells comprises immune checkpoint inhibitors, e.g,, agents such as antibodies that inhibit the activity of CTLA4 (Cytotoxic T-Lymphocyte-Associated protein 4, CD152), PD1 (also known as PD-1;
  • PD-IJ Programmed Death 1 receptor
  • PD-I..2 LAG-3 (Lymphocyte Activation Gene-3)
  • 0X40 A2AR (Adenosine A2A receptor)
  • B7-H3 CD276
  • B7-H4 VTCN1
  • BTLA B and T Lymphocyte Attenuator, CD272
  • IDO Indoleamine 2, 3 ⁇ dioxygena.se
  • cytokine can comprise one or more mutations in the amino acid sequence compared to wild-type sequence.
  • Variants of cytokine can be used to modulate a. target signaling pathway.
  • variants of cytokine include, but are not limited to, mutated variant or truncated variant.
  • a combination of agents that activate T cells are added to cultures.
  • Combinations of agents can comprise a combination of two or more of any of the agents listed above, in some embodiments, activation of T cells comprises protocols to reverse T cell exhaustion, e.g. pulsatile stimulation, addition of kinase inhibitors such as dasatinib, etc.
  • the culture when PDOs are cultured with agents that activate T cells, the culture can be maintained for any period of time to activate T cells.
  • culturing time with agents that activate T cells can be for up to 2 days, up to .3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 1 1 days, up to 12 days, up to 13 days, up to 14 days, up to 15 days, or more than 15 days.
  • culturing time with agents that activate T cells can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least .17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 2.3 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days.
  • T cell activation can be assessed.
  • Activated T ceils can be identified and quantitated based on a number of criteria, e.g., expression of CD3, CD25, CD69, CD137, CD107a, Granzyme B (GZMB), Perforin 1 (PRF1 ), PD1 , etc.
  • GZMB Granzyme B
  • PRF1 Perforin 1
  • PD1 PD1
  • activated T cells can be isolated based on expression of these activation markers.
  • non-acti.vated PDO cultures e.g., cultures that are not treated with a T cell activation agent, can be used as a control.
  • the PDO culture is an ALI culture.
  • PDOs or ALI organoids can be cryopreserved.
  • PDOs or ALI organoids can be cryorecovered.
  • PDOs or ALI organoids can be passaged to maintain organoid culture.
  • tire PDO cultures provide the immune cells comprising a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs), that can further be expanded and later used for an individual in need of treatment such as in adoptive cell therapy.
  • TILs tumor infiltrating lymphocytes
  • the immune cells e.g., TILs, comprises T cells. EXPANSION OF AIR-LIQUID INTERFACE TUMOR INFILTRATING LYMPHOCYTES (ALI TILS)
  • PDO cultures also called ALI culture in the present disclosure
  • these PDOs can be utilized to expand immune cells such as Tumor Infiltrating Lymphocytes (TILs) using Rapid Expansion Protocol (REP).
  • TILs Tumor Infiltrating Lymphocytes
  • REP Rapid Expansion Protocol
  • immune cells from the ALI culture are first maintained in the pre-Rapid Expansion Protocol (pre-REP) step.
  • pre-REP pre-Rapid Expansion Protocol
  • ALI derived immune ceils are treated with cytokines, e.g., IL-2, to activate these immune cells, e.g., TIL.
  • ALI derived immune cells are treated with two different concentrations of cytokines during two different time periods, In some instances, the first concentration of cytokines used during the first time period in the pre-REP step is lower than the second concentration of cytokines used during the second time period in the pre-REP step.
  • ALI derived immune cells are subjected to a REP step.
  • immune cells undergo rapid expansion, resulting in a large population of immune cells, e.g., TILs.
  • ALI derived immune cells are cultured with a cytokine, e.g., IL-2, an antibody, e.g., anti-CD3 antibody, and feeder cells, e.g,, PBMC-derived feeder cells. These immune cells are cultured for a period of time unti l a large population of immune cells are achieved. These immune cells can then be harvested and used for immunotherapy or for other applications.
  • the PDO-derived TILs or ALI- derived TILs are further reprogrammed to have stem cell-like properties, such as improved lifespan, self-renewal capacity, and effector differentiation potential, thereby augmenting antitumor activity of these cells.
  • these immune cells e.g., TILs are reprogrammed into stem cel 1-1 ike properties during the REP step, such as by activating Notch signaling pathway, inhibiting interferon gamma (IFNy) signaling pathway, or a combination of both.
  • the immune cells, e.g., TILs comprise T cells.
  • pre-REP Pre-Rapid Expansion Protocol
  • immune cells e.g., TILs
  • Activation of immune cells occurs in a. pre-REP step, and this process trains immune cells, e.g., TILs, to be more effective at expansion during REP step and better at targeting tumor cells.
  • the present disclosure provides a method of generating air-liquid interface (ALI) derived immune ceils, wherein the method comprises (a) obtaining one or more tumor samples from a subject, wherein the one or more tumor samples comprise immune cells;
  • incubating the one or more tumor samples using in vitro culture process comprises a first time period and a second time period.
  • the one or more agents are added to the culture medium at a first concentration during the first time period.
  • the one or more agents are added to the culture medium at a second concentration during the second time period.
  • the first concentration is lower than the second concentration.
  • the in vitro culture process comprises an air-liquid interlace setup.
  • the present disclosure provides systems and methods for enhancing immune cells obtained from patient derived organoids (PDOs).
  • the immune cells comprise tumor-reactive immune cells, e.g., TILs.
  • the pre-Rapid Expansion Protocol or pre-REP step involves treating the immune cells using different concentrations of cytokine, e.g., IL-2, during a first time period and a second time period.
  • the pre-REP step described herein discloses treating the immune cells during the first time period using a low concentration of cytokine before treating the immune cells with a higher concentration of cytokine during the second time period.
  • Treating immune cells, e.g., TILs, with a low concentration o f cytokine during the first time period in the pre-REP step helps maintain the immune cells in vitro and also prevents the immune cells from dying.
  • immune cells, e.g., TILs, obtained using the protocol described herein have a better tumor killing capacity and are more reactive to tumor cells when compared to immune cells, e.g., TILs, obtained from the standard protocol, which uses one concentration of the cytokine.
  • the present disclosure provides systems and methods to generate and expand effective immune cells for cancer immunotherapy and other applications, such as immune cells that are more effective for cancer immunotherapy than the standard protocol.
  • the immune cells comprise tumor infiltrating lymphocytes (TILs).
  • TILs comprise T cells.
  • the immune cells e.g., TILs, comprises T cells.
  • the T cells comprise activated T cells, naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4+ T cells, helper T cells, e.g. T H 1, T H 2, T H 9, TH 17, TH22, T FH : memory T cells, e.g. central memory T cells, T stem cell memory cells (TSCM), effector memory T cells, NKT cells, or yd T cells.
  • TSCM T stem cell memory cells
  • the one or more agents comprises a cytokine.
  • the cytokine used in the pre-REP protocol comprises IL-2, an IL-2 variant, IL-7, an IL-7 variant, IL-15, an IL-15 variant, IL-18, an IL-18 variant, IL-21 , an IL-21 variant, or combination thereof.
  • Variants of cytokine can comprise one or more mutations in the amino acid sequence compared to wild-type sequence. Variants of cytokine can. be used to modulate a target signaling pathway. Examples of variants of cytokine include, but are not limited to, mutated variant or truncated variant
  • the concentration of cytokine added in the pre-REP step during the first time period is at least about 10 IU/mL, at least about 20 IU/mL, at least about 30 IU/mL, at least about 40 IU/mL, at least about 50 IU/mL, at least about 60 IU/mL, at least about 70 IU/mL, at least about 80 I U/mL, at least about 90 IU/mL.
  • the concentration of cytokine added in the pre-REP step during the first time period is at most about 50 IU/mL, at most about 60 IU ml , at most about 70 I U/mL, at most about 80 IU/mL, at most about 90 IU/mL, at most about .100 IU/mL, at most about 500 IU/mL, at most about 1000 IU/mL, at most about 2000 IU/mL, at most about 3000 IU ml. at most about 4000 IU/mL, at most about 5000 IU ml.
  • the concentration of cytokine added in the pre-REP step during the first time period is about 10 IU/mL, about 20 IU/mL, about 30 IU/mL, about 40 IU/mL, about 50 IU/mL, about 60 IU ml.,, about 70 IU/mL, about 80 IU ml... about 90 IU/mL, about 100 IU ml...
  • the concentration of cytokine added in the pre-REP step during the first time period is at least about 10 IU ml... In some embodiments, the concentration of cytokine added in the pre-REP step during the first time period is about 50 I U/mL, In. some embodimen ts, the concentration of cytokine added in the pre- REP step during the first time period is about 150 IU/mL.
  • the concentration of cytokine added in the pre-REP step during the first time period is from about 10 IU/mL to about 12000 IU nil. from about 20 IU/mL to about 12000 IU ml., from about 30 IU/mL to about .12000 IU InL. from, about 40 IU/mL to about 12000 IU ml, from about 50 IU/mL to about 12000 IU ml , from about 60 IU/mL to about 12000 IU ml, from about 70 IU nil. to about 12000 IU nil , from about 80
  • IU ml to about 12000 IU ml. from about 90 IU/mL to about 12000 IU /mL, from about 100 JU/mL to about 12000 IU /mL, from about 500 lU/mL to about 12000 JU /mL, from about 1000 lU/rnL to about 12000 IU ml.., from about 2000 IU ml, to about 12000 IU ml, from about 3000 lU/ml, to about 12000 JU /mL, from about 4000 JU/mL to about 12000 IU /mL, from about 5000
  • the concentration of the cytokine added during the first time period in the pre-REP step is about 10 lU/ml, to about 10000 JU ml,, about 20 IU ml, to about 10000 IU nil , about 30 IL ml. to about 10000 JU/mL, about 40 IL ml. to about 10000 IlJ/mL, about 50 lU/mL to about 10000 IU nd .. about 60 IU mL to about 10000 IU nil , about 70 IU ml.
  • the concentration of cytokine added in the pre-REP step during the first time period is about 50 IU nd. to about 10000 lU/ml,
  • the first time period of the pre-REP step is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days. In some embodiments, the first time period of the pre-REP step is at most about !
  • the first time period of the pre-REP step is from about 1 to about 14 days, about 2 to about 14 days, about 3 to about 14 days, about 4 to about 14 days, about 5 to about 14 days, about 6 to about 14 days, about 7 to about 14 days, about 8 days, at most about 9 days, at most about 10 days, at most about 1 1 days, at most about 12 days, at most about 13 days, at most about 14 days, or at most about 15 days.
  • the first time period of the pre-REP step is from about 1 to about 14 days, about 2 to about 14 days, about 3 to about 14 days, about 4 to about 14 days, about 5 to about 14 days, about 6 to about 14 days, about 7 to about 14 days, about 8 to about 14 days, or about 9 to about 14 days.
  • the first time period of the pre-REP is about 7 days to about 14 days. In various embodiments, the first time period of the pre-REP step is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days. In some embodiments, the first time period of the pre-REP step is at least about 1 day. In some embodiments, the first time period of the pre-REP step is about 7 days. In some embodiments, the first time period of the pre-REP step is about 7 to about 14 days.
  • the concentration of cytokine added in the pre-REP step during the second time period is at least about 10 IU/mL, at least about 20 IUML, at least about 30 I U mL, at least about 40 IU/mL. at least about 50 11 ml... at least about 60 IU nd.., al least about 70 IU/mL, at least about 80 IU/mL, at least about 90 IU/mL, at least about 100 IU/mL, at least about 500 IUML, at least about 1000 IUML, at least about 2000 IU ml., al least about 3000 IU nd . at least about 4000 IU/mL, at least about 5000 IE ml..
  • the concentration of cytokine used in the pre-REP step during the second time period is at most about 50 IUML, at most about 60 IUML, at most about 70 IUML, at most about 80 IUML. at most about 90 IU mL, at most about 100 IU/mL, al most about 500 IU ml , at most about 1000 IUML, at most about 2000 IU/mL, at most about
  • the concentration of cytokine added in the pre-REP step during the second time period is about 10 IU ml... about 20 IUML, about 30 11 ; nd... about 40 IU nd... about 50 IUML, about 60 IUML, about 70 LU/mL, about 80 IU/mL, about 90 IUML, about 100 IU/mL, about 500 IU ml. about WOO JU ml. . about 2000 IUML, about 3000 IU nd... about 4000 IU ml.
