Systems and methods for enhancing tumor reactive immune populations with organoids

Abstract

Systems and methods are provided for enhancing tumor reactive immune populations, such as tumor infiltrating lymphocytes from organoids of patient origin. In addition, provided herein are systems and methods for amplifying and reprogramming tumor reactive immune populations, such as tumor infiltrating lymphocytes, to improve anti-tumor activity. The systems and methods described herein can be used for adoptive cell therapy.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 374,891, filed on September 7, 2022, and U.S. Provisional Application No. 63 / 499,685, filed on May 2, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Adoptive cell therapy utilizes cells from the immune system, such as tumor-reactive immune cells, for cancer or tumor treatment. This form of immunotherapy obtains cells, such as tumor-reactive immune cells, from a patient. These cells are then expanded, or in some cases engineered or reprogrammed, to enhance their ability to target and eliminate cancer cells. These cells are then reinfused into the patient for cancer treatment. The process of expanding tumor-reactive immune cells and the quality of tumor-reactive immune cells remain limited. Summary of the Invention

[0004] There is a need for enhancing tumor-reactive immune cells for cancer and tumor treatment that has not been met. The present disclosure meets this unmet need.

[0005] In one aspect, the present disclosure provides a method for generating immune cells from a subject, the method comprising: (a) obtaining one or more tumor samples from the subject, wherein the one or more tumor samples contain 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 a culture medium, and wherein one or more agents are added to the culture medium; and (c) collecting immune cells from the one or more tumor samples, thereby generating immune cells from the subject.

[0006] In some embodiments, the incubation in (b) includes a first time period and a second time period. In some embodiments, the first time period is at least about 1 day. In some embodiments, the first time period is about 7 to about 14 days. In some embodiments, the second time period is at least about 1 day. In some embodiments, the second time period is about 11 days.

[0007] In some embodiments, one or more agents are added to the culture medium at a first concentration during the first time period. In some embodiments, 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.

[0008] In some embodiments, the in vitro culture process includes a gas-liquid interface setup.

[0009] In some embodiments, the method further comprises cryopreserving the immune cells from (c). In some embodiments, the method further comprises (d) expanding the immune cells obtained from (c) for a period of time using one or more agents. In some embodiments, the method further comprises (e) collecting the immune cells from (d). In some embodiments, the method further comprises, after (e), (f) cryopreserving the immune cells.

[0010] In some embodiments, the immune cells comprise tumor infiltrating lymphocytes. In some embodiments, the tumor infiltrating lymphocytes comprise T cells. In some embodiments, the T cells comprise activated T cells; naive CD8+ T cells; cytotoxic CD8+ T cells; naive CD4+ T cells; helper T cells, such as T H 1, T H 2, T H 9, T H 17, T H 22, T FH ; memory T cells, such as central memory T cells, T stem cell memory cells (TSCM), effector memory T cells, NKT cells, or γδ T cells.

[0011] In some embodiments, the one or more agents in (b) comprise cytokines. In some embodiments, the cytokines comprise IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, or combinations thereof. In some embodiments, the concentration of the cytokine added during the first time period in (b) is at least about 10 IU / mL. In some embodiments, the concentration of the cytokine added during the first time period in (b) is about 50 IU / mL. In some embodiments, 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.

[0012] In some embodiments, the expansion of the immune cells in (d) does not include an air-liquid interface setting. In some embodiments, the period of time in (d) is at least 1 day. In some embodiments, the period of time in (d) is about 14 days. In some embodiments, the one or more agents in (d) comprise cytokines, antibodies, modulators, or any combination thereof.

[0013] In some embodiments, one or more agents further comprise irradiated feeder cells. In some embodiments, the irradiated feeder cells are irradiated allogeneic PBMC-derived feeder cells. In some embodiments, the ratio of immune cells to irradiated feeder cells is about 1:100.

[0014] In some embodiments, the cytokines include IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, or any combination thereof. In some embodiments, the cytokines are added at a concentration of at least about 2000 IU / mL. In some embodiments, the cytokines are added at a concentration of about 3000 IU / mL.

[0015] In some embodiments, the antibody includes an anti-CD3 antibody. In some embodiments, the concentration of the anti-CD3 antibody is at least about 10 ng / mL. In 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.

[0016] In some embodiments, the modulators include Notch signaling pathway modulators, interferon gamma (IFNγ) modulators, or a combination thereof. In some embodiments, the Notch signaling pathway modulators include Notch activators. In some embodiments, the Notch activators include antibodies, small molecules, or a combination thereof. In some embodiments, the IFNγ modulators include IFNγ inhibitors. In some embodiments, the IFNγ inhibitors include antibodies, small molecules, or a combination thereof.

[0017] In some embodiments, the method further comprises providing an additional reagent during a first time period or a second time period, wherein the additional reagent comprises one or more of PD-1, CD39, 4-1BB positive T cells, or a CXCR3-binding chemokine. In some embodiments, the CXCR3-binding chemokine includes CXCL9 or CXCL10.

[0018] In some embodiments, the method further comprises providing an additional reagent during a first time period or a second time period, wherein the additional reagent comprises interferon.

[0019] In some embodiments, the method further comprises providing an additional reagent during a first time period or a second time period, wherein the additional reagent comprises a checkpoint inhibitor.

[0020] In some embodiments, the method further comprises providing an additional reagent during a first time period or a second time period, wherein the additional reagent comprises TLR3, TLR7, TLR9, or other TLR agonists.

[0021] In some embodiments, the method further comprises providing an additional reagent during a first time period or a second time period, wherein the additional reagent comprises a regulator of RIG-I-like receptors, a regulator of NOD-like receptors, a regulator of C-type lectin receptors, a regulator of STING, or a combination thereof.

[0022] In some embodiments, the method further comprises combining the tumor organoids with immune cells obtained from other sources. In some embodiments, the other sources include peripheral blood cells or organoids grown from lymphoid tissue.

[0023] In some embodiments, the method further comprises stimulating antigen presentation.

[0024] In some embodiments, the method further comprises depletion of immunosuppressive cell types. In some embodiments, the immunosuppressive cell types include Tregs, myeloid-derived suppressor cells, TAMs, vascular endothelial cells, or CAFs.

[0025] In some embodiments, the method further comprises negative selection of bystander tumor-reactive immune cells.

[0026] In some embodiments, the method further comprises knocking down exhaustion regulators. In some embodiments, the knockdown exhaustion regulator is TOX.

[0027] In some embodiments, the method further comprises reprogramming immune cells. In some embodiments, the immune cells are reprogrammed by activating the Notch signaling pathway during (d), inhibiting the interferon gamma (IFNγ) signaling pathway during (d), or both.

[0028] In some embodiments, the method further comprises identifying a T cell receptor (TCR) to identify TCRs enriched in the air-liquid interface culture. In some embodiments, the TCRs are identified using sequencing techniques.

[0029] In some embodiments, the method comprises providing one or more tumor antigens during a first time period or a second time period. In some embodiments, providing one or more tumor antigens comprises providing cells expressing the tumor antigens.

[0030] Incorporated by reference

[0031] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the present invention are set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and the accompanying drawings, which set forth illustrative embodiments in which the principles of the invention are utilized, in the drawings:

[0033] Figure 1 Depicts an exemplary timeline of the pre-Rapid Expansion Protocol (pre-REP) steps for the Standard Tumor Infiltrating Lymphocyte (STD TIL) protocol and the air-liquid interface Tumor Infiltrating Lymphocyte (ALI TIL) protocol.

[0034] Figure 2 Shows a graph demonstrating that co-culture of air-liquid interface Tumor Infiltrating Lymphocytes (ALI TIL) with autologous tumor epithelial cells exhibits better tumor killing ability than co-culture of standard Tumor Infiltrating Lymphocytes (or STD TIL) with autologous tumor epithelial cells. The Y-axis represents the percentage of viable tumor epithelial cells.

[0035] Figures 3A-3C Shows that ALI TIL is more reactive to tumor cells compared to STD TIL. Figure 3A Shows the experimental outline. Figure 3B and Figure 3C Shows a graph demonstrating that, based on Figure 3B the percentage of CD3+ cells secreting IFNγ shown in Figure 3C and the percentage of CD3+ cells expressing CD107a shown in , ALI Tumor Infiltrating Lymphocytes (ALI TIL) are more reactive to tumor cells compared to standard TIL (STD TIL). Data from samples from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated three times. Non-parametric ANOVA was performed, corrected by Geisser-Greenhouse correction for comparison between different conditions; *≤Pv = 0.05.

[0036] Figures 4A-4D Shows that ALI TIL expresses higher levels of HLA-DR compared to STD TIL. Figure 4A Shows the experimental outline. Figure 4B , Figure 4C and Figure 4D Show graphs illustrating the expression levels of PD1, HLA-DR, and CD137, respectively.Figure 4C ALI tumor-infiltrating lymphocytes (ALI TIL) were shown to express higher levels of HLA-DR compared to standard TIL (STD TIL). Results are shown as fold change. Data from samples derived from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated three times. Non-parametric ANOVA was performed, corrected by Geisser-Greenhouse correction, for comparison between different conditions; *≤Pv = 0.05.

[0037] Figure 5 ALI tumor-infiltrating lymphocytes (ALI TIL) were shown to express a higher percentage of HLA-DR+. Data from samples derived from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated three times. Non-parametric ANOVA was performed, corrected by Geisser-Greenhouse correction, for comparison between different conditions; *≤Pv = 0.05.

[0038] Figures 6A-6C Examples of ALI tumor organoid establishment are shown. Figure 6A Examples of ALI tumor organoids generated from tissues obtained from kidney, lung, esophagus, and uterus are shown. Figure 6B Examples of ALI tumor organoids generated from tissues obtained from colon and glioblastoma (GBM) are shown. The top panel shows the sample growing as an ALI organoid. The bottom panel shows the submerged organoid. Figure 6C Sequencing results of three colorectal cancer (CRC) organoid lines are shown, presented as CNV plots and mutated genes (APC, TP53, KRAS).

[0039] Figures 7A-7C Generation and phenotypic characterization of ALI TIL are presented. Figure 7A A schematic diagram depicting the 2-step ALI TIL process and cryopreservation of the product is shown. Figure 7B The number of ALI TIL cells obtained from 14 preparations (left) and the fold expansion distribution of 14 products (right) are shown. Figure 7C Flow cytometry analysis results for detection of T cells, lineage, and memory subsets using CD3, γδTCR, CD4, CD8, CD45RA, and CD62L are shown. Results are plotted as the percentage of the parent for each individual sample, with mean and SEM.