  • the concentration of cytokine added in the pre-REP step during the second time period is at least about 4000 IU/mL. In some embodiments, the concentration of cytokine added in the pre-REP step during the second time period i s about 6000 IU nd..
  • the concentration of cytokine added in the pre-REP step during the second time period is from about 10 IU/mL to about 12000 1U ml, from about 20 IUML to about 12000 IU nd,, from about 30 If ml. to about 12000 IU . ml , from about 40
  • IU ml to about 12000 IU ML from about 2000 IU/mL to about 12000 IU ML. from about 3000 IU/mL to about 12000 IU /mL, from about 4000 IU/mL to about 12000 IU /mL, from about 5000 IU/mL to about 12000 IU ml, from about 6000 II' ml., to about 12000 IU ml... from about 7000 IU/mL to about 12000 JU /mL, from about 8000 IU/mL to about 12000 IU /mL, from about 9000 IU/mL to about 12000 IU /mL, or from about 10000 IU/mL to about 12000 JU /mL.
  • the concentration of the cytokine added during the second time period in the pre-REP step is from about 10 IU/mL to about 10000 IU/mL, from about 20 IU ml. to about 10000 IU/mL, from about 30 IU/mL to about 10000 IU nd.. from about 40 ll ml to about 10000 IU/mL, from about 50 IU/mL, to about 10000 IU/mL, from about 60
  • IU ml. to about 10000 IU/mL from about 500 IU/mL to about 10000 IU ml , from about 1000 IU/mL to about 10000 IU/mL, from about 2000 IU/mL to about 10000 IU/mL, from about 3000 IU/mL to about 10000 IU/mL, from about 4000 IU/mL to about 10000 IU/mL, from about 5000 IU/mL to about 10000 IU/mL, from about 6000 IU/mL to about 10000 IU/mL, from about 7000 IU/mL to about 10000 lU inL.
  • the concentration of the cytokine added during the second time period in the pre-REP step is from about 5000 IU/mL to about 10000 IU/mL.
  • the second time period of the pre-REP step is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, or at least about 24 days.
  • the second time period of the pre-REP step is at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 1 1 days, at most about 12 days, at most about 13 days, at most about 14 days, at most about 15 days, at most about .16 days, at most about 17 days, at most about 18 days, at most about 19 days, at most about 20 days, at most about 21 days, at most about 22 days, at most about 23 days, or at most about 24 days, In.
  • the second time period of the pre-REP step is from about 1 to about 24 days, from about 2 to about 24 days, from about 3 to about 24 days, from about 4 to about 24 days, from about 5 to about 24 days, from about 6 to about 24 days, from about 7 to about 24 days, from about 8 to about 24 days, from about 9 to about 24 days, from about i 0 to about 24 days, from about 1 1 to about 24 days, from about 12 to about 24 days, from about 13 to about 24 days, from about 14 to about 24 days, from about 15 to about 24 days, from about 16 to about 24 days, from about 17 to about 24 days, from about 18 to about 24 days, from about 19 to about 24 days, from about 20 to about 24 days, from about 21 to about 24 days, from about 22 to about 24 days, or from about 23 to about 24 days.
  • the second time period of the pre-REP step is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, or about 24 days.
  • the second time period of the pre-REP step is at least about I day. In some embodiments, the second time period of the pre-REP step is about 11 days. In some embodiments, the second time period of the pre-REP step is about 7 to 14 days. In some embodiments, the second time period of the pre-REP step is about 11 to 14 days.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise one or more of PD-.1 , CD.39, 4-lBB-positive T cells, or CXCR3-binding chemokines.
  • the CXCR3-binding chemokines comprise CXCL9 or CXCLIO.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the addi tional reagents comprise interferons.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise checkpoint inhibitors.
  • the method further comprises providing additional reagents during the first time period or the second time period, wherein the additional reagents comprise TLR3, TLR7, TLR9, or other TI..R agonists. In some embodiments, the method further comprises providing additional reagents during the first time period or the second time period, wherein tiie additional reagents comprise modulators of RIG-l-like receptors, modulators of NOD-like receptors, modulators ofC-type lectin receptors, modulators of STING, or combination thereof. [0102] In some embodiments, the method further comprises combining tumor organoids with immune cells obtained from other sources, In some embodiments, the other sources comprise peripheral blood cells or organoids grown from lymphoid tissue.
  • the method further comprises stimulating antigen presentation.
  • tiie method further comprises depletion of immune inhibitory 7 cell types.
  • the immune inhibitory cell types comprise Tregs, myeloid derived suppressor cells, TAMs, vascular endothelial cells, or CAFs,
  • the method further comprises negati ve selection of bystander tumor reactive immune cells.
  • the method further comprises knock-down exhaustion regulator.
  • the knock-down exhaustion regulator is TON.
  • the method comprises providing one or more tumor antigens during the first time period or the second time period, In some embodiments, the providing of one or more tumor antigens comprises providing ceils expressing the tumor antigens.
  • the immune cells are collected for expansion using Rapid Expansion Protocol (REP). In some embodiments, after the pre-REP, the immune cells are collected for other applications. In some embodiments, after the pre-REP, the immune cells are cryopreserved. In some embodiments, the immune cells are cryorecovered after cryo preservation, In some embodiments, the method further comprises cry op reserving the immune cells after collecting the immune cells from the one or more tissue samples. In some embodiments, the immune cells comprise tumor infiltrating lymphocytes. In some embodiments, the tumor infiltrating lymphocytes comprise T cells.
  • the immune cells e.g., TILs
  • the immune cells undergo activation as described in the pre-REP step
  • the immune cells are subjected to expansion.
  • the immune ceils e.g., TILs
  • the immune ceils are rapidly expanded by exposing the TILs to a cytokine, an antibody, a modulator, feeder cells, or any combination thereof
  • these immune cells can be reprogrammed into stem cell-like properties in this REP step, such as via Notch signaling pathway activation and interferon gamma ( IFN y) inhibition.
  • IFN y interferon gamma
  • the present disclosure provides a method of generating ALI derived immune cells, wherein the method further comprises (d) expanding the immune cells after collecting the immune cells from the one or more tissue samples using one or more agents for a time period. In some embodiments, the method further comprises (c) collecting the immune cells after expanding tiie immune cells obtained from tiie isolation of the immune cells from the one or more tissue samples. In some embodiments, the method further comprises, after collecting the immune cells after the expansion, (f) cryopreserving the immune cells.
  • the immune cells comprise tumor-reactive immune cells, e.g., TILs. In some embodiments, the immune cells comprise tumor infiltrating lymphocytes.
  • the tumor infiltrating lymphocytes comprise T cells
  • the T cells comprise activated T cells, naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4-E T cells, helper T cells, e.g. TH I , TH2, TH9, TH 17, TH22, TFH; memory T cells, e.g. central memory T cells, T stem cell memory cells (TSCM ), effector memory T cells, NKT cells, or yd T cells.
  • T cells comprise activated T cells, naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4-E T cells, helper T cells, e.g. TH I , TH2, TH9, TH 17, TH22, TFH; memory T cells, e.g. central memory T cells, T stem cell memory cells (TSCM ), effector memory T cells, NKT cells, or yd T cells.
  • TSCM T stem cell memory cells
  • the immune cells are rapidly expanded through the REP step.
  • the expanding of the immune cells does not comprise an airliquid interface setup.
  • the immune cells, e.g., TILs are expanded in the non air-liquid interface (non-ALI) culture system.
  • the immune cells, e.g., TILs, used in the REP step is collected from the pre-REP step as described above.
  • the REP step comprises culturing the immune cells, e.g., TILs, in a non-ALI culture.
  • the one or more agents used in the REP step comprise a cytokine, an antibody, a modulator, or any combination thereof.
  • the REP culture comprises cytokines, an antibody, a modulator, feeder cells, or any combination thereof.
  • the immune cells e.g., TILs, comprises T cells.
  • the cytokines used in the REP culture comprise IL-2, an IL-2 variant, IL-7, an IL-7 variant, IL-15, an IL-15 variant, IL-18, an IL-18 variant, IL-21 , an IL-21 variant, or combination thereof.
  • Variants of cytokine can comprise one or more mutations in the amino acid sequence compared to wild-type sequence. Variants of cytokine can be used to modul ate a target signaling pathway. Examples of variants of cytokine include, but are not limited to, mutated variant or truncated variant.
  • the cytokine is added in the REP step at a concentration of at least about 10 IU/mL, at least about 20 IU/mL, at least about 30 IU/mL, at least about 40 lU/mL, at least about 50 IIJ/mL, at least about 60 ILJ/mL, at least about 70 IU/mL, at least about 80 IU/mL, at least about 90 IU/mL, at least about 100 IU/mL, at least about 500 IU/mL, at least about 1000 IU ml.., at least about 2000 IL nil...
  • the cytokine is added at a concentration of at least about 2000 IIJ/mL.
  • the cytokine is added in the REP step at a concentration of at most about 50 IU/mL, at most about 60 IU/mL, at most about 70 IU/mL, at most about 80 IU/mL, at most about 90 IU/mL, at most about 100 IU/mL, at most about 500 IU/mL, at most about 1000 IU nd ., at most about 2000 IU/mL, at most about 3000 IU/mL, at most about 4000 IL ml., at most about 5000 IU/mL, at most about 6000 IU nd ., at most about 7000 IU/mL, at most about 8000 IU/mL, at most about 9000 IU/mL, at most about 10000 IU ml...
  • the cytokine is added in the REP step at a concentration of about 10 IUM L, about 20 IUML, about 30 IUML, about 40 IUML, about 50 IUML, about 60 IUML, about 70 IUML, about 80 IUML, about 90 IUML, about 100 IUML, about 500 IUML, about 1000 IU ml... about 2000 IUML, about 3000 IUML, about 4000 IU ml... about 5000 IUML, about 6000 IUML, about 7000 IUML, about 8000 IUML, about 9000 IU.ML, or about 10000 IU ml...
  • the cytokine is added in the REP step at a concentration of at least about 1000 IU ML. In some embodiments, the cytokine is added in the REP step at a concentration of about 3000 IU ml...
  • the cytokine is added in the REP step at a concentration of from about 10 IUML to about 12000 IU ML, from about 20 IUML to about .12000 IU ML, from about 30 IU ml. to about 12000 IU /mL, from about 40 IU ml.
  • the cytokine is added in the REP step at a concentration of from about 10 lU/mL to about 10000 IUML, from about 20 IUML to about 10000 IU ML, from about 30 IUML to about 10000 IUML, from about 40 IU.
  • ML to about 10000 IUML from about 50 IUML to about 50000 IU.ML, from about 60 IU ml to about 10000 IU ml... from about 70 IUML to about 10000 IUML, from about 80 IU ml.
  • the cytokine added in the REP step at a concentration of from about 2000 IU.ML to about 10000 IUML.
  • the time period of the REP step is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, or at least about 24 days.
  • the time period of the REP step is at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 11 days, at most about 12 days, at most about 13 days, at most about 14 days, at most about 15 days, at most about 16 days, at most about 17 days, at most about 18 days, at most about 19 days, at most about 20 days, at most about 2.1 days, at most about 22 days, at most about 23 days, or at most about 24 days.
  • the time period of the REP is from about 1 to about 24 days, from about 2 to about 24 days, from about 3 to about 24 days, from about 4 to about 24 days, from about 5 to about 24 days, from about 6 to about 24 days, from about 7 to about 24 days, from about 8 to about 24 days, from about 9 to about 24 days, from about 10 to about 24 days, from about 11 to about 24 days, from about 12 to about 24 days, from about 13 to about 24 days, from about 14 to about 24 days, from about I 5 to about 24 days, from about 16 to about 24 days, from about 17 to about 24 days, from about 18 to about 24 days, from about 19 to about 24 days, from about 20 to about 24 days, from about 21 to about 24 days, from about 22 to about 24 days, or from about 23 to about 24 days.
  • the time period of the REP is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, or about 24 days.
  • the time period of the REP is at least about 7 days. In some embodiments, the time period of the REP is about 14 days. In some embodiments, the time period of the REP is from about 7 to about 14 days.