[0040] Figure 8A and 8B ALI TIL tumor reactivity and cytotoxicity assays are shown. Figure 8AShows flow cytometry analysis of ALI TILs from CRC co-cultured with autologous tumor organoids. Increased IFNγ, CD107a, and 4-1BB (or CD137) were observed. Figure 8B Shows results obtained from confocal imaging at 0 hours (0h) and 24 hours (24h) of the ALI TIL and autologous tumor organoid co-culture experiment. Tumor cell killing over time was observed.

[0041] Figure 9A and 9B Shows results of single-cell RNA sequencing analysis of CRC and melanoma ALI-TILs. Figure 9A Shows the number of unique clonotypes (upper row) and clonotype frequency distribution (lower row) relative to the number of sequenced cells. Figure 9B Shows the expression levels of 397 immune genes identified in 10 clusters using SeqGeq. Detailed implementation

[0042] Adoptive cell immunotherapy uses a patient's own immune cells for cancer treatment. The immune cells are isolated from the patient's own blood or tumor tissue, grown and expanded in the laboratory, and then transfused back into the patient to treat the patient's cancer. In some cases, the immune cells are engineered to enhance their ability to target cancer cells, such as in chimeric antigen receptor (CAR) T cell therapy.

[0043] Tumor-infiltrating lymphocytes are another type of adoptive cell immunotherapy that has been developed for the treatment of solid tumors. Lymphocytes or white blood cells (such as T cells or B cells) are part of the immune system that helps the body resist infections and eliminate abnormal cells (such as cancer or tumors). Once the lymphocytes recognize abnormal cells and infiltrate into the tumor, these cells are called tumor-infiltrating lymphocytes (TILs or TIL). TILs are known to be able to kill cancer cells.

[0044] One of the main benefits of using TILs is that since TILs are directly obtained from the tumor, they can recognize targets on cancer cells. In addition, a set of TILs can be obtained from the tumor that can recognize multiple unique targets on cancer cells, thereby predicting and preventing tumor adaptation to therapy.

[0045] To effectively utilize TILs as a cell therapy for cancer or tumor treatment, TILs are first collected from the tumor during a biopsy procedure. Next, in vitro TIL expansion is performed to obtain a large number of these immune cells suitable for administration to the patient.

[0046] The present disclosure provides systems and methods for enhancing tumor-reactive immune populations using patient-derived organoids (PDOs). There are two main steps for enhancing and expanding tumor-reactive immune cells: a pre-rapid expansion protocol (or pre-REP) step and a rapid expansion protocol (REP) step. In the standard (STD) protocol, the pre-REP step is treated with one concentration of IL-2 over the entire time period. In contrast, in the present disclosure, the pre-REP step method discloses treatment with low-concentration IL-2 for an additional time period (e.g., an additional week) compared to the standard protocol. When compared to TILs obtained from the standard protocol, the TILs obtained from PDOs (also referred to as "air-liquid interface TILs" or "ALITILs") using the protocols described herein exhibit better tumor killing ability and are more reactive to tumor cells. Thus, the ALI TILs obtained from the present disclosure can provide effective immune cells for cancer immunotherapy (e.g., adoptive cell therapy). Additionally, the present disclosure provides a method for reprogramming TILs into stem cell memory immune cells by inhibiting the IFNγ signaling pathway and activating the Notch signaling pathway during the REP step. The stem cell-like properties of the reprogrammed immune cells provide longer lifespan, self-renewal ability, and effector differentiation potential, thereby generating anti-tumor or anti-cancer TILs with enhanced anti-tumor or anti-cancer activity.

[0047] Unless otherwise defined, all professional terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms with commonly understood meanings are defined herein for clarity and / or for convenience of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0048] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a strict limitation on the scope of the present disclosure. Accordingly, a range description should be considered to have expressly disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a range description such as from 1 to 6 should be considered to have expressly disclosed sub-ranges 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 the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0049] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0050] The terms "determine", "measure", "evaluate", "assess", "assay", and "analyze" are used interchangeably herein to refer to forms of measurement. The term includes determining the presence of an element (e.g., detecting). These terms can include quantitative, qualitative, or both quantitative and qualitative determinations. An assessment can be relative or absolute.

[0051] The terms "subject", "individual", or "patient" are used interchangeably herein. A "subject" can be a biological entity containing genetic material that is expressed. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, cell, and progeny thereof of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. The mammal can be a human, primate, non-human primate, horse, cow, pig, dog, cat, rodent, such as a mouse, rat, hamster, etc. A subject can include, but is not limited to, humans, cows, dogs, mice, rats, rabbits, guinea pigs, chickens, fish, birds, reptiles, camelids, cows, chimpanzees, sheep, goats, and non-human primates. A subject may be diagnosed or suspected of being at high risk of a disease. In some cases, a subject is not necessarily diagnosed as or suspected of being at high risk of the disease.

[0052] The term "in vivo" is used to describe an event that occurs within a subject.

[0053] The term "in vitro" is used to describe an event that occurs outside of a subject. An in vitro assay is not performed on a subject. Instead, it is performed on a sample that is separated from the subject. An example of an in vitro assay performed on a sample is an "in vitro" assay.

[0054] The term "in vitro" is used to describe an event that occurs in a container containing laboratory reagents such that the reagent is separated from the biological source from which the material was obtained. An in vitro assay can include cell-based assays, where live or dead cells are used. An in vitro assay can also include cell-free assays, where intact cells are not used.

[0055] As used herein, the term "about" a number means that number plus or minus 10% of that number. The term "about" a range means that range minus 10% of its minimum value and plus 10% of its maximum value.

[0056] As used herein, the term "immune cell" refers to cells of hematopoietic origin that play a role in the immune response. In some cases, immune cells include 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. In some embodiments, immune cells include a complex mixture of immune cells, such as tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment.

[0057] As used herein, the term "T cell" refers to mammalian immune effector cells that can be characterized by expression of CD3 and / or the T cell antigen receptor. In some embodiments, T cells include naive CD8 + T cells; cytotoxic CD8 + T cells; naive CD4 + T cells; helper T cells such as T H 1, T H 2, T H 9, T H 17, T H 22, T FH ; memory T cells such as central memory T cells, T stem cell memory cells (T SCM ), effector memory T cells, NKT cells, γδ T cells.

[0058] As used herein, the term "adoptive cell therapy" refers to an immunotherapy that uses a patient's own immune cells (such as T cells or B cells) to help the body fight disease (such as cancer or a tumor). In some cases, adoptive cell therapy is also referred to as adoptive cell transfer, cellular adoptive immunotherapy, or T cell transfer therapy.

[0059] As used herein, the terms “cancer” or “tumor” are used interchangeably and refer to cells that exhibit autonomous, uncontrolled 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 include pre-cancerous cells (e.g., benign cells), malignant cells, pre-metastatic cells, metastatic cells, and non-metastatic cells. Cancers are known to occur in almost every tissue. Additionally, cancer is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness, or malignancy of the affected tissue or cell aggregate. Different stages / grades / malignancies of cancer include stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, and primary cancer. As used herein, the term “cancer cell” refers to a cancer cell or derived from a cancer cell, such as a clone of a cancer cell. Multiple types of cancers are known to those of skill in the art, including solid tumors such as carcinoma, sarcoma, glioblastoma, melanoma, lymphoma, myeloma, etc. Examples of cancers include ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, esophageal cancer, uterine cancer, and brain cancer.

[0060] As used herein, the terms “tumor infiltrating lymphocytes” or “tumor infiltrating lymphocyte” or “TILs” or “TIL” are used interchangeably and refer to immune cells found in a tumor, such as lymphocytes. In some embodiments, TILs can recognize and destroy cancer cells. In some embodiments, TILs are obtained from a patient's tumor. In some embodiments, TILs are obtained from a patient-derived tumor sample. In some embodiments, TILs are obtained from patient-derived organoids. Examples of TILs include, but are not limited to, T lymphocytes such as CD4+ or CD8+ T cells, B lymphocytes, macrophages, or NK cells.

[0061] As used herein, the term "cell culture" or "culture" refers to the maintenance of cells in an artificial in vitro environment. In some cases, the term "cell culture" is a general term that can be used to include not only the culture of individual cells, but also the culture of tissues or organs or samples that are part of a tissue or organ and derived from a patient. As used herein, the term "culture system" is used herein to refer to the culture conditions in which cells, tissues, organs, or samples derived from a patient's tissue or organ grow. The culture system promotes extended tissue or cell expansion, along with proliferation, multi-lineage differentiation, and the reproduction of the ultrastructure of cells and tissues. In some cases, the culture system refers to an air-liquid interface (ALI) 3D culture system. In some cases, the culture system also refers to non-ALI culture, in which the cells of interest can be expanded, for example, on feeder layer cells.

[0062] As used herein, the term "air-liquid interface" or "ALI" refers to the interface to which tumor cells, tissues, or samples derived from a patient's tissue or organ in the cultures described herein are exposed. In some cases, the primary tissue can be mixed with a gel solution and then poured onto a gel layer formed in a container that has a lower semi-permeable support (such as a membrane). In some cases, the container is placed in an outer container containing a culture medium such that the gel containing the tissue is not submerged in the culture medium. In some cases, the gel solution containing the primary tissue is exposed to air from the top and to the liquid culture medium from the bottom.

[0063] As used herein, the term "container" refers to a glass, plastic, or metal container that can provide a sterile environment for culturing cells.

[0064] As used herein, the term "organoid" refers to the three-dimensional growth of tumor tissue in culture that retains the characteristics of the tumor in vivo. In some cases, the organoid reproduces the ultrastructure of cells and tissues, immune cell interactions, etc. In some cases, the organoids used in the methods described herein are generally cultured from tumor biopsy sections. In some cases, organoids can be generated using any method known in the art, depending on the application. The organoid culture methods described in the present disclosure include the immersion method, the air-liquid interface method, the droplet and bioreactor methods, etc.

[0065] As used herein, the term "gel matrix" refers to a semi-solid extracellular matrix in the conventional sense. The gels described herein include, but are not limited to, various combinations of one or more of collagen gels, Matrigel, extracellular matrix proteins, fibronectin, collagen and laminin, nidogen, fibronectin and heparan sulfate; or human placenta extracellular matrix.

[0066] As used herein, the term "ultrastructure" refers to the three-dimensional structure of cells or tissues observed in vivo. Examples of ultrastructures include, but are not limited to, the ultrastructure of cells (which can be their polarity or morphology in vivo), or the ultrastructure of tissues (which can be the arrangement of different cell types within the tissue relative to each other).