  • the antibody used in the REP culture comprises an anti-CD3 antibody.
  • the anti-CD3 antibody is a monoclonal antibody, In some embodiments, the anti-CD3 monoclonal antibody is obtained from Clone OKT3 or Clone UCHT1 , In some embodiments, a concentration of the anti-CD3 antibody added is at least about 1 ng/mL, at least about 3 ng/mL, at least about 5 ng/mL, at least about 7 ng/niL, at least about 10 ng ml. .
  • the concentration of the anti-CD3 antibody added is at most about 1 ng/mL, at most about 3 ng ml... at most about 5 ng/mL, at most about 7 ng. ml... at most about 10 ng ml...
  • ng/mL at most about 15, ng/mL, at most about 20 ng/mL, at most about 25 ng/mL, at most about 30 ng/mL, at most about 35 ng/mL, at most about 40 ng/mL, at most about 45 ng/mL, at most about 50 ng/mL, at most about 55 ng/mL, at most about 60 ng/mL, at most about 65 ng/mL, at most about 70 ng/mL, at most about 75 ng/mL, at most about 80 ng/mL, at most about 85 ng/mL, at most about 90 ng/mL, at most about 95 ng/mL, or at most about 100 ng mL.
  • the concentration of the ant.i-CD3 an tibody added is about 1 ng/mL, about 3 ng/mL, about 5 ng/mL, about 7 ng/mL, about 10 ng/mL, about 15, ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng'mL, about 50 ng mL. about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng. nil...
  • the concentration of the anti-CD3 antibody added is about 10 ng ml. In some embodiments, a concentration of the anii-CD3 antibody added is about 30 ng/mL.
  • the one or more agents in REP step further comprise irradiated feeder cells.
  • the irradiated feeder cells are irradiated allogeneic PBMC- derived feeder cells.
  • the ratio of the immune cells to the irradiated feeder cells is about 1:10, about 1:50, about 1 : 100, about 1 : 150, or about 1:200. In some embodiments, the ratio of the immune cells to the irradiated feeder cells is about 1:100.
  • the feeder cells used in the REP culture comprise peripheral blood mononuclear cell (PBMC) feeders.
  • the feeder cells used in the REP culture are derived from PBMC feeders.
  • the PBMC feeders are irradiated feeder cells.
  • the PBMC feeders are allogeneic PBMC feeder cells.
  • tire PBMC feeders are irradiated allogeneic PBMC feeder cells.
  • the immune cells are expanded in the REP step for up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 1 .1 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about .19 days, up to about 20 days, up to about 21 days or greater.
  • the immune cells are expanded in the REP step for at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 1 1 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, or greater.
  • the immune cells, e.g., TILs are expanded in the REP step for about 14 days.
  • immune cells e.g., TILs
  • TILs can be assayed for functional activity.
  • the immune cells e.g., TILs
  • the immune cells e.g., TILs
  • the immune cells e.g., TILs, comprises T cells.
  • the method further comprises combining tumor organoids with immune cells obtained from, other sources.
  • the other sources comprise peripheral blood ceils or organoids grown from lymphoid tissue.
  • the method further comprises stimulating antigen presentation.
  • the method further comprises depletion of immune inhibitory cell types.
  • the immune inhibitory cell types comprise Tregs, myeloid derived suppressor cells, TAMs, vascular endothelial cells, or CAFs.
  • the method further comprises negative selection of bystander tumor infiltrating immune cells.
  • the method further comprises knockdown exhaustion regulator.
  • the knock-down exhaustion regulator is TOX.
  • the method further comprises reprogramming T cells.
  • the immune cells e.g., TILs
  • the TILs can be reprogrammed to have stem cell-like properties by inhibiting interferon-gamnia. (IFNy) signaling pathway, activating Notch signaling pathway, or both.
  • the T cells are reprogrammed by activation of Notch signaling pathway during the R EP step.
  • the T cells are reprogrammed by inhibiting IFNy signaling. In some embodiments, the T cells are reprogrammed by both activating Notch signaling pathway and inhibiting IFNy signaling pathway using the modulator.
  • the modulator can be an IFNy modulator, a Notch modulator, or combination thereof. In some embodiments, the IFNy modulator can inhibit IFNy signaling pathways. In some embodiments, the IFNy modulator comprises an IFNy inhibitor. In some embodiments, the Notch modulator can activate Notch signaling pathways. In. some embodiments, tire Notch modulator comprises a Notch activator.
  • the method further comprises identifying T cell receptors (TCR) to identify TCRs enriched in. the air-liquid interface culture.
  • TCR T cell receptors
  • the identifying TCRs is performed using sequencing technology.
  • the method comprises providing one or more tumor antigens during the first time period or the second time period, In some embodiments, providing the tumor antigens comprises providing cells expressing the tumor antigens.
  • the immune cells e.g., TILs
  • the immune cells can be cryopreserved.
  • the immune cells e.g., TILs
  • methods and systems described herein can comprise reprogramming isolated, expanded, or cryopreserved TILs (e.g., ALI TILs) into a stem-like state.
  • the reprograming occurs during the REP step.
  • the reprogramming occurs before the REP step.
  • the reprogramming occurs after the REP step.
  • the isolated or cryopreserved TILs comprise (i) central memory T cells (TCM), which can be characterized by CD45RA- CCR7+CD62L+ expression, (ii) tissue resident memory T cells (TRM), which can be identified by CD69+CDI03+ expression, (iii) effector memory T cells (TEM), which can be identified by CD45RA-CCR.7-CD62L- expression, (iv) stem cell memory T ceils (TSCM ), which can be characterized by CD45RA+CCR7-tCD62LtCD95 t expression, (v) Naive T celis, which can be characterized by CD45R A+CCR7 t CD62L v expression, and (vi) terminally differentiated effector memory cells (TEMRA), which can be identified by CD45RO-.
  • TCM central memory T cells
  • TRM tissue resident memory T cells
  • TEM effector memory T cells
  • TSCM stem cell memory T ceils
  • Naive T celis which can be characterized by CD45
  • methods and systems described herein can reprogram one or more isolated TILs, e.g., T cells, into cells having a stem-like property (e.g., sell-renewal capacity), such as memory T celis (TSCMs) or central memory T cells (TCMs),
  • TSCMs memory T celis
  • TCMs central memory T cells
  • the reprogrammed immune celis e.g., T celis
  • the methods and systems described herein can reprogram one or more terminally differentiated immune cells, e.g., T cells (e.g., effector memory' T cells), into stem-like T ceils, such as memory T ceils (TSCMs).
  • T cells e.g., effector memory' T cells
  • TSCMs memory T ceils
  • the reprogrammed immune cells, e.g., T ceils can have enhanced persistence, proliferation, and'or efficacy in adoptive cellular therapy for different cancer types.
  • the reprogrammed immune cells, e.g, T cells can be used in adoptive cellular therapy to treat cancer.
  • the cancer comprises glioblastoma.
  • the reprogrammed immune cells, e.g., T cells can be used in adoptive cellular therapy to treat glioblastoma.
  • the method further comprises reprograming the immune cells, e.g., T cells.
  • the immune cells e.g., T cells
  • methods and systems described herein can comprise treating isolated or cryopreserved TILs (e.g., ALI TILs) with one or more agents that facilitate the reprogramming of the immune cells, e.g., T, cells into TSCMs.
  • the one or more agents comprise modulators.
  • the modulators can enhance a.
  • the modulators can inhibit a signaling pathway that is known to inhibit the maintenance and diversity of stemlike immune cells, e.g., T cells, or to induce the terminal differentiation of memory immune cells, e.g., memory T cells, (e.g., IFNy signaling pathway).
  • the modulators comprise a Notch signaling pathway modulator, an interferon gamma (IFNy) modulator, or a combination thereof.
  • the one or more agents comprise an IFNy modulator.
  • the IFNy modulator can inhibit IFNy signaling pathways.
  • the IFNy modulator comprises an IFNy inhibitor.
  • the IFNy inhibitor comprises an antibody, a small molecule, or a combination thereof.
  • the IFNy inhibitor comprises IFNy neutralizing antibodies or fragments thereof In some embodiments, the IFNy neutralizing antibodies can bind to the receptor. In some embodiments, the IFNy neutralizing antibody can bind to circulating IFNy.
  • the one or more agents comprise a protein tyrosine phosphatase that dephosphorylates the IFNy receptor.
  • the one or more agents that block IFNy signaling pathways comprise an inhibitor that targets proteins or signaling molecules downstream of IFNy signaling pathway. In some embodiments, the one or more agents comprise an inhibitor of JAK/STAT signaling pathways. In some embodiments, the one or more agents comprises a small molecule inhibitor.
  • the treatment of immune cells, e.g., TILs, with the one or more agents described herein can result in expression changes of IFNy induced genes, such as Gbp5, h-fl, and Ccl2 within 8 hours, 24 hours, 48 hours, or longer, after the treatment.
  • the one or more agents comprise the Notch signaling pathway modulator.
  • the Notch signaling pathway modulator can activate Notch signaling pathways.
  • the Notch signaling pathway modulator comprises a Notch activator.
  • the Notch activator comprises an antibody, a small molecule, or a combination thereof.
  • the Notch activator comprises agonistic antibodies that target and activate the Notch receptor.
  • the one or more agents can be Delta-like 1 ligand.
  • the one or more agents can enhance expression of the downstream molecules of the Notch signaling pathway, in some embodiments, the one or more agents can be a small molecule agonist.
  • the one or more agents can be an agonistic monoclonal antibody (mAb).
  • the treatment of immune cells, e.g., TILs, wi th the one or more agents described herein can result in expression changes of Notch induced genes, such as Myc. Deltexl, and Hesi within 8 hours, 24 hours, 48 hours, or longer, after the treatment.
  • methods and systems described herein can comprise treating immune cells, e.g., TILs, with one or more agents to inhibit IFNy signaling and activate Notch signaling.
  • the one or more agents comprise one or more modulators.
  • the one or more modulators comprise a first modulator that inhibits IFNy signaling and a second modulator that activates Notch signaling.
  • the one or more modulators comprise Notch signaling pathway modulator, interferon gamma (IFNy) modulator, or combination thereof.
  • the one or more modulators are added separately.
  • the one or more modulators are added sequentially.
  • the one or more modulators are added simultaneously.
  • the one or more agents described herein can be a mAb cocktail that simultaneously targets IFNy inhibition and Notch activation.
  • the mAb cocktail can be applied throughout or for the last about I day, throughout or for the last about 2 days, throughout or for the last about 3 days, throughout or for the last about 4 days, throughout or for the last about 5 days, throughout or for the last about 6 days, throughout or for the last about 7 days, throughout or for the last about 8 days, throughout or for the last about 9 days, throughout or for the last about 10 days, throughout or for the last about 1 1 days, throughout or for the last about 12 days, throughout or for the last about 13 days, or throughout or for the last about 14 days of the REP step during culture.
  • the method may further comprise treating TIL culture during the REP step with one or more cytokines
  • the cytokines can be IL- 2, an IL-2 variant, IL-7, an IL-7 variant, IL- 15, an IL- 15 variant, IL- 18, an IL- 18 variant, IL-21, an IL-21 varian t, or combination thereof.
  • Variants of cytokine can comprise one or more mutations in the amino acid sequence compared to wild-type sequence. Variants of cytokine can be used to modulate a target signaling pathway. Examples of variants of cytokine include, but are not limited to, mutated variant or truncated variant.
  • the method described herein can yield a TSCM population of about 2 % to about 50 % of the TIL. population after the reprogramming. In some cases, the method described herein can yield a TSCM population of about 2 % to about 4 %, about 2 % to about 6 %, about 2 % to about 8 %, about 2 % to about 10 %, about 2 % to about 15 %, about 2 % to about 20 %, about 2
  • about 2 % to about 30 about 2 % to about 35 about 2 % to about 40 about 2 % to about 50 %, about 4 % to about 6 %, about 4 % to about 8 %, about 4 % to about 10 %, about 4 % to about 15 %, about 4 % to about 20 %, about 4 % to about 25 %, about 4 % to about 30 %, about 4 % to about 35 %, about 4 % to about 40 %, about 4 % to about 50 %, about
  • the method described herein can yield a TSCM population of about 2 %, about 4 %, about 6 %, about 8 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 To, or about 50 % of the TIL populat ion, after the reprogramming.