[0067] As used herein, the term "biological sample" refers to liquid samples of biological origin (e.g., blood, sputum, semen, mucus, urine, cerebrospinal fluid), solid tissue samples (such as biopsy samples or tissue cultures or cells derived therefrom and their progeny), e.g., clinical samples or tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, etc. "Biological sample" includes samples obtained from a patient's cancer cells, e.g., samples containing polynucleotides and / or polypeptides obtained from a patient's cancer cells (e.g., cell lysates or other cell extracts containing polynucleotides and / or polypeptides); and samples containing cancer cells from a patient. Biological samples include cancer cells from a patient and may also include non-cancer cells. In some cases, the sample has been manipulated in any way after acquisition, such as by treatment with a reagent; washing; or enrichment for a particular cell population. The term sample also includes samples that have been enriched for a particular type of molecule (e.g., nucleic acids, polypeptides, etc.).

[0068] As used herein, the terms "air-liquid interface immune cells" or "ALI immune cells" are used interchangeably and refer to in vitro isolated immune cells obtained from the systems and methods described herein. These immune cells are obtained from PDO cultures derived from one or more tumor samples of a subject and are activated and expanded using the systems and methods described herein. In some cases, "immune cells" refer to cells of hematopoietic origin that play a role in the immune response. In some cases, immune cells include 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. In some embodiments, immune cells include a complex mixture of immune cells, such as tumor-infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. In some cases, "air-liquid interface immune cells" refer to "air-liquid interface tumor-infiltrating lymphocytes", "ALITILs", or "ALI TIL".

[0069] As used herein, the term "treatment" or "treating" refers to a drug or other intervention regimen used to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefits and / or prophylactic benefits. Therapeutic benefits may refer to eliminating or ameliorating symptoms or the underlying condition being treated. Additionally, a therapeutic benefit can be achieved by eliminating or ameliorating one or more physiological symptoms associated with the underlying condition, such that an improvement in the subject is observed, even though the subject may still have the underlying condition. Prophylactic effects include delaying, preventing, or eliminating the onset 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. For prophylactic benefits, a subject at risk of developing a particular disease or reporting the presence of one or more physiological symptoms of a disease may receive treatment even if a diagnosis of the disease has not been made.

[0070] Immunotherapy

[0071] Immunotherapy uses immune cells to protect the body from infections and diseases. There are several types of cancer immunotherapy. For example, immune checkpoint therapy uses immune cells (such as TIL) and immune checkpoint inhibitors that block negative immune checkpoint proteins (such as CTLA-4, PD-1L, or PD-1), enabling T cells to continue working and destroy cancer cells. Cancer vaccines stimulate immune cells to recognize and destroy cancer cells. Monoclonal antibodies can attach to specific proteins on the surface of cancer cells or immune cells and enhance the ability of immune cells to fight cancer. Cytokine therapy uses cytokines (such as interferons and / or interleukins) to trigger an immune response against cancer. Adoptive cell therapy uses the patient's own immune cells to fight cancer after being expanded or modified in vitro.

[0072] Adoptive cell therapy focuses on increasing the number and / or enhancing the effectiveness of immune cells. In adoptive cell therapy, the patient's immune cells are isolated, expanded, or modified in vitro and then transfused back into the patient to help the immune system fight cancer. For example, chimeric antigen receptor (CAR) T cell therapy modifies T cells to make them better at recognizing and attacking cancer cells. Similarly, 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 the blood and only select those cells that recognize specific characteristics of cancer cells. These cells are then expanded and transfused back into the patient. Tumor-infiltrating lymphocyte (TIL) therapy uses the patient's lymphocytes that are isolated from the tumor, expanded in vitro, and then transfused back into the patient.

[0073] Adoptive cell therapy involves obtaining immune cells, such as TILs, from a patient's tumor. In some cases, the tumor can be obtained from the patient by biopsy or surgical resection. In some cases, the patient's tumor is derived and cultured as an organoid in an in vitro environment, such as a patient-derived organoid (PDO). Once immune cells (such as TILs) are obtained from the PDO or directly from the tumor and cultured in vitro, these immune cells (such as TILs) are then cultured in a pre-rapid expansion protocol (pre-REP) step and then expanded in a rapid expansion protocol step by T cell receptor (TCR) engagement and cytokine therapy. These immune cells (such as TILs) are then collected for experimentation or treatment.

[0074] In one aspect, the present disclosure provides systems and methods for enhancing tumor-reactive immune populations using patient-derived organoids (PDOs). In another aspect, in the present disclosure, the methods provided herein involve a pre-rapid expansion protocol or pre-REP step that includes an additional period (such as one week) of low-concentration IL-2 treatment. When compared to immune cells (such as TILs) obtained from a standard protocol, immune cells (such as TILs) obtained from the PDOs used in this protocol (also referred to as "air-liquid interface immune cells, such as TILs" or "ALI immune cells, such as TILs") show better tumor-killing ability and are more reactive to tumor cells. Thus, the ALI immune cells (such as ALI TILs) obtained from the present disclosure are more effective for cancer immunotherapy than TILs obtained from a standard protocol (such as one without the additional period of low-concentration IL-2 treatment). Additionally, the present disclosure provides systems and methods for reprogramming TILs (such as TILs obtained from PDOs) to have stem cell-like properties. In some cases, the reprogramming of TILs can be performed in the REP step.

[0075] In some embodiments, the systems and methods described herein are improvements compared to standard methods of generating or expanding immune cells (such as TILs). In some embodiments, the systems and methods described herein for generating and / or expanding immune cells (such as TILs) provide improvements for immunotherapy. In some embodiments, the immune cells (such as TILs) obtained from the present disclosure provide improvements in tumor-killing ability and reactivity. In some embodiments, the immune cells (such as TILs) obtained from the present disclosure are also reprogrammed into a stem cell memory immune cell phenotype that has stem cell-like properties, such as a longer lifespan, self-renewal ability, and effector differentiation potential, thereby enhancing anti-tumor activity.

[0076] In some cases, the tissues used in the present disclosure include adrenocortical carcinoma, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancer, central nervous system (CNS) cancer, glioblastoma (GBM), peripheral nervous system (PNS) cancer, breast cancer, cervical cancer, childhood non-Hodgkin lymphoma, colorectal cancer, endometrial cancer, esophageal cancer, Ewing tumor family (e.g., Ewing sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, Hodgkin lymphoma, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumor, non-Hodgkin lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disease, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, melanoma skin cancer, non-melanoma skin cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell cancer, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid cancer, bronchial adenoma, choriocarcinoma, head and neck cancer, teratocarcinoma or Waldenström macroglobulinemia tissue.

[0077] In some cases, the system and method provide immune cells (e.g., TIL) for adoptive cell therapy. In some cases, the immune cells (e.g., TIL) obtained from the system and method described herein are only used for adoptive cell therapy. In some cases, the immune cells (e.g., TIL) obtained from the system and method described herein are used in combination with other immunotherapies (e.g., immune checkpoint therapy, monoclonal antibody therapy, cytokine therapy, etc.). In some cases, the immune cells (e.g., TIL) obtained from the system and method described herein are used in combination with chemotherapy. In some cases, the immune cells (e.g., TIL) obtained from the system and method described herein are used in combination with other cancer therapies (e.g., drug therapy).

[0078] Patient-derived organoids (PDO)

[0079] Patient-derived organoids (PDO) are three-dimensional in vitro cultures that recapitulate the cellular and ultrastructural features of the tumor or tissue sample from which they are derived. In some embodiments, PDOs include immune cells associated with the in vivo tumor or tissue, such as tumor-infiltrating lymphocytes, parenchymal components, stromal components, epithelial cells, etc. In some embodiments, PDOs are cultured in an air-liquid interface (ALI) system. In some embodiments, PDOs can be used for drug screening assays, in vitro disease modeling, or the generation and / or expansion of immune cells (e.g., TIL) for immunotherapy (e.g., adoptive cell therapy).

[0080] The air-liquid interface (ALI) method provides a culture system that allows the propagation of organoids with both tumor epithelial and stromal components. In some embodiments, the ALI method utilizes a Boyden chamber (cell culture insert) commonly used in cell migration assays. In some embodiments, cells are embedded in an extracellular matrix (ECM) gel on the upper surface of the cell culture insert, with a porous membrane below, which greatly increases the oxygen supply to the cells compared to the epithelial submersion-only organoid method. In some cases, the cells obtain nutrients and growth factors from the culture medium placed in the outer dish by diffusion through the porous membrane on the lower surface. The ALI method provides many benefits for organoid culture - the ALI method not only maintains stromal and immune cells in tissue samples, but this system can also retain the tumor microenvironment for a long time. For example, an example of the ALI method for culturing PDOs is described in Neal et al., Cell. December 13, 2018; 175(7):1972-1988.e16, which is incorporated herein by reference in its entirety.

[0081] In some cases, alternative organoid cultures can be used. In some cases, such as in alternative organoid cultures, for example, droplet and bioreactor methods, tissues are embedded in cell culture matrix droplets, such as basement membrane extract (BME) or ECM gel, and then transferred to a rotating bioreactor. In some cases, continuous agitation can improve the uptake of nutrients and oxygen.

[0082] A. Methods for generating PDO cultures

[0083] In one aspect, the present disclosure provides culture systems and methods for generating and expanding tumor-specific immune cells from organoid cultures of solid tumors, including stromal and immune cells associated with in vivo tumors, to activate and expand tumor-infiltrating lymphocytes (TIL) cells specific for tumor-associated antigens, such as T cells. In some embodiments, the PDO cultures and methods described herein include tumor cells, immune cells, etc. associated with in vivo tumors or tissues. In some embodiments, the PDO cultures and methods described herein reproduce the cell structure and ultrastructure of the tumor or tissue sample from which they are derived.

[0084] In some embodiments, a biological sample is obtained to generate a PDO culture. In some embodiments, the biological sample includes a tumor tissue sample. In some embodiments, the tumor tissue sample can be obtained by any convenient method, such as by biopsy, for example, during endoscopy, during surgery, by needle, etc. In some embodiments, the tumor tissue sample is obtained using aseptic techniques. In some embodiments, the tumor tissue sample includes human tissue, particularly cancers and other lesions, such as solid tumor micro-biopsy samples, such as needle or fine needle aspirates. In some embodiments, the tumor tissue sample is collected at a single time point. In some embodiments, the tumor tissue sample is collected at multiple time points. In some embodiments, the tumor tissue sample can be as small as 10 7 cells, 10 6 cells, 10 5 cells or smaller. In some embodiments, the tumor tissue sample can be a tumor biopsy section of about 0.1 mm 2 , about 1 mm 2 , about 10 mm 2 , etc.