  • the methods described herein can yield a TSCM population of at least about 2 %, at least about 4 %, at least about 6 %.
  • the methods described herein can yield a TSCM population of at most about 4 %, at most about 6 %, at most about 8 %, at most about 10 %, at most about 15 %, at most about 20 %, at most about 25 %, at most about 30 %, at most about 35 %, or at least about 40 % of the TIL popula tion, alter the reprograming.
  • the methods described herein can yield a TSCM population of at most about 4 %, at most about 6 %, at most about 8 %, at most about 10 %, at most about 15 %, at most about 20 %, at most about 25 %, at most about 30 %, at most about 35 %, at most about 40 %, or at most about 50 % of the TI L population, after the reprogramming.
  • the methods described herein can yield a TCM population of about 2 % to about 50 % of the TIL population, after the reprogramming. In some cases, the methods described herein can yield a TCM population of about 2 % to about 4 %, about 2 % to about 6 %, about 2 % to about 8 %, about 2 % io about 10 %, about 2 % to about 15 %, about 2 % to about 20 %, about 2
  • the method described herein can yield a TCM population of about 2 %, about 4 %, about 6 %, about 8 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, or about 50 % of the TIL. population, after the reprogramm ing.
  • the method described herein can. yield a TCM population of at least about 2 %, at least about 4 >4>, at least about 6 %, at least about 8 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, or at least about 40 % of the TIL population, after the reprograming.
  • the methods described herein can yield a TCM population of at most about 4 %, at most about 6 %, at most about 8 %, at most about 10 %, at most about 15 %, at most about 20 %, at most about 25 %, at most about 30 %, at most about 35 %, at most about 40 %, or at most about 50 % of the TIL population, after the reprogramming,
  • the method further comprises identifying T cell receptors (TCRs) to identify TCRs enriched in the air-liquid interface culture.
  • TCRs T cell receptors
  • the identifying TCRs is performed using sequencing technology.
  • the immune cells e.g., rALI Ill s
  • the immune cells can be cryopreserved.
  • the immune cells e.g., rALI TILs, are cryorecovered after cryopreservation.
  • the immune ceils generated from the systems and methods described herein demonstrate a better tumor killing capacity compared to standard immune cells
  • the immune ceils generated from the systems and methods described herein are more reactive to tumor ceils compared to standard immune cells (e.g., those immune cells generated from a standard protocol, as described herein)
  • the immune cells comprise lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • the immune cells comprise tumor infiltrating lymphocytes.
  • the tumor infiltrating lymphocytes comprise T cells
  • the T cells comprise naive CD8* T cells, cytotoxic CD8' T cells, naive CD4 ⁇ T cells, helper T cells, e.g. THI , TH2, TH9, TH 17, TH22, TFH; memory T cells, e.g. central memory T ceils, T stem cell memory cells (TSCM), effector memory T cells, NKT cells, y5 T ceils.
  • naive T cells or terminally differentiated T cells can be reprogrammed to stem-like T cells (e.g., T stem cell memory cells).
  • the present disclosure provides a composition for treating a tumor comprising in vitro isolated immune ceils, wherein the immune cells comprise tumor infiltrating lymphocytes expressing IFNy, CD107a, or HLA-DR, wherein the tumor infiltrating lymphocyte population express higher IFNy compared to tumor infiltrating lymphocytes obtained from standard protocol.
  • the immune cells comprise tumor infiltrating lymphocytes, I ll s, or reprogrammed lymphocytes.
  • the population of the Aid TILs or reprogrammed ALI TILs (rALI TILs) obtained from the systems and methods described herein express higher IFNy, CD107a, and/or HLA-DR compared to population of the STD TILs in response to speci fic stimulus.
  • the ALI TILs or rALI TILs express higher IFNy and. or CD 107a compared to the STD TILs.
  • At least about 5%, at least about 3%, at least about 5%, at least about 10%, at least about .15%, at least about 20%, at least about 23%, at least about 30%), at least about 35%), at least about 40%), at least about 45%), at least about 50%, at least about 55%, or at least about 60%> of ALI TIL or rALI TIL population obtained from methods and system described herein are IFNy expressing cells.
  • at least about 5% of ALI TIL or rALI TIL population obtained from methods and system described herein are IFNy expressing cells. In some instances, up to about 10%, up to about 15%.
  • ALI TIL or rALI TIL population obtained from methods and system described herein are IFNy expressing cells.
  • up to about 20% of ALI TIL or rALI TIL population obtained from methods and system described herein are IFNy expressing cells.
  • ALI TIL or rALI TIL population obtained from methods and system described herein are IFNy expressing cells.
  • ALI TIL or rALI TIL population obtained from methods and system described herein are IFNy expressing cells
  • about 5%) to about 20%> of ALI TIL or rALI TIL population obtained from methods and system, described herein are IFNy expressing cells.
  • up to about ].()%> up to about 15*%, up to about 20*%, up to about 23%, up to about 30%, up to about 35%, up to about 40*%, up to about 45%, up to about 50%, up to about 55*%, or up to about 60% of ALI TIL or rALI TIL population obtained from methods and system described herein are CD 107a expressing cells. In some instances, up to about 20% of ALI TIL or rALI TIL population obtained from methods and system described herein are CD 107a expressing cells.
  • about 3'% to about 60%), about 5%) to about 60%, about 10%) to about 60%, about 15% to about 60%, about 20% to about 60%, about 25%) to about 60%), about 30%> to about 60%), 35% to about 60%, about 40% to about 60%, about 45%) to about 60%, about 50%) to about 60%), or about 55*% to about 60% of ALI TIL or rALI TIL population obtained from methods and system described herein are CD 107a expressing cells.
  • about 3% to about 20%), 5% to about 20%, about 10% to about 20%, or about 15%) to about 20% of ALI TIL or rALI TIL population obtained from methods and system described herein are CD 107a expressing cells.
  • about 3% to about 20% of ALI TIL or rALI TIL population obtained from methods and system described herein are CD 107a expressing cells.
  • population obtained from methods and system described herein are HLA-DR expressing cells.
  • at least about 10% of AU TIL, or rALI TIL, population obtained from methods and system described herein are HLA-DR expressing cells.
  • up to about 10%>, up to about 15%, up to about 20%, up to about 23%, up to about 30%, up to about 35%, up to about 40" •>, up to about 45%, up to about 50%>, up to about 55%, up to about 60%, up to about 65%, or up to about 7014 of ALI TIL or rALI TIL population obtained from methods and system described herein are HLA-DR expressing cells.
  • up to about 2014 of AU TIL or rALI TIL population obtained from methods and system described herein are HLA-DR expressing cells.
  • HLA-DR expressing cells 35% to about 60%, about 40% to about 60%, about 45% to about 60%, about 50% to about 60%, or about 55% to about 60% of AU TIL or rALI TIL population obtained from methods and system described herein are HLA-DR expressing cells.
  • expression markers oflFNy, CD 107a, and/or HLA-DR can be characterized at least at about 5 hours, at least at about 6 hours, at least at about 7 hours, at least at about 8 hours, at least at about 9 hours, at least at about 10 hours, at least at about 1 1 hours, at least at about 12 hours, at least at about 13 hours, at least at about 14 hours, at least at about 15 hours, at least at about 16 hours, at least at about 17 hours, at least at about 18 hours, at least at about 19 hours, at least at about 20 hours, at least at about 21 hours, at least at about 22 hours, at least at about 23 hours, at least at about 24 hours, at least at about 25 hours, at least at about 26 hours, at least at about 27 hours, at least at about 28 hours, at least at about 29 hours, at least at about 30 hours, at least at about 31 hours, at least at about 32 hours, at least at about 33 hours, at least at about 34 hours, at least at about 35 hours, at least at about 36 hours
  • the AU TILs or rALI TILs express higher level of Human Leukocyte Antigen - DR isotype (HLA-DR.) compared to the STD TILs.
  • expression markers of HLA-DR can be characterized at least at about 5 hours, at least at about 6 hours, at least at about 7 hours, at least at about 8 hours, at least at about 9 hours, at least at about 10 hours, at least at about I 1 hours, at least at about 12 hours, at least at about I 3 hours, at least at about 14 hours, at least at about 15 hours, at least at about 16 hours, at least at about 17 hours, at least at about 18 hours, at least at about 19 hours, at least at about 20 hours, at least at about 21 hours, at least at about 22 hours, at ieast at about 23 hours, at least at about 24 hours, at ieast at about 25 hours, at least at about 26 hours, at least at about 27 hours, at ieast at about 28 hours, at least at about 29 hours, at least
  • population of the AU TILs or rALI TILs obtained from systems and methods described herein express higher HLA-DR compared to population of the SI D TILs.
  • the HLA-DR expression level of ALI TILs or rALI TILs population obtained from methods and systems described herein is at least about 10%, at least about 15%, at least about 20%, at least about 23%, at least about 30%, at least about 35%, at ieast about 40%, at least about 45%, at least about 50%, at ieast about 55%, or at least about 60% higher compared to the STD TILs.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40° at least about 45%, at least about 50%, at least about 55%, at ieast about 60%, or at ieast about 65% of ATI TIL or rALI TIL population obtained from methods and system described herein are HLA-DR expressing cells.
  • standard refers to immune cells obtained from a method involving obtaining and culturing immune cells, e.g., TILs, from fresh tumor tissue and treated with one concentration of cytokine during the pre-REP step. Briefly, on day 0 of a STD pre-REP step, the tumor tissue fragments are transferred to G-Rexl O plate. Each well is added with 5-30 tumor tissue fragments in 10 to 40 mL of culture medium comprising RPMI, 10% FBS, gentamicin, and 6000 JU ml.. of IL-2. The tumor tissue fragments are then cultured at 37°C in 5% CO2.
  • the present disclosure provides a composition for treating a tumor comprising in vitro isolated immune cells or reprogrammed immune cells, wherein the immune cells comprise tumor infiltrating lymphocytes expressing IFNy, CD 107a, or HL A-DR, wherein at least 5% of the tumor infiltrating lymphocyte population express IFNy, In some embodiments, 20% of the tumor infiltrating lymphocyte population express I FNy. In some embodiments, 5% to 20% of the tumor infiltrating lymphocyte population express IFNy. In some embodiments, at least 3% of the tumor infiltrating lymphocyte population express GDI 07a. In some embodiments, 20% of the tumor .infiltrating lymphocyte population express CD 107a. In some embodiments, 3% to 20% of the tumor infiltrating lymphocyte population express CD107a.
  • the immune cells are derived from one or more tumor samples obtained from a subject.
  • the subject comprises a human, cow, dog, mouse, rat, rabbit, guinea pig, chicken, fish, bird, reptile, camelid, bovine, chimpanzee, sheep, goat, and non-human primate.
  • the subject is a human.
  • At least about 10% of the tumor infiltrating lymphocyte population express the HLA-DR. In some embodiments, at least about 65% of the tumor infiltrating lymphocyte population express tire HLA-DR.
  • the expanded immune cells, e.g., TILs, or reprogrammed immune cells, e.g., rALI TILs, obtained from the systems and methods described herein are assayed for functional activity,
  • the assay for functional activity comprises T cell cytotoxicity assays, IL-2 response, etc., as known in the art.
  • the immune cells, e.g,, TILs, or reprogrammed immune cells, e.g., rALI TILs are assessed for the presence of markers indicative of activation, e.g.
  • the immune cells e.g., TILs
  • the immune cells are assessed for the HLA-DR, PD-I, CD45, or EPC AM markers.
  • the immune cells, e.g., TILs, or reprogrammed immune cells, e.g., rALI TILs can be selected for an activated phenotype prior to administration to a patient or for other applications.
  • the immune cells, e.g., TILs, or reprogrammed immune cells, e.g., rALI TILs comprises T cells.
  • a subject in need of a treatment according to the method described herein may be a subject in need of adoptive cellular therapy to treat the subject for cancer or tumor.
  • the immune cells e.g., I ll s, or reprogrammed immune cells, e.g., rALI TILs
  • the cancer or tumor comprises glioblastoma, colon tumor, lung tumor, gastric tumor, brain tumor, kidney tumor, esophageal tumor, uterine tumor, skin tumor, pancreatic tumor, or breast tumor.
  • the cancer or tumor is glioblastoma.
  • a subject is treated using adoptive cellular therapy employing an expanded and/or reprogrammed cell population that has been activated, expanded, and/or reprogrammed by the methods and/or systems disclosed herein.
  • cells may be collected from a subject, activated and expanded, and reintroduced into the subject as part of the adoptive cellular therapy.
  • the cells are further reprogrammed to have stem cell-like properties prior to reintroduce into the subject.
  • the cells collected from the subject may be collected from any convenient and appropriate source for the adoptive cellular therapy, such as peripheral blood (e.g., the subject’s peripheral blood), a biopsy (e.g., a tumor biopsy from the subject), and the like.
  • the cells collected are immune cells.
  • the immune cells comprise tumor infiltrating lymphocytes (TILs), e.g., TILs collected from a tumor of a subject.
  • TILs tumor infiltrating lymphocytes
  • the cells collected are blood cells, e.g., NK cells collected from a subject’s blood (e.g., a. subject having cancer or a subject having an infection).
  • the present disclosure provides a therapeutic method comprising introducing into a recipient in need thereof of an expanded cell population, e.g., immune cells, as described above.
  • the expanded cell population, e.g., immune cells are reprogrammed to have stem cell-like properties, as described above.
  • the cell population is autologous or allogeneic with respect to the recipient.
  • the expanded immune cells e.g., TILs, or reprogrammed immune cells, e.g., r.ALI TILs
  • therapeutic formulations compri sing the expanded immune cells or reprogrammed immune cells can be frozen, or prepared for administration with physiologically acceptable earners, excipients or stabilizers ( Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of aqueous solutions.
  • the expanded immune cells or reprogrammed immune cells are formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context comprises the particular disorder being treated, the particular mammal being treated, the clinical condit ion of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners,
  • the expanded immune cells or reprogrammed immune cells can be administered by any suitable means.
  • the administration comprises intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal , intrathecal, intratumoral, intravesical, or subcutaneous administration,
  • the administration comprises parenteral infusion.
  • the expanded immune ceils or reprogrammed immune cells can be administered via parenteral infusions.
  • the parenteral infusions comprise intramuscular, intravenous (bolus or slow' drip), intraarterial, intraperitoneal, intrathecal, intratumoral, intravesical, or subcutaneous administration.
  • the present disclosure provides a composition for treating a tumor comprising in vitro isolated immune cells, wherein the immune cells comprise tumor infiltrating lymphocytes expressing IFNy, CD 107a, or HLA-DR, wherein at least 5% of the tumor infiltrating lymphocyte population express IFNy,
  • the tumor includes, but not are limited to, Colon Tumor, Lung Tumor, Gastric Tumor, Atypical Teratoid/Rhabdoid Tumor, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma, Glioblastoma multiforme (GB.M).
  • Bronchial Tumors e.g., Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Lymphoma, etc, ), Embryonal Tumors, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Islet Cell Tumors (e.g., Pancreatic Neuroendocrine Tumors, etc.), Kidney Cancer (e.g., Renal Cell, Wilms Tumor, Childhood Kidney Tumors, etc.), Ovarian Cancer (e.g., Epithelial, Germ.
  • Central Nervous System e.g., Atypical Teratoid Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Lymphoma, etc, ), Embryonal Tumors, Extracranial
  • TILs tumor infiltrating lymphocytes
  • the composition for administration depends on the formulation desired, phannaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected and not affect the biological activity of the combination. Examples of diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation can comprise other carrier's, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • the composition further comprises acceptable carriers, excipients, or stabilizers.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed.
  • acceptable carriers, excipients, or stabilizers comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimeihylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or
  • compositions are prepared as injectables, either as liquid solutions or suspensions.
  • composition in solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • proteins can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • the pharmaceutical compositions are formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U .S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • an effective dose of the immune cells can then be administered to a patient, including without limitation the patient from which the PDOs were derived from.
  • the effective dose can be at least about 10 2 cells, at least about 10 3 cells, at least about 10 4 cells, at least about HF cells, at least about 10 6 ceils, at least about 10' cells, at least about 10 8 cells, at least about 10 9 cells, at least about 10 10 cells, at least about .10 11 cells, at least about 10 12 cells, at least about 1 () L> cells, or more per administration.
  • the effective dose can be at least about 10 ⁇ cells to about 10 13 cells, at least about 10 3 cells to about 10 3 cells, at least abou 1t0 4 cells to about 10 13 cells, at least about 10 5 cells to about 10 3 cells, at least about 10 6 cells to about 1 O’ ’ cells, at least about 10 7 cells to about 10 13 cells, at least about 10 s cells to about 10 13 cells, at least about 10 9 cells to about 10 3 cells, at least about 10 10 cells to about 10 13 ’ cells, at least about 10 f ’ cells to about 10 13 cells, or at least about 10 12 cells to about 10 13 cells per administration.
  • the effective dose can be at least about 10 2 cells to about 10 11 cells, at least about 10' cells to about 10 11 cells, at least about 10 4 cells to about 10 11 cells, at least about 10 5 cells to about 10 11 cells, at least about 10 6 cells to about 10 11 cells, at least about 10 cells to about 10” cells, at least about 10 8 cells to about 10 11 cells, at least about 10 9 cells to about 10 11 cells, or al least about 10 10 cells to about 10 11 cells per administration.
  • the effective dose can be delivered systemically, by intratumoral injection or other administrative routes as described above.
  • an enhanced immune response may be manifest as an increase in the cytolytic response of T cells towards the target cells present in the recipient.
  • patient who will receive the expanded immune cells may also recei ve chemotherapy.
  • the composition further comprises acceptable carriers, excipients, or stabilizers.
  • the composition can be administered with other therapeutic treatments.
  • the other therapeutic treatments comprise chemotherapeutic agents, immune checkpoint inhibitors, cancer therapeutics, targeted therapeutics, immunomodulators, cytokines, antibiotics, or antiviral agents.
  • the present disclosure provides composition that can be administered with other therapeutic treatments.
  • the other therapeutic treatments comprise chemotherapeutic agents, immune checkpoint inhibitors, cancer therapeutics, targeted therapeutics, immunomodulators, cytokines, antibiotics, or antiviral agents.
  • cancer cells prevent immune cells from attacking by braking in the immune system or using signals from the tumor that weaken the immune response.
  • immune checkpoint inhibitors are utilized along with adoptive cellular therapy.
  • the expanded immune ceils or reprogrammed immune cells are treated with immune checkpoint inhibitors prior to administration to the patient.
  • treatment of a subject or patient for a condition employing a composition and or cells of the subject disclosure can be combined with one or more additional active agents
  • useful additional active agents comprise active agents for treating cancer.
  • treatment can be combined with other active agents, including antibiotics, cytokines, and antiviral agents, Ln
  • antibiotics comprises penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.
  • the penicillins are used in combination with p-lactama.se inhibitors, cephalosporins, e.g.
  • the cytokines comprise interferon y, tumor necrosis factor a, interleukin 12, etc.
  • the antiviral agents comprises acyclovir, gancyclovir, etc,
  • chemotherapeutic agents that can be administered in combination with the expanded immune cells or reprogrammed immune cells.
  • the chemotherapeutic agents comprise abifrexate, adriamycin, adruciL amsacrine, asparaginase, anthracyclincs, azacitidine, azathioprine, bienu, blenoxane, busulfan, bleomycin, camptosar, camptothecins, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, Cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil
  • targeted therapeutics that can be administered in combination with the expanded immune cells or reprogrammed immune cells comprise tyrosine-kinase inhibitors, such as Imatinib mesylate (Gleevec, also known as STI-571), Gefithiib (Iressa, also known as ZD 1839), Erlotinib (marketed as Tarceva), Sorafenib (Nexavar), Sunitinib (Sutent), Dasatimb (Spiycel), Lapatimb (Tykerb), Nilotinib (Tasigna), and Bortezomib (Velcade); Janus kinase inhibitors, such as tofaeitinib; ALK inhibitors, such as crizotinib; Bel-2 inhibitors, such as obatoclax, venclexta, and gossypol; FLT3 inhibitors, such as midostaurin (Rydapt), IDH inhibitor
  • the expanded immune cells or reprogrammed immune cells can be administered in combination with an immunomodulator, such as a cytokine, a lymph okine, a monokine, a stem cell growth factor, a lympbotoxin (LT), a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), a transforming growth factor (TGF), such as TGF-o or TGF-0, insulin-like growth factor (IGF), erythropoietin, thrombopoietin, a tumor necrosis factor (TNF) such as TNF-a or TNF-p, vascular endothelial growth factor, integrin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), an interferon such as interferon-a, interferon-p, or inter feron-y.
  • an immunomodulator such as a cytokin
  • an interleukin such as IL- I , IL- lcc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21 or IL-25, LIE, kit-ligand, FLT-3, endostatin, and. LT.
  • IL- I interleukin
  • tumor specific monoclonal antibodies that can be administered in combination with the expanded immune cells or reprogrammed immune cells comprise Ipilimumab targeting CTLA-4 (as used in the treatment of Melanoma, Prostate Cancer, RCC); Tremelimumab targeting CTLA.-4 (as used in the treatment of CRC, Gastric, Melanoma, NSCLC); Nivolumab targeting PD-1 (as used in the treatment of Melanoma, NSCLC, RCC); MK-3475 targeting PD-1 (as used in the treatment of Melanoma); Pidilizumab targeting PD-1 (as used in the treatment of Hematologic Malignancies); BMS-936559 targeting PD-L1 (as used in the treatment of Melanoma, NSCLC, Ovarian, RCC); MEDI4736 targeting PD-L.1;
  • MPDL33280A targeting PD-L 1 (as used in the treatment of Melanoma); Rituximab targeting CD20 (as used in the treatment of Non -Hodgkin's lymphoma); Ibritumomab tiuxetan and tositumomab (as used in the treatment of Lymphoma); Brentuximab vedotin targeting CD30 (as used in the treatment of Hodgkin's lymphoma); Gemtuzumab ozogamicin targeting CD33 (as used in the treatment of Acute myelogenous leukaemia); Alemtuzumab targeting CD52 (as used in the treatment of Chronic lymphocytic leukaemia); IGN101 and adecatum.um.ab targeting EpCAM (as used in the treatment of Epithelial tumors (breast, colon and lung)); Labetuzumab targeting CEA (as used in the treatment of Breast, colon and lung tumors
  • lung, pancreas, and head and neck tumors 8106 targeting Tenascin (as used in the treatment of Glioma, breast and prostate tumors); Blinatumomab (Blincyto; Amgen) targeting CD3 (as used in the treatment of ALL); pembrolizutnab targeting PD-1 as used in cancer immunotherapy; 9E10 antibody targeting c-Myc; and etc.
  • the expanded immune cells e.g.. TILs, or reprogrammed immune cells, e.g., rALI TILs, as described in the present disclosure can be used for oilier applications aside from immunotherapy.
  • the expanded immune cells, e.g., TILs, or reprogrammed immune cells, e.g., rALI TILs can be used for functional in vitro assays for disease modeling or for determining patient specific responsiveness to immunotherapy agent.
  • the expanded immune cells, e.g., TILs, or reprogrammed immune cells, e.g., rALI TILs can be used for screening assays.
  • the expanded immune cells e.g., TILs, or reprogrammed immune cells, e.g., rALI TILs
  • the expanded immune cells can be used for various applications, experiments modified from methods known in the art.
  • FIG. 1 depicts the timeline of pre-Rapid Expansion Protocol (pre-REP) in Standard Tumor Infiltrating Lymphocytes (STD TIL) protocol and in air-liquid interface Tumor Infiltrating Lymphocytes (AU TIL) protocol.
  • Standard (“STD”) refers to immune cells obtained from a method involving obtaining and culturing TIL from fresh tumor tissue and treated with one concentration of cytokine during the pre-REP step. Briefly, on Day 0 of the STD pre-REP step, the tumor tissue fragments are transferred to G-Rexl O plate. Each well is added with 5-30 tumor tissue fragments in 10 to 40 mL of culture medium comprising RPMI, 10% FBS, gentamicin, and 6000 11.
  • the tumor ti ssue fragments are then cultured at 37°C in 5% CO2.
  • Half medium is removed and replaced with fresh medium and 6000 lU/mL of I L-2.
  • half medium is changed every 2 to 3 days.