[0085] After removing the tissue sample, the tissue is immersed in a cold buffer solution, such as PBS, Ham's F12, MEM, culture medium, etc. In some embodiments, the tissue pieces can be minced to a size of less than about 1 mm^3, and can be less than about 0.5 mm^3, or less than about 0.1 mm^3. The minced tissue is mixed with: a gel matrix, such as a collagen gel solution, such as Cellmatrix Type I-A Collagen (Nitta Gelatin Inc.); Matrigel solution, etc. Next, the gel matrix containing the tissue is spread on a layer of gel in a container ("base layer"), and the container has a semi-permeable support, such as a membrane, at the bottom to support the base gel layer and allow the gel matrix containing the tissue to solidify. The container is placed in an outer container containing a suitable culture medium, such as HAMs F-12 medium, which is supplemented with fetal bovine serum (FCS) at a concentration of about 1 to about 25%, usually about 5 to about 20%, etc. In some cases, an alternative serum to FCS can be used in the methods described herein. In some cases, a portion of the tumor tissue sample is preserved for different applications or experiments, such as histology or sequencing. In some embodiments, a portion of the tumor tissue sample is frozen for different applications or experiments. Various methods known in the art can be used to preserve the tumor tissue sample.

[0086] The arrangement of the PDO cultures as described above allows nutrients to pass from the bottom through the membrane and the basal gel layer to the gel layer containing the tissue. In some embodiments, the level of the maintenance medium is maintained such that the top of the gel (e.g., the gel layer containing the tissue sample) is not submerged in the liquid but is exposed to air. As a result, the tissue grows in the gel with an air-liquid interface environment. Examples of air-liquid interface culture systems are described, for example, in Ootani et al., Nat Med. Jun. 2009;15(6):701-6, which is incorporated herein by reference in its entirety. In some embodiments, the air-liquid interface organoid cultures can be transferred into other formats, such as porous for screening or submerged in 2D or 3D geometries, where the cells are placed beneath the tissue culture medium.

[0087] In some embodiments, 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 longer. In some embodiments, the tissue (e.g., primary tissue) is obtained from a solid tumor. In some cases, one or more tumor samples are obtained from a subject to generate PDO cultures. The tumor tissue can be from any mammalian species, such as human, non-human primate, horse, cow, pig, dog, cat, rodent (e.g., mouse, rat, hamster, etc.). In some cases, the subject includes, but is not limited to, human, cow, dog, mouse, rat, rabbit, guinea pig, chicken, fish, bird, reptile, camelid, cow, chimpanzee, sheep, goat, and non-human primate. In some cases, the subject is human. In some embodiments, the PDO cultures can be passaged for maintenance. In some embodiments, the PDO cultures can be cryopreserved. In some embodiments, the PDO cultures can be cryopreserved and then cryorecovered for downstream applications.

[0088] In some embodiments, the PDO cultures described herein include homologous immune cells, such as endogenous immune cells present in the biopsy sample; allogeneic T cells; and so on. Immune cells that can be co-cultured with the PDO include, but are not limited to, T cells, macrophages, B cells, natural killer cells (NK cells), etc., including any of the T cell subsets.

[0089] In some cases, the PDO culture may contain exogenous agents that are added to activate T cells present in the culture. In some embodiments, an agent that activates T cells can be added to the culture. In some embodiments, the agent that activates T cells includes immune checkpoint inhibitors, such as those that inhibit CTLA4 (cytotoxic T lymphocyte-associated protein 4, CD152), PD1 (also known as PD-1; programmed death 1 receptor), PD-L1, PD-L2, LAG-3 (lymphocyte activation gene-3), OX40, A2AR (adenosine A2A receptor), B7-H3 (CD276), B7-H4 (VTCN1), BTLA (B and T lymphocyte attenuator, CD272), IDO (indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin-like receptor), TIM 3 (T cell immunoglobulin and mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), IL-2R (interleukin-2 receptor), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), cytokines, such as agents that are antibodies to IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, etc. having activity. Compared to the wild-type sequence, cytokine variants can contain one or more mutations in the amino acid sequence. Cytokine variants can be used to modulate target signaling pathways. Examples of cytokine variants include, but are not limited to, mutant variants or truncated variants. In some embodiments, a combination of agents that activate T cells is added to the culture. The combination of agents can include a combination of two or more of any of the agents listed above. In some embodiments, activation of T cells includes a protocol for reversing T cell exhaustion, such as pulse stimulation, addition of kinase inhibitors (such as dasatinib), etc.

[0090] In some cases, when culturing PDOs with an agent that activates T cells, the culture can be maintained for any period of time to activate T cells. In some cases, the culture time with an agent that activates T cells can be 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 11 days, up to 12 days, up to 13 days, up to 14 days, up to 15 days, or more than 15 days. In some cases, the culture time with an agent that activates 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 11 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 23 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. After culturing with one or more T cell activators, T cell activation can be evaluated. Activated T cells can be identified and quantified according to various criteria (such as the expression of CD3, CD25, CD69, CD137, CD107a, granzyme B (GZMB), perforin 1 (PRF1), PD1, etc.). In some cases, activated T cells can be isolated based on the expression of these activation markers. In some cases, an unactivated PDO culture (such as a culture not treated with a T cell activator) can be used as a control.

[0091] In some cases, the PDO culture is an ALI culture. In some cases, PDOs or ALI organoids can be cryopreserved. In some cases, PDOs or ALI organoids can be cryo - thawed. In some cases, PDOs or ALI organoids can be passaged to maintain the organoid culture. In some cases, the PDO culture provides immune cells containing a complex mixture of immune cells (such as tumor - infiltrating lymphocytes (TILs)), and these immune cells can be further expanded and subsequently used in individuals in need of treatment (such as adoptive cell therapy). In some embodiments, the immune cells (such as TILs) include T cells.

[0092] Expansion of air - liquid interface tumor - infiltrating lymphocytes (ALI TILs)

[0093] Once a PDO culture (also referred to as an ALI culture in the present disclosure) is established, these PDOs can be used to expand immune cells, such as tumor-infiltrating lymphocytes (TILs), using a rapid expansion protocol (REP). Before expanding the cells in the REP step, the immune cells from the ALI culture are first maintained in a pre-rapid expansion protocol (pre-REP) step. In this pre-REP step, the ALI-derived immune cells are treated with cytokines (such as IL-2) to activate these immune cells (such as TILs). In some cases, during the pre-REP step, the ALI-derived immune cells are treated with two different concentrations of cytokines during two different time periods. In some cases, the first cytokine concentration used during the first time period in the pre-REP step is lower than the second cytokine concentration used during the second time period in the pre-REP step.

[0094] After the pre-REP step, the ALI-derived immune cells are subjected to the REP step. In this REP step, the immune cells undergo rapid expansion, resulting in a large number of immune cells, such as TILs. In some cases, in this REP step, the ALI-derived immune cells are cultured with cytokines (such as IL-2), antibodies (such as anti-CD3 antibodies), and feeder cells (such as PBMC-derived feeder cells). These immune cells are cultured for a period of time until a large number of immune cells are obtained. These immune cells can then be harvested and used for immunotherapy or other applications. In some embodiments, the PDO-derived TILs or ALI-derived TILs are further reprogrammed to have stem cell-like properties, such as a longer lifespan, self-renewal ability, and effector differentiation potential, thereby enhancing the anti-tumor activity of these cells. In some embodiments, these immune cells (such as TILs) are reprogrammed to have stem cell-like properties during the REP step, such as by activating the Notch signaling pathway, inhibiting the interferon gamma (IFNγ) signaling pathway, or a combination of both. In some embodiments, the immune cells (such as TILs) include T cells.

[0095] B. Pre-rapid expansion protocol (pre-REP)

[0096] Activation of immune cells (such as TILs) occurs in the pre-REP step, which trains the immune cells (such as TILs) to expand more effectively and better target tumor cells during the REP step.

[0097] In one aspect, the present disclosure provides a method for generating immune cells from an air-liquid interface (ALI) source, 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 a culture medium, and wherein one or more agents are added to the culture medium; and (c) collecting immune cells from the one or more tumor samples. In some embodiments, incubating the one or more tumor samples using an in vitro culture process comprises a first time period and a second time period. In some embodiments, one or more agents are added to the culture medium at a first concentration during the first time period. In some embodiments, 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. In some embodiments, the in vitro culture process comprises an air-liquid interface setup.

[0098] In one aspect, the present disclosure provides systems and methods for enhancing immune cells obtained from patient-derived organoids (PDOs). In some embodiments, the immune cells include tumor-reactive immune cells, such as TILs. In the present disclosure, a pre-rapid expansion protocol or pre-REP step involves treating immune cells with different concentrations of cytokines (such as IL-2) during a first time period and a second time period. In some cases, the pre-REP step described herein discloses treating immune cells with a low concentration of cytokines during the first time period and then treating the immune cells with a higher concentration of cytokines during the second time period. Treating immune cells (such as TILs) with a low concentration of cytokines during the first time period in the pre-REP step helps maintain the immune cells in vitro and also prevents immune cell death. In some cases, the immune cells (such as TILs) obtained using the protocol described herein have better tumor-killing ability and are more reactive to tumor cells compared to immune cells (such as TILs) obtained using a standard protocol with one concentration of cytokine. Accordingly, the present disclosure provides systems and methods for generating and expanding effective immune cells for cancer immunotherapy and other applications, such as immune cells that are more effective for cancer immunotherapy than the standard protocol. In some embodiments, the immune cells include tumor-infiltrating lymphocytes (TILs). In some embodiments, the TILs include T cells. In some embodiments, the immune cells (such as TILs) include T cells. In some embodiments, the T cells include activated T cells; naive CD8+ T cells; cytotoxic CD8+ T cells; naive CD4+ T cells; helper T cells, such as T H 1, T H 2, T H 9, T H 17, T H 22, TFH ; memory T cells, such as central memory T cells, T stem cell memory cells (TSCM), effector memory T cells, NKT cells or γδ T cells.

[0099] In some embodiments, one or more agents include cytokines. In various embodiments, the cytokines used in the pre-REP regimen include IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, or combinations thereof. Compared to the wild-type sequence, cytokine variants may include one or more mutations in the amino acid sequence. Cytokine variants can be used to modulate target signaling pathways. Examples of cytokine variants include, but are not limited to, mutant variants or truncated variants.

[0100] In various embodiments, the concentration of the cytokine added during the first time period in the pre-REP step 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 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 IU / mL, at least about 3000 IU / mL, at least about 4000 IU / mL, at least about 5000 IU / mL, at least about 6000 IU / mL, at least about 7000 IU / mL, at least about 8000 IU / mL, at least about 9000 IU / mL, or at least about 10000 IU / mL. In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is 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 / 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, at most about 6000 IU / mL, at most about 7000 IU / mL, at most about 8000 IU / mL, at most about 9000 IU / mL, at most about 10000 IU / mL, at most about 11000 IU / mL, or at most about 12000 IU / mL. In various embodiments, the concentration of the cytokine added during the first time period in the pre-REP step 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, about 150 IU / mL, about 500 IU / mL, about 1000 IU / mL, about 2000 IU / mL, about 3000 IU / mL, about 4000 IU / mL, about 5000 IU / mL, about 6000 IU / mL, about 7000 IU / mL, about 8000 IU / mL, about 9000 IU / mL or about 10000 IU / mL. In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is at least about 10 IU / mL. In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is about 50 IU / mL. In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is about 150 IU / mL.

[0101] In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is from about 10 IU / mL to about 12000 IU / mL, from about 20 IU / mL to about 12000 IU / mL, from about 30 IU / mL to about 12000 IU / mL, 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 / mL to about 12000 IU / mL, from about 80 IU / mL to about 12000 IU / mL, from about 90 IU / mL to about 12000 IU / mL, from about 100 IU / mL to about 12000 IU / mL, from about 500 IU / mL to about 12000 IU / mL, from about 1000 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 IU / mL to about 12000 IU / mL, from about 7000 IU / mL to about 12000 IU / 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 IU / mL.

[0102] In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is from about 10 IU / mL to about 10,000 IU / mL, from about 20 IU / mL to about 10,000 IU / mL, from about 30 IU / mL to about 10,000 IU / mL, from about 40 IU / mL to about 10,000 IU / mL, from about 50 IU / mL to about 10,000 IU / mL, from about 60 IU / mL to about 10,000 IU / mL, from about 70 IU / mL to about 10,000 IU / mL, from about 80 IU / mL to about 10,000 IU / mL, from about 90 IU / mL to about 10,000 IU / mL, from about 100 IU / mL to about 10,000 IU / mL, from about 500 IU / mL to about 10,000 IU / mL, from about 1,000 IU / mL to about 10,000 IU / mL, from about 2,000 IU / mL to about 10,000 IU / mL, from about 3,000 IU / mL to about 10,000 IU / mL, from about 4,000 IU / mL to about 10,000 IU / mL, from about 5,000 IU / mL to about 10,000 IU / mL, from about 6,000 IU / mL to about 10,000 IU / mL, from about 7,000 IU / mL to about 10,000 IU / mL, from about 8,000 IU / mL to about 10,000 IU / mL, or from about 9,000 IU / mL to about 10,000 IU / mL. In some embodiments, the concentration of the cytokine added during the first time period in the pre-REP step is from about 50 IU / mL to about 10,000 IU / mL.

[0103] In various embodiments, 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 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, or at most about 15 days. In some embodiments, the first time period of the pre-REP step is from about 1 to about 14 days, from about 2 to about 14 days, from about 3 to about 14 days, from about 4 to about 14 days, from about 5 to about 14 days, from about 6 to about 14 days, from about 7 to about 14 days, from about 8 to about 14 days, or from about 9 to about 14 days. In some embodiments, the first time period of the pre-REP step is from 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 from about 7 to about 14 days.

[0104] In various embodiments, the concentration of the cytokine added during the second time period in the pre-REP step 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 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 IU / mL, at least about 3000 IU / mL, at least about 4000 IU / mL, at least about 5000 IU / mL, at least about 6000 IU / mL, at least about 7000 IU / mL, at least about 8000 IU / mL, at least about 9000 IU / mL, or at least about 10000 IU / mL. In some embodiments, the concentration of the cytokine used during the second time period in the pre-REP step is 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 / 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, at most about 6000 IU / mL, at most about 7000 IU / mL, at most about 8000 IU / mL, at most about 9000 IU / mL, at most about 10000 IU / mL, at most about 11000 IU / mL, or at most about 12000 IU / mL. In various embodiments, the concentration of the cytokine added during the second time period in the pre-REP step 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, about 500 IU / mL, about 1000 IU / mL, about 2000 IU / mL, about 3000 IU / mL, about 4000 IU / mL, about 5000 IU / mL, about 6000 IU / mL, about 7000 IU / mL, about 8000 IU / mL, about 9000 IU / mL or about 10000 IU / mL. In some embodiments, the concentration of the cytokine added during the second time period in the pre-REP step is at least about 4000 IU / mL. In some embodiments, the concentration of the cytokine added during the second time period in the pre-REP step is about 6000 IU / mL.

[0105] In some embodiments, the concentration of the cytokine added during the second time period in the pre-REP step is from about 10 IU / mL to about 12,000 IU / mL, from about 20 IU / mL to about 12,000 IU / mL, from about 30 IU / mL to about 12,000 IU / mL, from about 40 IU / mL to about 12,000 IU / mL, from about 50 IU / mL to about 12,000 IU / mL, from about 60 IU / mL to about 12,000 IU / mL, from about 70 IU / mL to about 12,000 IU / mL, from about 80 IU / mL to about 12,000 IU / mL, from about 90 IU / mL to about 12,000 IU / mL, from about 100 IU / mL to about 12,000 IU / mL, from about 500 IU / mL to about 12,000 IU / mL, from about 1,000 IU / mL to about 12,000 IU / mL, from about 2,000 IU / mL to about 12,000 IU / mL, from about 3,000 IU / mL to about 12,000 IU / mL, from about 4,000 IU / mL to about 12,000 IU / mL, from about 5,000 IU / mL to about 12,000 IU / mL, from about 6,000 IU / mL to about 12,000 IU / mL, from about 7,000 IU / mL to about 12,000 IU / mL, from about 8,000 IU / mL to about 12,000 IU / mL, from about 9,000 IU / mL to about 12,000 IU / mL, or from about 10,000 IU / mL to about 12,000 IU / mL.

[0106] In some embodiments, the concentration of the cytokine added during the second time period in the pre-REP step is from about 10 IU / mL to about 10,000 IU / mL, from about 20 IU / mL to about 10,000 IU / mL, from about 30 IU / mL to about 10,000 IU / mL, from about 40 IU / mL to about 10,000 IU / mL, from about 50 IU / mL to about 10,000 IU / mL, from about 60 IU / mL to about 10,000 IU / mL, from about 70 IU / mL to about 10,000 IU / mL, from about 80 IU / mL to about 10,000 IU / mL, from about 90 IU / mL to about 10,000 IU / mL, from about 100 IU / mL to about 10,000 IU / mL, from about 500 IU / mL to about 10,000 IU / mL, from about 1000 IU / mL to about 10,000 IU / mL, from about 2000 IU / mL to about 10,000 IU / mL, from about 3000 IU / mL to about 10,000 IU / mL, from about 4000 IU / mL to about 10,000 IU / mL, from about 5000 IU / mL to about 10,000 IU / mL, from about 6000 IU / mL to about 10,000 IU / mL, from about 7000 IU / mL to about 10,000 IU / mL, from about 8000 IU / mL to about 10,000 IU / mL, or from about 9000 IU / mL to about 10,000 IU / mL. In some embodiments, the concentration of the cytokine added during the second time period in the pre-REP step is from about 5000 IU / mL to about 10,000 IU / mL.

[0107] In various embodiments, 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. In some embodiments, 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 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 21 days, at most about 22 days, at most about 23 days, or at most about 24 days. In some embodiments, 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 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 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. In some embodiments, 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. In some embodiments, the second time period of the pre-REP step is at least about 1 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 from about 7 to 14 days. In some embodiments, the second time period of the pre-REP step is from about 11 to 14 days.

[0108] In some embodiments, the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises one or more of PD-1, CD39, 4-1BB positive T cells, or a CXCR3-binding chemokine. In some embodiments, the CXCR3-binding chemokine comprises CXCL9 or CXCL10. In some embodiments, the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises interferon. In some embodiments, the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises a checkpoint inhibitor. In some embodiments, the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises TLR3, TLR7, TLR9, or other TLR agonists. In some embodiments, the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises a modulator of RIG-I-like receptors, a modulator of NOD-like receptors, a modulator of C-type lectin receptors, a modulator of STING, or a combination thereof.

[0109] In some embodiments, the method further comprises combining the tumor organoids with immune cells obtained from other sources. In some embodiments, the other sources include peripheral blood cells or organoids grown from lymphoid tissue. In some embodiments, the method further comprises stimulating antigen presentation. In some embodiments, the method further comprises depletion of immunosuppressive cell types. In some embodiments, the immunosuppressive cell types include Tregs, myeloid-derived suppressor cells, TAMs, vascular endothelial cells, or CAFs.

[0110] In some embodiments, the method further comprises negative selection of bystander tumor-reactive immune cells. In some embodiments, the method further comprises knockdown of exhaustion regulators. In some embodiments, the knockdown exhaustion regulator is TOX.

[0111] In some embodiments, the method comprises providing one or more tumor antigens during the first time period or the second time period. In some embodiments, providing one or more tumor antigens comprises providing cells expressing the tumor antigens.

[0112] In some embodiments, after pre-REP, immune cells are collected using a rapid expansion protocol (REP) for expansion. In some embodiments, after pre-REP, immune cells are collected for other applications. In some embodiments, after pre-REP, immune cells are cryopreserved. In some embodiments, immune cells are thawed after cryopreservation. In some embodiments, the method further comprises cryopreserving immune cells after collection from 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.

[0113] C. Rapid Expansion Protocol (REP)

[0114] After the immune cells (e.g., TIL) have undergone activation as described in the pre-REP step, the immune cells are then expanded. During REP, the immune cells (e.g., TIL) can be rapidly expanded by exposing the TIL to cytokines, antibodies, modulators, feeder cells, or any combination thereof. Additionally, these immune cells (e.g., TIL) can be reprogrammed to stem cell-like properties during this REP step (such as by Notch signaling pathway activation and interferon gamma (IFNγ) inhibition).