  • cells are collected before being treated with cocktails for Rapid Expansion Protocol (REP) treatment.
  • REP Rapid Expansion Protocol
  • the starting samples are patient derived organoids (PDOs), which are generated from tissue samples. These PDOs are cultured in air-liquid interface (ALI) environment. These PDOs comprise various immune populations, e.g,, tumor infiltrating immune cells, lymphocytes, T cells, etc., which can then be used for expansion.
  • PDOs or ALI organoids are treated with 50 IL ml. of 11.-2 for 1-2 weeks before being treated with higher concentrations of IL-2 (6000 IL' ml. ) for 1 1 days, At the end of this, cells are harvested for REP treatment.
  • FIG. 2 shows that ALI Tumor Infiltrating Lymphocytes (ALI TILs) demonstrate a better tumor killing capacity compared to standard TILs (STD TILs).
  • ALI TILs and STD TILs were co-culture with autologous tumor epithelial cells derived from submerged Organoids for 48 hours. Single Cell tumor Organoids alone were used as a control. At 48 hours post-incubation, cells were stained with fiuorochrome-conjugated antibodies for CD45, EPC AM, Annexin V, Zombie NIR live dead and other T cell markers. Flow Cytometry analysis for live tumor cell count was performed after staining.
  • FIGS. 3A-3C show that ALI Tumor Infiltrating Lymphocytes (ALI TILs) are more reactive to tumor cells compared to standard TILs (STD TILs).
  • FIG. 3A shows the experimental scheme. Briefly, ALI TILs and STD TILs were co-cultured with autologous tumor epithelial cells derived from submerged Organoids for 16 hours. ALI TILs or STD TILs alone were used as a control. At 16 hours post-incubation, cells were stained with fluorochrome-conjugated antibodies for IFNy secretion, CD 107a, CD45, EPCAM, Annexin V, Zombie NIR live dead and other T cell markers for Flow Cytometry analysis.
  • FIG. 3B and FIG. 3C to assess the tumor-reactive of TILs, the percentage of CD3+ cells secreting IFNy and the % of CD3+ cells expressing CD ! 07a on their surface were assessed at 16 hours post-incubation. Data is shown for samples derived from 3 different patients (CRC- 1 TM colorectal cancer patient 1 , CRC-2TM colorectal cancer patient 2, MLNTM melanoma patient). 3 replicates per experiment were performed. Nonparametric ANOVA tested corrected by Geisser Greenhouse Correction was performed to compare between different conditions; * ⁇ PvTM0.()5.
  • FIGS. 4A-4D show that ALI Tumor Infiltrating Lymphocytes (AL1 TILs) express higher levels of HLA-DR compared to standard TILs (STD TILs).
  • FIG. 4A. show's the experimental scheme. Briefly, ALI TILs and S I D TILs were co-cultured with autologous tumor epithelial cells derived from submerged Organoids for 48 hours. ALI TILs or STD TILs alone were used as a control.
  • FIG. 5 show's that ALI Tumor Infiltrating Lymphocytes (ALI TILs) express higher percentage of HLA-DR* compared to standard TILs (STD TILs).
  • the experimental timeframe and condition were similar as described in FIGS. 4A-4D. The result is presented in percentage instead of Fold Change as in FIG. 4C.
  • FIGS. 6A-6C illustrate an example of the establishment of ALI tumor organoids.
  • FIG. 6 A show's representative brightfield images of ALI tumor organoids generated from tissue obtained from kidney, lung, esophagus, and uterus.
  • FIG. 6B show's an example of ALI tumor organoids generated from tissue obtained from colon and glioblastoma (GBM).
  • Top figures show samples grown as air-liquid interface (ALI) organoids, which can be used for TIL expansion and. or reprogramming.
  • Bottom figures show submerged organoids, which can be used for assays.
  • FIG. 6C shows sequencing results for three colorectal cancer (CRC) organoid lines, which are shown as CNV plots and mutated genes (AFC, TP53, KRAS).
  • CRC colorectal cancer
  • FIGS. 7A-7C demonstrate the generation and phenotypic characterization of ALI TILs.
  • FIG. 7 A depicts a schematic representation of the 2-siep ALI TIL process and cry opreservation of the product comprising ALI TILs.
  • FIG. 7B shows cell number of ALI TILs obtained from 14 preps (left) and the fold-expansion distribution of the 14 products (right ).
  • FIG. 7C shows results of flow cytometry analysis using CD3, ySTCR, CD4, CDS, CD45RA, and (. 1)621. to detect T cells, lineage, and memory subsets of twelve ALI-TIL preps. Results are plotted as percent parent for each individual sample with average and SEM.
  • TEM effector memory T cells
  • TCM central memory T cells
  • TN/TSCM stem cell memory T cells
  • TEMRA terminally differentiated effector memory cells
  • FIGS. 8A and 8B show ALI TIL tumor reactivity and cytotoxicity detection.
  • FIG. 8A shows flow cytometry analysis of ALI TIL from CRC co-cultured with autologous tumor organoids for 16 hours at 1:1 effector: target ratio. Increased IFNy, CD 107a, and 4- IBB were observed. PMA/ionomycin was used as activation control and W2/36 to block MHCI presentation and confirm antigen specificity. Cells were stained for live/dead, EPCAM, CD3, CDS, and CD 107a & IFNy or 4-1 BB and analyzed by flowy using a Quanteon instrument ( Agilent, Santa Clara, CA ).
  • FIG. 8B shows results obtained from confocal imaging at 0 hour (Oh) and 24 hours (24h) of co-culturing experiment of ALI TILs obtained from CRC and autologous tumor organoids for 24 hours at a 10:1 effectontarget ratio. Tumor cell killing was observed over time.
  • FIGS. 9 A and 9B display the results of single-cell RNA seq analysis of CRC and melanoma ALI-TILs.
  • FIG. 9 A shows numbers of unique clonotypes relative to number of sequenced cells (upper row) and distribution of elonotype frequencies (lower row). Unique paired a
  • FIG. 9B shows expression levels of 397 immune genes which identify 10 clusters using SeqGeq.
  • Example 1 Air-Liquid Interface Tumor Infiltrating Lymphocytes (ALI TILs) demonstrated a better tumor killing capacity compared to standard Tumor Infiltrating
  • ALI air-liquid interface
  • TILs tumor infiltrating lymphocytes
  • collagen liquid mixture was prepared according to the manufacturer’s instructions.
  • the collagen liquid mixture was prepared and kept on ice.
  • 1 mL of collagen liquid mixture was added into the 0.4 pm insert.
  • the insert containing collagen mixture was then left at room temperature for at least 30 min to solidify. The rest of collagen liquid mixture was kept on ice until later use in organoid culture step.
  • the numbers of inserts are prepared according to the size of the tissue sample. Total volume per insert is 2 mL.
  • the tissue sample for organoid culture was prepared by mincing the tissue using scissors until the tissue became paste-like texture and almost-liquid form. There were no chunks in this step, and the tissue was kept hydrated with appropriate volume of medium throughout the process. At this step, some of minced tissue can be frozen down in -80°C. Alternatively, all of the ti ssue or the rest of the tissue can be used for organoid culture.
  • the minced tissue was resuspended and reconstitute in collagen liquid mixture that was previously prepared. Each insert was then added with 1 mL of minced tissue dissolved in collagen liquid mixture directly on top of the collagen matrix -inserts that was previously prepared. The inserts containing minced tissue were then incubated at 37°C for 30-45 min to allow collagen liquid mixture to solidify,
  • tissue samples Prior to establishing the organoid culture, parts of the tissue samples can be kept for histology and/or sequencing experiments later. In order to store tire tissue samples, the tissue was washed with 5 times (5x) volume of F12 ⁇ Normoein in a. 10 cm culture dish. Optional ly, the tissue can be transferred to a petri dish on ice and cut into smaller pieces for IHC experiments. The tissue can be fixed in 4% PF A or 10% Formalin overnight at 4°C.
  • the collagen liquid mixture was prepared as described above and kept on ice.
  • collagenase solution was prepared by adding 950 ⁇ L of Fl 2 medium with 50 pL of Collagenase stock, which was prepared by adding 5mL of PBS i nto 1 vial of Collagenase.
  • the tissue in collagen gel insert was collected using a scraper, and each insert was added with 50- 100 pL of collagenase solution so each insert was added with 300U of collagenase.
  • the tissue in collagen gel and collagenase solution were then incubated at 37°C in a gently shaker for 45 min.
  • the pellet was resuspended in collagen liquid mixture.
  • Each insert was then added with 1 mL of pellet'collagen liquid mixture, on top of the collagen layer.
  • the inserts were then left in room temperature for 30 min to solidi fy.
  • the ALI organoids can be cryopreserved.
  • the freezing medium comprising 90% FBS and 10% DMSO was prepared prior cry op reservation.
  • the medium was aspirated from the culture dish.
  • the collagen gel containing ALI organoids was collected using a cell scraper or Pl 000 pipette tip before transferring into a 15 mL conical tube.
  • the volume of collagenase IV was calculated according to the number of inserts; about 50-100 pL collagenase I V and 500 p.L of culture medium were used per insert.
  • the ALI organoids were then shake at 37®C for 30-60 min until collagen gel was dissolved.
  • 3x volume medium was added to the tube. The tube was then spun at 400xg for 3 min.
  • Pre-REP Pre- Rapid Expansion Protocol
  • ALI derived TILs In order to prepare tissues and cells for Rapid Expansion of ALI derived TILs, after establishing organoids from tumor tissue, 50 IL ml of IL- 2 was added to the culture medium. The ALI organoids were then cultured for 7 to 14 days. Between day 7 to day 14, the ALI organoids were collected using a scraper. Collagenase IV was used in this step to collect ALI organoids. Next, to prepare TIL initiation between day 7 to day 14, the organoid pellets were resuspended into one well of G-Rex 24 Wel l Plate, and 6000 KJ ml . of IL-2 in RPMI medium was add to each well. The total volume of each well was about 6 mL.
  • 6000 IL ml. of IL-2 was added to the culture every three days. On day 11 after TIL initiation (or day 18 or day 24 after the start of 50 IL ml of IL-2 treatment, cells were harvested and were filtered through a 100 pm strainer. Next, cells were spun at 600xg for 3 min. Cells were then ready for REP step. For comparison, standard TILs (STD TILs) from standard pre-REP protocol by culturing with
  • 60001U/mL IL-2 for 11 days were harvested separately. Alternatively, these cells can be counted and freeze down using freezing medium as described previously.
  • Table 2 Materials and reagents for REP step of ALI derived TILs
  • ALI TILs 5 mil hon cells of ALI TILs were obtained from the Pre-REP step, and cells were suspended in 100 ml, of 50/50 medium.
  • 0.5 million ALI TILs were counted and mixed with 50 million irradiated PBMCs before plating on to each well of G- Rex 6M Well plate. This is day 0 of REP.
  • ALI TILs were added with 3000 lU/mL of IL-2 every 3 days.
  • ALI TILs were collected and filtered through 100 m cell strainer. ALI TILs can be frozen down or used for the experimenVtreatment.
  • ALI TILs were replaced with STD TILs.
  • ALI TILs and STD TILs were co-culture for 48 hours w ith autologous tumor epithelial cells derived from submerged Organoids. Single Cell tumor Organoids alone were used as a control. At 48 hours post-incubation, cells were collected for Flow Cytometry analysis.
  • FIG. 1 depicts the timeline and treatment of pre-Rapid Expansion Protocol (pre-REP) step of , ALI TILs and STD TILs.
  • pre-REP pre-Rapid Expansion Protocol
  • ALI TILs ALI TILs
  • STD TILs STD TILs
  • TILs Tumor Infiltrating Lymphocytes
  • PDO organoids were established from primary tumor as described in the previous example.
  • FIG. 3A depicts coculture experimental timeline.
  • Data w r as shewn for samples derived from 3 different patients CRC-1TM colorectal cancer patient 1, CRC-2TM colorectal cancer patient 2, MLNTM melanoma patient). .3 replicates per experiment were performed.
  • ALI TILs and STD TILs were co-cultured with autologous tumor epithelial cells derived from submerged Organoids for 48 hours. ALI TILs or STD TILs alone were used as a control. 48hours post-incubation, cells were collected for Flow Cytometry analysis.