[0115] In one aspect, the present disclosure provides a method of generating ALI-derived immune cells, wherein the method further comprises (d) expanding the immune cells after a period of time after collecting the immune cells from one or more tissue samples using one or more agents. In some embodiments, the method further comprises (e) collecting the immune cells after expanding the immune cells obtained by isolating immune cells from one or more tissue samples. In some embodiments, the method further comprises (f) cryopreserving the immune cells after collecting the expanded immune cells. In some embodiments, the immune cells comprise tumor-reactive immune cells, such as TIL. In some embodiments, the immune cells comprise tumor-infiltrating lymphocytes. In some embodiments, the tumor-infiltrating lymphocytes comprise T cells. In some embodiments, the T cells comprise activated T cells; naïve CD8+ T cells; cytotoxic CD8+ T cells; naïve CD4+ T cells; helper T cells, such as TH1, TH2, TH9, TH17, TH22, TFH; memory T cells, such as central memory T cells, T stem cell memory cells (TSCM), effector memory T cells, NKT cells, or γδ T cells.

[0116] In some embodiments, immune cells (e.g., TILs) are rapidly expanded by the REP step. In some embodiments, the expansion of immune cells does not include an air-liquid interface setting. In some embodiments, immune cells (e.g., TILs) are expanded in a non-air-liquid interface (non-ALI) culture system. In some embodiments, the immune cells (e.g., TILs) used in the REP step are collected from the pre-REP step as described above. In some embodiments, the REP step includes culturing immune cells (e.g., TILs) in non-ALI culture. In some embodiments, one or more agents used in the REP step include cytokines, antibodies, modulators, or any combination thereof. In some embodiments, the REP culture includes cytokines, antibodies, modulators, feeder cells, or any combination thereof. In some embodiments, immune cells (e.g., TILs) include T cells.

[0117] In some embodiments, the cytokines used in the REP culture include IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, or a combination thereof. Compared to the wild-type sequence, cytokine variants may contain one or more mutations in the amino acid sequence. Cytokine variants can be used to modulate target signaling pathways. Examples of cytokine variants include, but are not limited to, mutant variants or truncated variants.

[0118] In some embodiments, the concentration of the cytokine added in the REP step 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 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 IU / mL, at least about 3000 IU / mL, at least about 4000 IU / mL, at least about 5000 IU / mL, at least about 6000 IU / mL, at least about 7000 IU / mL, at least about 8000 IU / mL, at least about 9000 IU / mL, or at least about 10000 IU / mL. In some embodiments, the cytokine is added at a concentration of at least about 2000 IU / mL. In some embodiments, the concentration of the cytokine added in the REP step is 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 / 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, at most about 6000 IU / mL, at most about 7000 IU / mL, at most about 8000 IU / mL, at most about 9000 IU / mL, at most about 10000 IU / mL, at most about 11000 IU / mL or at most about 12000 IU / mL. In various embodiments, the concentration of the cytokine added in the REP step 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, about 500 IU / mL, about 1000 IU / mL, about 2000 IU / mL, about 3000 IU / mL, about 4000 IU / mL, about 5000 IU / mL, about 6000 IU / mL, about 7000 IU / mL, about 8000 IU / mL, about 9000 IU / mL or about 10000 IU / mL. In some embodiments, the concentration of the cytokine added in the REP step is at least about 1000 IU / mL. In some embodiments, the concentration of the cytokine added in the REP step is about 3000 IU / mL.

[0119] In some embodiments, the concentration of the cytokine added in the REP step is from about 10 IU / mL to about 12000 IU / mL, from about 20 IU / mL to about 12000 IU / mL, from about 30 IU / mL to about 12000 IU / mL, 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 / mL to about 12000 IU / mL, from about 80 IU / mL to about 12000 IU / mL, from about 90 IU / mL to about 12000 IU / mL, from about 100 IU / mL to about 12000 IU / mL, from about 500 IU / mL to about 12000 IU / mL, from about 1000 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 IU / mL to about 12000 IU / mL, from about 7000 IU / mL to about 12000 IU / 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 IU / mL.

[0120] In some embodiments, the concentration of the cytokine added in the REP step is from about 10 IU / mL to about 10,000 IU / mL, from about 20 IU / mL to about 10,000 IU / mL, from about 30 IU / mL to about 10,000 IU / mL, from about 40 IU / mL to about 10,000 IU / mL, from about 50 IU / mL to about 10,000 IU / mL, from about 60 IU / mL to about 10,000 IU / mL, from about 70 IU / mL to about 10,000 IU / mL, from about 80 IU / mL to about 10,000 IU / mL, from about 90 IU / mL to about 10,000 IU / mL, from about 100 IU / mL to about 10,000 IU / mL, from about 500 IU / mL to about 10,000 IU / mL, from about 1000 IU / mL to about 10,000 IU / mL, from about 2000 IU / mL to about 10,000 IU / mL, from about 3000 IU / mL to about 10,000 IU / mL, from about 4000 IU / mL to about 10,000 IU / mL, from about 5000 IU / mL to about 10,000 IU / mL, from about 6000 IU / mL to about 10,000 IU / mL, from about 7000 IU / mL to about 10,000 IU / mL, from about 8000 IU / mL to about 10,000 IU / mL, or from about 9000 IU / mL to about 10,000 IU / mL. In some embodiments, the concentration of the cytokine added in the REP step is from about 2000 IU / mL to about 10,000 IU / mL.

[0121] In some embodiments, 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. In some embodiments, 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 21 days, at most about 22 days, at most about 23 days, or at most about 24 days. In some embodiments, 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 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. In some embodiments, 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. In some embodiments, 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.

[0122] In some embodiments, the antibody used in the REP culture includes an anti-CD3 antibody. In some embodiments, 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, the concentration of the added anti-CD3 antibody is at least about 1 ng / mL, at least about 3 ng / mL, at least about 5 ng / mL, at least about 7 ng / mL, at least about 10 ng / mL, at least about 15 ng / mL, at least about 20 ng / mL, at least about 25 ng / mL, at least about 30 ng / mL, at least about 35 ng / mL, at least about 40 ng / mL, at least about 45 ng / mL, at least about 50 ng / mL, at least about 55 ng / mL, at least about 60 ng / mL, at least about 65 ng / mL, at least about 70 ng / mL, at least about 75 ng / mL, at least about 80 ng / mL, at least about 85 ng / mL, at least about 90 ng / mL, at least about 95 ng / mL, or at least about 100 ng / mL. In some embodiments, the concentration of the added anti-CD3 antibody 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, 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. In some embodiments, the concentration of the added anti-CD3 antibody 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 / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL or about 100 ng / mL. In some embodiments, the concentration of the added anti-CD3 antibody is about 10 ng / mL. In some embodiments, the concentration of the added anti-CD3 antibody is about 30 ng / mL.

[0123] In some embodiments, one or more agents in the REP step further include irradiated feeder cells. In some embodiments, the irradiated feeder cells are feeder cells derived from irradiated allogeneic PBMCs. In some embodiments, the ratio of immune cells to 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 immune cells to irradiated feeder cells is about 1:100.

[0124] In some embodiments, the feeder cells used in REP culture include peripheral blood mononuclear cell (PBMC) feeder cells. In some embodiments, the feeder cells used in REP culture are derived from PBMC feeder cells. In some embodiments, the PBMC feeder cells are irradiated feeder cells. In some embodiments, the PBMC feeder cells are allogeneic PBMC feeder cells. In some embodiments, the PBMC feeder cells are irradiated allogeneic PBMC feeder cells.

[0125] In some embodiments, immune cells (e.g., TILs) 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 11 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 longer. In some embodiments, immune cells (e.g., TILs) 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 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, or longer. In some embodiments, immune cells (e.g., TILs) are expanded in the REP step for about 14 days.

[0126] In some embodiments, once expanded, the immune cells (e.g., TILs) can be assayed for functional activity. In some embodiments, once expanded, the immune cells (e.g., TILs) can be administered to a patient for immunotherapy. In some embodiments, once expanded, the immune cells (e.g., TILs) can be collected for other applications, such as in vitro modeling, drug screening, etc. In some embodiments, the immune cells (e.g., TILs) include T cells.

[0127] In some embodiments, the method further comprises combining the tumor organoids with immune cells obtained from other sources. In some embodiments, the other sources include peripheral blood cells or organoids grown from lymphoid tissue. In some embodiments, the method further comprises stimulating antigen presentation. In some embodiments, the method further comprises depletion of immunosuppressive cell types. In some embodiments, the immunosuppressive cell types include Tregs, myeloid-derived suppressor cells, TAMs, vascular endothelial cells, or CAFs.

[0128] In some embodiments, the method further comprises negative selection of bystander tumor-infiltrating immune cells. In some embodiments, the method further comprises knockdown of exhaustion regulators. In some embodiments, the knockdown exhaustion regulator is TOX.

[0129] In some embodiments, the method further comprises reprogramming T cells. In some embodiments, during the REP step, immune cells (e.g., TILs) can be reprogrammed to have stem cell-like properties by inhibiting the interferon-γ (IFNγ) signaling pathway, activating the Notch signaling pathway, or both. In some embodiments, T cells are reprogrammed by activating the Notch signaling pathway during the REP step. In some embodiments, T cells are reprogrammed by inhibiting IFNγ signaling. In some embodiments, T cells are reprogrammed by activating the Notch signaling pathway and inhibiting the IFNγ signaling pathway both using a modulator. In some embodiments, the modulator can be an IFNγ modulator, a Notch modulator, or a combination thereof. In some embodiments, the IFNγ modulator can inhibit the IFNγ signaling pathway. In some embodiments, the IFNγ modulator includes an IFNγ inhibitor. In some embodiments, the Notch modulator can activate the Notch signaling pathway. In some embodiments, the Notch modulator includes a Notch activator.

[0130] In some embodiments, the method further comprises identifying a T cell receptor (TCR) to identify TCRs enriched in the air-liquid interface culture. In some embodiments, identifying the TCR is performed using sequencing techniques. In some embodiments, the method comprises providing one or more tumor antigens during a first time period or a second time period. In some embodiments, providing the tumor antigens comprises providing cells expressing the tumor antigens.

[0131] In some embodiments, after the REP step, the immune cells (e.g., TILs) can be cryopreserved. In some embodiments, the immune cells (e.g., TILs) can be cryopreserved and then thawed after cryopreservation.

[0132] D. Reprogramming air-liquid interface tumor-infiltrating lymphocytes (rALI TIL)

[0133] In some embodiments, the methods and systems described herein can include reprogramming isolated, expanded, or cryopreserved TILs (e.g., ALITILs) into a stem cell-like state. In some embodiments, the reprogramming occurs during the REP step. In some embodiments, the reprogramming occurs prior to the REP step. In some embodiments, the reprogramming occurs after the REP step. In some embodiments, the isolated or cryopreserved TILs (e.g., T cells) include (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+CD103+ expression, (iii) effector memory T cells (TEM), which can be identified by CD45RA−CCR7−CD62L− expression, (iv) stem cell memory T cells (TSCM), which can be characterized by CD45RA+CCR7+CD62L+CD95+ expression, (v) naive T cells, which can be characterized by CD45RA+CCR7+CD62L+ expression, and (vi) terminally differentiated effector memory cells (TEMRA), which can be identified by CD45RO− / CCR7− expression. In some embodiments, the methods and systems described herein can reprogram one or more isolated TILs (e.g., T cells) into cells with stem cell-like properties (e.g., self-renewal ability), such as memory T cells (TSCM) or central memory T cells (TCM). In some embodiments, the reprogrammed immune cells (e.g., T cells) can have a longer lifespan, enhanced self-renewal ability, and / or the ability to differentiate into other cell lineages. In some embodiments, 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 cell-like T cells, such as memory T cells (TSCM). In some embodiments, the reprogrammed immune cells (e.g., T cells) can have enhanced persistence, proliferation, and / or efficacy in adoptive cell therapies against different cancer types. In some embodiments, the reprogrammed immune cells (e.g., T cells) can be used in adoptive cell therapies to treat cancer. In some embodiments, the cancer includes glioblastoma. In some embodiments, the reprogrammed immune cells (e.g., T cells) can be used in adoptive cell therapies to treat glioblastoma.

[0134] In some embodiments, the method further comprises reprogramming immune cells, such as T cells. In some embodiments, the immune cells, such as T cells, are reprogrammed by activating the Notch signaling pathway during the REP step, inhibiting interferon gamma (IFNγ) signaling during the REP step, or both. In some embodiments, the methods and systems described herein can comprise treating isolated or cryopreserved TILs (such as ALITILs) with one or more agents that promote the reprogramming of immune cells (such as T cells) into TSCM. In some embodiments, the one or more agents comprise modulators. In some embodiments, the modulator can enhance a signaling pathway (such as the Notch signaling pathway) known to convert activated T cells into stem cell memory T cells (TSCM). In some embodiments, the modulator can inhibit a signaling pathway (such as the IFNγ signaling pathway) known to inhibit the maintenance and diversity of stem cell-like immune cells (such as T cells) or induce the terminal differentiation of memory immune cells (such as memory T cells). In some embodiments, the modulator comprises a Notch signaling pathway modulator, an interferon gamma (IFNγ) modulator, or a combination thereof.

[0135] In some embodiments, the one or more agents comprise an IFNγ modulator. In some embodiments, the IFNγ modulator can inhibit the IFNγ signaling pathway. In some embodiments, the IFNγ modulator comprises an IFNγ inhibitor. In some embodiments, the IFNγ inhibitor comprises an antibody, a small molecule, or a combination thereof. In some embodiments, the IFNγ inhibitor comprises an IFNγ neutralizing antibody or a fragment thereof. In some embodiments, the IFNγ neutralizing antibody can bind to the receptor. In some embodiments, the IFNγ neutralizing antibody can bind to circulating IFNγ. In some embodiments, the one or more agents comprise a protein tyrosine phosphatase that dephosphorylates the IFNγ receptor. In some embodiments, the one or more agents that block the IFNγ signaling pathway comprise inhibitors that target proteins or signaling molecules downstream of the IFNγ signaling pathway. In some embodiments, the one or more agents comprise an inhibitor of the JAK / STAT signaling pathway. In some embodiments, the one or more agents comprise small molecule inhibitors. In some embodiments, treating immune cells (such as TILs) with one or more of the agents described herein can result in a change in the expression of IFNγ-induced genes (such as Gbp5, Irf1, and Ccl2) within 8 hours, 24 hours, 48 hours, or longer after treatment.

[0136] In some embodiments, one or more agents include Notch signaling pathway modulators. In some embodiments, the Notch signaling pathway modulator can activate the Notch signaling pathway. In some embodiments, the Notch signaling pathway modulator includes a Notch activator. In some embodiments, the Notch activator includes an antibody, a small molecule, or a combination thereof. In some embodiments, the Notch activator includes an agonistic antibody that targets and activates the Notch receptor. In some embodiments, one or more agents can be Delta-like 1 ligand. In some embodiments, one or more agents can enhance the expression of molecules downstream of the Notch signaling pathway. In some embodiments, one or more agents can be a small molecule agonist. In some embodiments, one or more agents can be an agonistic monoclonal antibody (mAb). In some embodiments, treating immune cells (e.g., TIL) with one or more agents described herein can result in a change in the expression of Notch-induced genes (such as Myc, Deltex1, and Hes1) at 8 hours, 24 hours, 48 hours, or longer after treatment.

[0137] In some embodiments, the methods and systems described herein can include treating immune cells (such as TIL) with one or more agents to inhibit IFNγ signaling and activate Notch signaling. In some embodiments, the one or more agents include one or more modulators. In some embodiments, the one or more modulators include a first modulator that inhibits IFNγ signaling and a second modulator that activates Notch signaling. In some embodiments, the one or more modulators include Notch signaling pathway modulators, interferon gamma (IFNγ) modulators, or combinations thereof. In some embodiments, the one or more modulators are added individually. In some embodiments, the one or more modulators are added sequentially. In some embodiments, the one or more modulators are added simultaneously. In some embodiments, the one or more agents described herein can be a mixture of mAbs that simultaneously target IFNγ inhibition and Notch activation. In some embodiments, the mAb mixture can be applied during the entire REP step or during the last approximately 1 day, last approximately 2 days, last approximately 3 days, last approximately 4 days, last approximately 5 days, last approximately 6 days, last approximately 7 days, last approximately 8 days, last approximately 9 days, last approximately 10 days, last approximately 11 days, last approximately 12 days, last approximately 13 days, last approximately 14 days of the REP step during culturing. In some embodiments, the method can further include treating the TIL culture with one or more cytokines during the REP step. In some embodiments, the cytokines can be IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, or combinations thereof. Compared to the wild-type sequence, the cytokine variants can contain one or more mutations in the amino acid sequence. The cytokine variants can be used to modulate the target signaling pathway. Examples of cytokine variants include, but are not limited to, mutant variants or truncated variants.

[0138] The methods described herein can generate a TSCM population that constitutes from about 2% to about 50% of the TIL population after reprogramming. In some cases, the methods described herein can generate a TSCM population that constitutes from about 2% to about 4%, from about 2% to about 6%, from about 2% to about 8%, from about 2% to about 10%, from about 2% to about 15%, from about 2% to about 20%, from about 2% to about 25%, from about 2% to about 30%, from about 2% to about 35%, from about 2% to about 40%, from about 2% to about 50%, from about 4% to about 6%, from about 4% to about 8%, from about 4% to about 10%, from about 4% to about 15%, from about 4% to about 20%, from about 4% to about 25%, from about 4% to about 30%, from about 4% to about 35%, from about 4% to about 40%, from about 4% to about 50%, from about 6% to about 8%, from about 6% to about 10%, from about 6% to about 15%, from about 6% to about 20%, from about 6% to about 25%, from about 6% to about 30%, from about 6% to about 35%, from about 6% to about 40%, from about 6% to about 50%, from about 8% to about 10%, from about 8% to about 15%, from about 8% to about 20%, from about 8% to about 25%, from about 8% to about 30%, from about 8% to about 35%, from about 8% to about 40%, from about 8% to about 50%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 35%, from about 10% to about 40%, from about 10% to about 50%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 35%, from about 15% to about 40%, from about 15% to about 50%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 35%, from about 20% to about 40%, from about 20% to about 50%, from about 25% to about 30%, from about 25% to about 35%, from about 25% to about 40, from about 25% to about 50%, from about 30% to about 35%, from about 30% to about 40%, from about 30% to about 50%, from about 35% to about 40%, from about 35% to about 50% or from about 40% to about 50% of the TCM population. The methods described herein can generate a TSCM population that constitutes 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 reprogramming. The methods described herein can generate a TSCM population that constitutes at least about 2%, at least about 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 reprogramming. The methods described herein can generate a TSCM population that constitutes 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 reprogramming.

[0139] The methods described herein can generate a TCM population that accounts for about 2% to about 50% of the TIL population after reprogramming. In some cases, the methods described herein can generate a TCM population that accounts for 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% to about 25%, 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 6% to about 8%, about 6% to about 10%, about 6% to about 15%, about 6% to about 20%, about 6% to about 25%, about 6% to about 30%, about 6% to about 35%, about 6% to about 40%, about 6% to about 50%, about 8% to about 10%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 30%, about 8% to about 35%, about 8% to about 40%, about 8% to about 50%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40%, about 10% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 15% to about 40%, about 15% to about 50%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 50%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40, about 25% to about 50%, about 30% to about 35%, about 30% to about 40%, about 30% to about 50%, about 35% to about 40%, about 35% to about 50% or about 40% to about 50% of the TIL population. The methods described herein can generate a TCM population that accounts for 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 reprogramming. The methods described herein can generate a TCM population that accounts for at least about 2%, at least about 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 reprogramming. The methods described herein can generate a TCM population that accounts for 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 reprogramming.

[0140] In some embodiments, the method further includes identifying a T cell receptor (TCR) to identify TCRs enriched in the air-liquid interface culture. In some embodiments, identifying the TCR is performed using sequencing techniques.

[0141] In some embodiments, after the REP step and reprogramming, the immune cells (e.g., rALITIL) can be cryopreserved. In some embodiments, the immune cells (e.g., rALI TIL) are thawed after cryopreservation.

[0142] Characteristics of air-liquid interface immune cells

[0143] In one aspect, compared to standard immune cells, the immune cells generated from the systems and methods described herein exhibit better tumor killing ability. In some embodiments, compared to standard immune cells (e.g., those generated from a standard protocol as described herein), the immune cells generated from the systems and methods described herein are more reactive to tumor cells. In some embodiments, the immune cells include 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. In some cases, the immune cells include tumor-infiltrating lymphocytes. In some embodiments, the tumor-infiltrating lymphocytes include T cells. In some embodiments, the T cells include naive CD8 + T cells; cytotoxic CD8 + T cells; naive CD4 + T cells; helper T cells such as T H 1, T H 2, T H 9, T H 17, T H 22, T FH ; memory T cells such as central memory T cells, T stem cell memory cells (T SCM ), effector memory T cells, NKT cells, γδ T cells. In some embodiments, naive T cells or terminally differentiated T cells can be reprogrammed into stem cell-like T cells (e.g., T stem cell memory cells).