  • FIG. 4A depicts an experimental timeline for co-culture experiment.
  • FIG. 4B-4D show flow cytometry result of PDI, HLA-DR, and ('DI 37.
  • FIG. 4C shows flow cytometry result of PDI, HLA-DR, and ('DI 37.
  • FIG. 4C shows higher level of HLA-DR compared to STD TILs. The result is presented on Fold Change. Further, as shown in FIG.
  • AIL TILs showed higher percentage of HLA-DR s- than STD TILs, at least about 50%, 23%, and 35%, in CR.C-.1 , CR.C-2, and Melanoma (MLN), respectively.
  • Data is shown for samples derived from 3 different patients (CRC-.1 :::: colorectal cancer patient 1 , CRC-2 ::: colorectal cancer patient 2, MLN ::::: melanoma patient). 3 replicates per experiment were performed. Nonparametric ANOVA tested corrected by Geisser Greenhouse Correction was performed to compare between different conditions;
  • FIG. 6A shows representative brighllield images of ALI tumor organoids from kidney, lung, esophagus, and uterus cancer.
  • the organoids were eventually maintained under submerged conditions that provide robust stem cell-based cancer models to allow extensive passaging and cryopreservation for subsequent in vitro and in vivo assessments (FIG. 6B).
  • FIG. 6B shows representative brighllield images of ALI tumor organoids from kidney, lung, esophagus, and uterus cancer.
  • FIG. 6B shows a colorectal cancer (CRC) tissue (left) and a GBM sample (right) grown as ALI organoids for TIL expansion (top) and submerged organoids for assays (bottom). Cancer origin of the CRC organoids was confirmed by whole genome, exome sequencing, which matched the clinical sequencing of the original tumor (FIG. 6C). As shown in FIG. 6C, sequencing results for 3 CRC organoid lines were shown as CNV plots and mutated genes (APC, TP53, KRAS),
  • FIG. 7 A show's a schematic representation of the 2 -step A LI-TIL process and cryopreservation of the product .
  • ALI-TILs obtained from 14 preps were counted and numbers factored to account for tissue sampling and pre-REP dilution. Estimated fun-scale yields are plotted for each individual prep with average and SEM (left). Box plot illustrates the foldexpansion distribution of tiie 14 REP products (right).
  • thawed cells were analyzed by FACS. Consistent with the TIL phenotypes, ALI-TILs were mainly comprised of ap T cells of the CD4 and CDS lineages, each made of -83% effector memory T cells (TEMs) and —16.5% central memory T cells (TCMs).
  • TEMs effector memory T cells
  • TCMs central memory T cells
  • FIG. 7C shows that twelve ALI-TILs preps (original 14 minus 2 CRCs for which material was insufficient) were analyzed by flow on an Agilent Quanteon analyzer (Santa Clara, CA), using CD3, yb TCR, CD4, CDS, CD45RA, and CD62L to detect T cells, lineage, and memory subsets. Results were analyzed with FlowJo ( Ashland, OR) and plotted as percent parent for each individual sample with average and SEM. This high-level analysis shows that TILs generated with the ALI process present with the expected phenotypic characteristics of a TIL product.
  • Tumor reactivity was tested by co-culturing the ALI-TILs from CRC organoids with autologous tumor cells established in a parallel organoid culture, grown as conventional organoids that exclusively possess tumor cells and not immune cells.
  • increased IFN-y, CD 107a, and 4- IBB were ail detected by flow- cytometry in 0.12 (6.3-fold induction relative to MHC block control), 0.8 (3,8x), and 0.4 (I.6x) % of T cells, respectively.
  • ALI-TILs from 1 CRC were co-cultured with autologous tumor organoids for 16 hours at a 1:1 effector: target ratio.
  • MHC major histocompatibility complex
  • EPC AM EPC AM
  • CD3, CD8, and CD107a & IFN-gamma or 4-1 BB analyzed by flow, using a Quanteon instrument (Agilent, Santa Clara, CA).
  • Data were processed with FlowJo (Ashland, OR), gating for indicated marker as shown. This result sho ws that le vel of responsive T cells can be used for clinically active TIL products. Further, tumor cell killing was assessed by longitudinal high-content confocal imaging (FIG.
  • ALI-TILs from 1 CRC were co-cultured with autologous tumor organoids for 24 hours at a 10:1 effector: target ratio. Tumor cell killing was monitored at 0 hour and 24 hours after co-culture using Molecular Devices I mageXpress (San Jose, CA). As shown in FIG. 8B, TILs/autologous organoids co-culture, staining, and detection conditions were established. Reduction in tumor organoids and increase in cell death were observed over time.
  • a pilot single-cell RNA seq experiment confirmed ALI-TIL product polyclonality and T cell subset diversity
  • TCR repertoire and gene expression profiles were generated for 4 ALI-TIL preps (3 CR.Cs and 1 melanoma) using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).
  • CITE-seq cellular indexing of transcriptomes and epitopes by sequencing
  • FIG. 9 A Shown in FIG, 9 A are # of unique clonotypes relative to # of sequenced ceils (upper row) and pie charts of distribution of clonotype frequencies decreasing from red to black with the fraction occupied by the top 20 sum of frequencies (lower row).
  • the cell surface markers data were consistent with the FACS analysis, confirming product purity and T cell subsets.
  • FIG* 9B unsupervised clustering of the gene expression data generated 10 T cell subsets, that appear to differentially express differentiation (LEF1 , CD27), activation (HLA-DR, CD25), effector (GZMA, PRF1, KLRC 1), exhaustion (LAGS, HAVCR2) markers in the CD4 (5 clusters), CD 8 (3 clusters), and mixed (2 clusters) lineages.
  • FIG. 1 unsupervised clustering of the gene expression data generated 10 T cell subsets, that appear to differentially express differentiation (LEF1 , CD27), activation (HLA-DR, CD25), effector (GZMA, PRF1, KL
  • TILs are isolated via mechanical dissociation of tumor tissue and subjected to two in vitro cultivation steps.
  • the steps comprise a pre-rapid expansion protocol (pre-REP) followed by the REP phase.
  • pre-REP pre-rapid expansion protocol
  • TILs are cultured in the presence of high-dose interleukin-2 (II., -2) to promote expansion, resulting in several fold increase (e.g., at least 10 fold increase) in cell numbers.
  • II. interleukin-2
  • the expanded TILs are cryopreserved and available for adoptive cell therapy (ACT).
  • GBM glioblastoma
  • immune competent GBM organoids are utilized and induce signaling pathway modifications during the in vitro TIL expansion process to promote differentiation into a TSCM phenotype.
  • the Interferon-gamma (IFN-y) signaling pathway is known to inhibit the maintenance and diversity of stem-like T cells, while Notch signaling has been identified as a potent regulator of T cell activation and can convert activated T cells into stem memory T cells (TSCMs), which possess increased self-renewal capacity and are critical for long-term immune memory and effective immunotherapy.
  • TSCMs stem memory T cells
  • TEMs terminally differentiated effector memory' T cells
  • IFN-y neutralizing antibodies prevent IFN-y from binding to its receptors and thereby inhibit downstream signaling pathways while agonistic antibodies targeting Notch receptors induce Notch signal activation.
  • mAbs monoclonal antibodies
  • Cryopreserved pre-REP TILs are used to culture pre-REP TILs in medium supplemented with IFNy inhibiting or Notch activating antibodies at multiple concentrations to identify the most effective mAh cocktail that can block the IFN-y signaling pathway and activate the Notch signaling pathway.
  • the expression levels of Myc, Deltex.1, and Hesl are measured to characterize the Notch activation level, as well as JFNy-induced genes Gbp5, Ml , and Ccl2 are measured to assess the level of IFNy signaling by qPCR 8 hours, 24 hours, and 48 hours post mAb administration.
  • TILs during the REP culture are exposed to the most effective mAb cocktail that simultaneously targets IFNy inhibition and Notch activation either throughout or for the last 7 days of the REP TIL phase.
  • II, -2 is partially substituted with IL- 7 and/or IL-15 in the culture to additional ly improve the TIL reprogramming process through IFN-y inhibition and Notch activation.
  • pre-REP TIL preparations comprising melanoma, colon cancer, lung cancer, and GBM pre-REP TIL sample is tested.
  • the cultured ceils are evaluated for the expression levels of markers, including CCR7, CD45RA, CD62L, CD69, CD95, and CD 103, that facilitate the identification of distinct T-cell memory subtypes are assessed.
  • TCM central memory T cells
  • TRM tissue resident memory T ceils
  • TEM effector memory T ceils
  • TSCM stem cell memory T cells
  • Naive T ceils which are characterized by CD45RA+CCR7+CD62L4- expression
  • TEMRA terminally differentiated effector memory cells
  • T cell function To characterize T cell function, differentiation, activation and exhaustion, the expression levels of immune checkpoint proteins such as PD-1, Tim-3, LAG-3, TIGIT, and CTLA-4, which are commonly associated with impaired T cell function and increased susceptibility to apoptosis during cancer progression (REF) are assessed.
  • the system yields a detectable TSCM subset of al least 5% and a TCM subset of at least 20%. These subsets are of particular interest because they are associated with enhanced persistence, proliferation, and efficacy in adoptive T cell therapy for cancer. By detecting these subsets, the effectiveness of the reprogramming protocol and identification of areas for further optimization to improve the yield of TSCMs are evaluated.
  • TILs donor- or patient-derived airliquid interface (All) immuno-competent tumor organoids is cultured.
  • TME in vivo tumor microenvironment
  • the ALI-T1L process favors the selection of T cells recognizing tumor neo antigens and leads to a shi ft of the T cell receptor (TCR.) repertoi re of TIL preparations towards enhanced neoantigen recognition capabilities. Consequently, these conditions enrich the TIL product for GBM specificity, function and achieve better outcomes in the fight against GBM.
  • AU GBM organoids containing TILs are cultured in GMB medium supplemented with a low dose of IL-2 to allow for the growth of GBM ALI TILs. Aller 1-7 days GMB ALI organoids are isolated by digesting the collagen block with Collagenase IV. Isolated organoids containing TILs are transferred into a 24-well G-Rex culture dish, followed by a pre-REP cultivation process. During this 1 1-14 day process, TILs are cultured in T cell medium enriched with high-dose IL-2. After 1 1- 14 days ALI pre-REP TILs are transferred into a 6M G-Rex well to initiate the REP phase.
  • This protocol involves stimulating TILs with anti-CD3 antibody, along with high-dose IL-2, in vitro for 11-14 days.
  • the ALI-TIL process for GBM through two successful runs can each yield a minimum of 250 million cells.
  • rALI-TILs reprogrammed ALI-TILs
  • rALI-TILs are expected to exhibit a T cell-dominant phenotype, with a minimum of 90% T cells represented by both CD4 and CDS lineages. This high degree of T cell representation is essential for the effective targeting of cancer cell.
  • rALI-TILs are from at least 4 different GBM samples and their T cell memory phenotype is compared to the matched control TIL preparations, including AL1, CTRL, and CTRL TILs. After TIL preparations have been generated, yields are determined.
  • the full-scale TIL yields is extrapolated, which provides insights of die potential for the TIL generation processes in clinical applications.
  • the TIL preparations at 20 million cells per vial are aliquoted and cryopreserved. This allows assessing the samples at a later time, if needed, and to continue the investigations into the efficacy of the optimized reprogramming protocol for GBM: rALI-TILs.
  • a detai led immune-profiling analysis of autologous TIL preparations including ALL rALIs, autologous control (CTRL), and rCTRL from 4 GBM samples is conducted.
  • Flow cytometry is used to comprehensively analyze individual the TIL preparations by assessing T cell subtypes, T cell memory and function.
  • the panel of markers comprise CCR7, CD45RA, CD62L, CD69, CD95, CD103, PD- 1 , Tim-3, LAG-3, TIGIT, and CTLA-4, which is used to differentiate between T cell memory' subsets, activation status, and functional potential, as well as to evaluate T cell exhaustion levels.
  • This analysis provides a comprehensive immune profile of GBM ALI-TILs and their autologous controls and identity differences in T-cell subsets and activation status.
  • the selection of the rALI-TIL process is based on a yield of at least 1 x 10 s cells, with a minimum of 90% T cells, and a minimum of 20% TCMs and 5% TSCMs indicating successful reprogramming.
  • a flow cytometry panel is established to characterize the expression levels of several cell surface markers linked to glioblastoma, including CD 133, CD44, CD45, and CD90. The goal is to identify cell surface markers that are uniquely expressed in autologous GBM cells but absent in their respective TIL preps.
  • an IFNy release assay is conducted on all 16 GBM TIL samples.
  • This assay is a widely used method to assess T cell activation and cytokine production in response to pan TCR stimulation.
  • the assay is performed by incubating individual TIL preparations with a pan TCR stimulant consisting of anti-CD3 and anti-CD28 antibodies. Following an incubation period of I to 3 days, the supernatant is collected, and the amount of I FNy released by the cells is measured using ELISA. Utilizing this approach allows evaluation of the TIL samples* responsiveness to pan TCR stimulation and to assess their overall T cell fi tness.
  • the secretion of over 200pg/ml IFNy serves as an indicator of a robust T cell fitness level.
  • GBOs are three- dimensional (3D) cultures that mimic the complex architecture and cellular heterogeneity of in vivo GBMs.
  • a GBO model is established by culturing 1 mm 3 patient derived GBO tissue fragments in suspension. The organoids are maintained in a serum-free medium and can be propagated for several months. To avoid necrotic cell death in the inner core, GBOs are propagated by cutting them into 0.5 mm diameter pieces every 1-2 weeks.
  • the goal is to establish and expand this GBO culture system from fresh glioblastoma tissues and cryopreserve them for future experiments. This ensures a continuous supply of GBOs that can be used to characterize the antitumor response of matched TIL preparations.
  • specific criteria is set. A GBO line is established if it can be passaged indefinitely, can be cryopreserved for future use, and exhibits antigen presentation in response to IFNy exposure.
  • the genotype of the established GBO line is matched to that of the original GBM sample by performing whole-genome sequencing. Meeting these criteria ensures the establishment of reliable and consistent GBO lines for future experiments.
  • the TRA an in vitro assay where different TIL preps are eo-cuitured with patient matched GBOs i s used.
  • IFN-y is a cytokine produced by activated T cells and is an important marker of T cell function. The presence of IFN-y in this assay indicates T cell activation and production of this cytokine in response to tumor antigens.
  • CD 107a is a lysosomal-associated membrane protein that is expressed on the surface of activated T cells. The presence of CD 107a on the cell surface indicates T cell degranulation and release of cytotoxic molecules, such as perforin and granzyme B, that are responsible for killing tumor cells.
  • 4-I.BB is a co-stimulatory molecule expressed on the surface of activated T cells. ('DI 37 signaling enhances T cell proliferation and survival and promotes the production of cytokines such as IFN-y.
  • the expression of CD 137 on T cells in the TRA is used as an additional marker of T cell activation and function.
  • TILs and GBO-derived tumor cells are co-cultured in a 1: 1 ratio for a period of 4-24 hours.
  • a PMA'Ionomycin T-cell stimulation cocktail is used as a positive control.
  • negative controls are used, including a supplement of MHC blocking antibody cocktail, which can block the recognition of tumor antigens by T cells, thereby preventing T cell activation.
  • TILs cultured in the absence of corresponding tumor cells also serve as negative controls.
  • all samples undergo flow cytometric analysis to evaluate the expression levels of IFN-y, CD107a, and CD 1.37 on T cells. This analysis provides a quantitative assessment of T cell reactivity against tumor cells and aid in identifying any variations regarding the tumor reactivity between GBM TIL preparations that are generated via different methods.
  • GMB TIL/GBO co-cultures at a 10: 1 ratio for a 24h, 48h and 72h are set. Negative controls, including MHC blocking antibodies, are used to ensure assay specificity.
  • the level of TIL- mediated tumor cell killing is evaluated through flow cytometric analysis. This analysis involves the measurement of various parameters, including the expression levels CD3 and GBO specific surface markers, which are used to label T cells and GBM tumor cells, respectively.
  • the levels of apoptosis and necrosis in tumor cells are measured using Dapi, Annexin V or propidium iodide staining, enabling discrimination of live, apoptotic, and necrotic ceils.
  • live cell confocal imaging is performed to monitor TIL-mediated GBO cell death over a period of 72h.
  • the TILs and GBOs are co-cultured at a 10: 1 ratio, and the GBM TILs are labeled with Cell Trace Violet while the GBOs are labeled with Cell Tracker Orange.
  • Sytox Green is added to flic co-culture medium, which is a nucleic acid stain that is impermeable to the cell membrane of living cells but enters dead cells and binds to nucleic acids, resulting in green fluorescence labeling of the dead cells.
  • Negative controls including MHC- blocking antibodies, are used to ensure assay specificity.
  • ODX organoid-derived xenograft
  • MOA mechanism of action
  • rALI-TILs The efficacy of rALI-TILs in controlling tumor growth in vivo is evaluated by monitoring their ability to control the growth of autologous ODX tumors.
  • organoid platform is used to generate autologous tumor xenograft models.
  • in vivo persistence is characterized by detecting transferred T cells in the tumor deposits and the circulation over time.
  • a minimum of four donor-derived GBO lines using a SQ xenograft model in immunocompromised NOG mice is used, The GBOs are dissociated into single cells and small cell clumps, then resuspended in a mixture of 50% GBO medium and 50*% Matrigel before transplantation.
  • Each GBO line is implanted in five mice, with doses varying from Ix lO 5 to lx KT, and tumor growth is monitored by caliper measurement weekly until the tumor reaches a size of 2000mm J , at which point the mice is sacrificed.
  • the tumor tissue is isolated from the mice, banked, and subjected to histological and molecular characterization.
  • the objective of this analysis is to validate that the genotype and phenotype of the ODX tumor tissue are similar to that of the corresponding primary GBM, thereby providing a reliable model for subsequent experiments.
  • orthotopic GBM xenograft models is established, which are more clinically relevant than subcutaneous models as they more closely replicate the tumor microenvironment of the brain. These models are used to study tumor invasion and better understand the behavior of GBM in its native environment.
  • GBO-derived tumor ceils are injected directly into the brain of immunocompromised mice using a stereotactic injection system through a small hole in the skull to ensure precise targeting of the injection site.
  • GBO tumor cells are resuspended in a mixture of 50% GBO medium and 50% Matrigel prior to transplantation. Following implantation, the mice are monitored for neurological symptoms, and tumor growth is measured using MR.L To minimize variability, the GBO dissociation protocol and transplantation methods is standardized. This involves using optimized digestive enzymic concentration and digestion time for each GBO line and employing a stereotactic injection system for orthotopic transplantations to minimize the risk of damage to brain tissue.
  • the first and second group receives infusions of 20 million GMB patient-matched rALI TILs and CTRL TILs, respectively, via the tail vein.
  • the third group serves as control. Tumor size is monitored weekly until one of the group reaches a tumor size of 2()()0mm ⁇ at which point the mice is sacrificed. At the termination of this GBO tumor xenograft experiment, all remaining tumors from the three groups are harvested and evaluated. Based on the hypothesis that rALI TILs present significantly enhanced cytotoxic anti-tumor activity, the administration of these cells elicits a substantial reduction in tumor volume relative to the control cohorts.
  • TIL invasion within the tumor microenvironment and T cell persistence in mouse blood samples is evaluated.
  • the blood samples at various time points after TIL infusion is collected and analyzed using flow cytometry to determine the percentage of human T cells in the mouse blood.
  • This data allows assessment of the in vivo persistence of GMB rALI TILs and autologous controls.
  • tumor samples at the termination of the GBO tumor xenograft experiment are collected and flow cytometry is performed and immunohistochemical staining to assess the extent of TIL infi ltration within the tumor microenvironment. This provides crucial, information on the localization of the TILs within the tumor and their potential impact on tumor growth.
  • the T cell receptor (TCR) repertoire of a TIL preparation represents the diversity and composition of I CRs expressed by T cells within the sample.
  • the TCRs expressed by each T cell recognize a specific antigen presented by major histocompatibility 7 complex (MHC) molecules, thereby refl ecting the speci ficity and diversity of the T cell response.
  • MHC major histocompatibility 7 complex
  • TCR sequencing on 1X10 4 single cells per GBM derived TIL preparation is performed.
  • a minimum of 4 GBM TIL sets, including autologous CTRL TIL, rCTRL TIL (reprogrammed CTRL TIL), ALI TIL and rALI TIL preparations per GBM samples are analyzed.
  • a distinct TCR clonotype composition between ALI and CTRL autologous samples may suggest that the ALI-TIL process selects for T ceils recognizing tumor antigens.
  • Altered clonotype frequencies in reprogrammed TILs compared to non-reprogrammed TILs may reflect the effect of pathway modulations during TIL reprogramming.
  • TIL production and selection methods for effective GBM immunotherapy can be further optimized.
  • Gene expression profiling of all G B M-derived TIL preparations at the singlecell level using single-cell RNA sequencing (scRNA-seq) and CITE-seq allows a thorough validation of the proposed reprogrammed stem cell-like state in TILs, exposed to pathway modulations.
  • Sequencing data, analyses using commercial (SeqGcq), academic (GLIPH258), and in-house (R. script) pipelines are used to analyze the sequencing data.
  • a commercial pipeline called SeqGeq is used to perform a comprehensive analysis of the sequencing data. This pipeline is useful for the characterization of TCR clonotypes and the RNAseq derived immune profiling data to validate the upregulation of TSCM and TCM markers and downregulation of exhaustion markers in reprogrammed TILs compared to the control group.
  • an academic pipeline called CLIP H258 is used to identify TCR clonotypes based on sequence homology. This pipeline is useful for identifying clonotypes that share common features and may recognize similar antigens.
  • an in-house R script pipeline is used to perform customized in-depth analyses of the sequencing data. This pipeline is instrumental to determine the TCR. clonotype overlap and diversity as well as altered clonotype frequencies between autologous TIL groups.

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Abstract

La présente invention concerne des systèmes et des procédés permettant d'augmenter les populations immunitaires réactives aux tumeurs, telles que les lymphocytes infiltrant les tumeurs, à partir d'organoïdes issus de patients. La présente invention concerne également des systèmes et des procédés permettant de multiplier et de reprogrammer les populations immunitaires réagissant aux tumeurs, tels que les lymphocytes infiltrant les tumeurs, afin d'améliorer l'activité antitumorale. Les systèmes et procédés décrits dans la présente invention peuvent être utilisés dans le cadre d'une thérapie cellulaire adoptive.
PCT/US2023/032111 2022-09-07 2023-09-06 Systèmes et procédés d'amélioration des populations immunitaires réactives aux tumeurs à l'aide d'organoïdes WO2024054518A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2020096986A2 (fr) * 2018-11-05 2020-05-14 Iovance Biotherapeutics, Inc. Sélection de lymphocytes t réactifs à une tumeur améliorés
WO2023043787A1 (fr) * 2021-09-15 2023-03-23 The Board Of Trustees Of The Leland Stanford Junior University Méthodes d'amplification de populations immunitaires réagissant aux tumeurs à l'aide d'organoïdes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020096986A2 (fr) * 2018-11-05 2020-05-14 Iovance Biotherapeutics, Inc. Sélection de lymphocytes t réactifs à une tumeur améliorés
WO2023043787A1 (fr) * 2021-09-15 2023-03-23 The Board Of Trustees Of The Leland Stanford Junior University Méthodes d'amplification de populations immunitaires réagissant aux tumeurs à l'aide d'organoïdes

Non-Patent Citations (2)

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Title
BESSER, M J ET AL.: "Modifying interleukin-2 concentrations during culture improves function of T cells for adoptive immunotherapy", CYTOTHERAPY, vol. 11, no. 2, 2009, pages 206 - 17, XP055154534, DOI: 10.1080/1465324080259039 *
NEAL JAMES T.; LI XINGNAN; ZHU JUNJIE; GIANGARRA VALERIA; GRZESKOWIAK CAITLIN L.; JU JIHANG; LIU IRIS H.; CHIOU SHIN-HENG; SALAHUD: "Organoid Modeling of the Tumor Immune Microenvironment", CELL, vol. 175, no. 7, 1 January 1900 (1900-01-01), Amsterdam NL , pages 1972, XP085560742, ISSN: 0092-8674, DOI: 10.1016/j.cell.2018.11.021 *

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