[0144] In another aspect, the present disclosure provides a composition for treating tumors, which comprises immune cells isolated in vitro, wherein the immune cells include tumor infiltrating lymphocytes expressing IFNγ, CD107a or HLA-DR, and wherein the population of tumor infiltrating lymphocytes expresses higher IFNγ compared to tumor infiltrating lymphocytes obtained from a standard protocol. In some cases, the immune cells include tumor infiltrating lymphocytes, TILs or reprogrammed lymphocytes. In some cases, the population of ALI TILs or reprogrammed ALI TILs (rALI TILs) obtained from the systems and methods described herein expresses higher IFNγ, CD107a and / or HLA-DR in response to a specific stimulus compared to the STD TIL population. In some cases, the ALI TILs or rALITILs express higher IFNγ and / or CD107a compared to the STD TILs.

[0145] In some cases, at least about 1%, 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 the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ. In some cases, at least about 5% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ. In some cases, 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%, or up to about 60% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ. In some cases, up to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ. In some cases, 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%, about 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 the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ. In some cases, about 5% to about 20%, about 10% to about 20%, or about 15% to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ. In some cases, about 5% to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing IFNγ.

[0146] In some cases, at least about 1%, 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 the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a. In some cases, at least about 3% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a. In some cases, 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%, or up to about 60% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a. In some cases, up to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a. In some cases, 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%, about 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 the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a. In some cases, about 3% to about 20%, 5% to about 20%, about 10% to about 20%, or about 15% to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a. In some cases, about 3% to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing CD107a.

[0147] In some cases, at least about 1%, 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%, at least about 60%, at least about 65% or at least about 70% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing HLA-DR. In some cases, at least about 10% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing HLA-DR. In some cases, 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 70% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing HLA-DR. In some cases, up to about 20% of the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing HLA-DR. In some cases, 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%, about 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 the ALI TIL or rALI TIL population obtained from the methods and systems described herein are cells expressing HLA-DR.

[0148] In some cases, the expression markers of IFNγ, CD107a, and / or HLA-DR can be characterized at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours, at least about 41 hours, at least about 42 hours, at least about 43 hours, at least about 44 hours, at least about 45 hours, at least about 46 hours, at least about 47 hours, at least about 48 hours, at least about 49 hours, at least about 50 hours, at least about 51 hours, at least about 52 hours, at least about 53 hours, at least about 54 hours, at least about 55 hours, at least about 56 hours, at least about 57 hours, at least about 58 hours, at least about 59 hours, at least about 60 hours, or more than about 60 hours after co-culture with tumor cells. In some cases, immunohistochemistry or flow cytometry can be used to characterize the expression markers.

[0149] In some cases, ALI TIL or rALI TIL express higher levels of the Human Leukocyte Antigen–DR isotype (HLA-DR) compared to STD TIL. In some cases, expression markers of HLA-DR can be characterized at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours, at least about 41 hours, at least about 42 hours, at least about 43 hours, at least about 44 hours, at least about 45 hours, at least about 46 hours, at least about 47 hours, at least about 48 hours, at least about 49 hours, at least about 50 hours, at least about 51 hours, at least about 52 hours, at least about 53 hours, at least about 54 hours, at least about 55 hours, at least about 56 hours, at least about 57 hours, at least about 58 hours, at least about 59 hours, at least about 60 hours or more than 60 hours after co-culture with tumor cells. In some cases, immunohistochemistry or flow cytometry can be used to characterize the expression markers.

[0150] In some cases, ALI TIL or rALI TIL populations obtained from the systems and methods described herein express higher HLA-DR compared to STD TIL populations. In some cases, the HLA-DR expression levels of ALI TIL or rALI TIL populations obtained from the methods and systems described herein are 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% higher compared to STD TIL. In some cases, 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 least about 60% or at least about 65% of the ALI TIL or rALI TIL populations obtained from the methods and systems described herein are cells expressing HLA-DR.

[0151] In some cases, "STD" refers to immune cells obtained from the following method, which involves obtaining and culturing immune cells (such as TIL) from fresh tumor tissue and treating them with a certain concentration of cytokines during the pre-REP step. Briefly, on day 0 of the STD pre-REP step, tumor tissue fragments are transferred to a G-Rex10 plate. 5 - 30 tumor tissue fragments are added to each well, and these fragments are in 10 to 40 mL of medium, which contains RPMI, 10% FBS, gentamicin, and 6000 IU / mL of IL-2. Then the tumor tissue fragments are cultured at 37 °C and 5% CO2. On days 5 and 10 of the normal pre-REP step, half of the medium is removed and replaced with fresh medium and 6000 IU / mL of IL-2. After day 10, half of the medium is replaced every 2 to 3 days. Certain types of immune cells are sorted from the tumor tissue fragments for the REP step. The protocol for the REP step has been previously described in this disclosure.

[0152] In one aspect, the present disclosure provides a composition for treating tumors, which comprises in vitro isolated immune cells or reprogrammed immune cells, wherein the immune cells include tumor-infiltrating lymphocytes expressing IFNγ, CD107a, or HLA-DR, and at least 5% of the tumor-infiltrating lymphocyte population expresses IFNγ. In some embodiments, 20% of the tumor-infiltrating lymphocyte population expresses IFNγ. In some embodiments, 5% to 20% of the tumor-infiltrating lymphocyte population expresses IFNγ. In some embodiments, at least 3% of the tumor-infiltrating lymphocyte population expresses CD107a. In some embodiments, 20% of the tumor-infiltrating lymphocyte population expresses CD107a. In some embodiments, 3% to 20% of the tumor-infiltrating lymphocyte population expresses CD107a.

[0153] In some embodiments, the immune cells are derived from one or more tumor samples obtained from a subject. In some embodiments, the subject includes humans, cows, dogs, mice, rats, rabbits, guinea pigs, chickens, fish, birds, reptiles, camelids, cows, chimpanzees, sheep, goats, and non-human primates. In some embodiments, the subject is a human.

[0154] In some embodiments, at least about 10% of the tumor-infiltrating lymphocyte population expresses HLA-DR. In some embodiments, at least about 65% of the tumor-infiltrating lymphocyte population expresses HLA-DR.

[0155] In some embodiments, functional activity assays are performed on the amplified immune cells (e.g., TILs) or reprogrammed immune cells (e.g., rALI TILs) obtained from the systems and methods described herein. In some embodiments, the functional activity assays include T cell cytotoxicity assays, IL-2 responses, etc., known in the art. In some embodiments, it is evaluated whether there are markers indicating activation in immune cells (e.g., TILs) or reprogrammed immune cells (e.g., rALI TILs), such as the expression of CD3, CD25, CD69, CD137, CD107a, granzyme B (GZMB), perforin 1 (PRF1); and so on. In some embodiments, the HLA-DR, PD-1, CD45, or EPCAM markers of immune cells (e.g., TILs) are evaluated. In some embodiments, immune cells (e.g., TILs) or reprogrammed immune cells (e.g., rALI TILs) can be selected for an activated phenotype before administration to a patient or for other applications. In some embodiments, the immune cells (e.g., TILs) or reprogrammed immune cells (e.g., rALI TILs) include T cells.

[0156] Treatment

[0157] In some cases, the subject in need of treatment according to the methods described herein can be a subject in need of adoptive cell therapy to treat the cancer or tumor of the subject. In some embodiments, immune cells (e.g., TILs) or reprogrammed immune cells (e.g., rALI TILs) are used for adoptive cell therapy to treat the cancer or tumor of the subject. In some embodiments, the cancer or tumor includes glioblastoma, colon tumor, lung tumor, stomach tumor, brain tumor, kidney tumor, esophageal tumor, uterine tumor, skin tumor, pancreatic tumor, or breast tumor. In some embodiments, the cancer or tumor is glioblastoma.

[0158] In some embodiments, a subject is treated using adoptive cell therapy that employs a population of amplified and / or reprogrammed cells that have been activated, amplified, and / or reprogrammed by the methods and / or systems disclosed herein. For example, cells can be collected from the subject, activated and amplified, and reintroduced into the subject as part of the adoptive cell therapy. In some embodiments, the cells are further reprogrammed to have stem cell-like properties before reintroduction into the subject. The cells collected from the subject can be collected from any convenient and suitable source for adoptive cell therapy, such as peripheral blood (e.g., the subject's peripheral blood), biopsy (e.g., a tumor biopsy from the subject), etc.

[0159] In some embodiments, the collected cells are immune cells. In some embodiments, the immune cells include tumor-infiltrating lymphocytes (TILs), such as TILs collected from a subject's tumor. In some embodiments, the collected cells are blood cells, such as NK cells (e.g., from a subject with cancer or a subject with an infection) collected from the subject's blood.

[0160] In one aspect, the present disclosure provides a method of treatment comprising introducing an expanded cell population (e.g., immune cells) into a recipient in need thereof, as described above. In some embodiments, the expanded cell population (e.g., immune cells) is reprogrammed to have stem cell-like properties, as described above. In some embodiments, the cell population is autologous or allogeneic relative to the recipient.

[0161] In some embodiments, the expanded immune cells (e.g., TILs) or the reprogrammed immune cells (e.g., rALI TILs) can be provided in a pharmaceutical composition suitable for therapeutic use (e.g., for human therapy). In some embodiments, a therapeutic preparation comprising the expanded immune cells or the reprogrammed immune cells can be frozen or prepared in an aqueous solution with a physiologically acceptable carrier, excipient, or stabilizer for administration (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)). In some cases, the expanded immune cells or the reprogrammed immune cells are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to the medical practitioner.

[0162] In some cases, the expanded immune cells or the reprogrammed immune cells can be administered by any suitable means. In some embodiments, administration includes intramuscular, intravenous (bolus or slow infusion), intraarterial, intraperitoneal, intrathecal, intratumoral, intravesical, or subcutaneous administration. In some embodiments, administration includes parenteral infusion. In some embodiments, the expanded immune cells or the reprogrammed immune cells can be administered via parenteral infusion. In some embodiments, parenteral infusion includes intramuscular, intravenous (bolus or slow infusion), intraarterial, intraperitoneal, intrathecal, intratumoral, intravesical, or subcutaneous administration.

[0163] In one aspect, the present disclosure provides a composition for treating a tumor, which comprises immune cells isolated in vitro, wherein the immune cells include tumor infiltrating lymphocytes expressing IFNγ, CD107a or HLA-DR, and at least 5% of the tumor infiltrating lymphocyte population expresses IFNγ. In some cases, the tumor includes, but is not limited to, colon tumors, lung tumors, stomach tumors, atypical teratoid / rhabdoid tumors, brain tumors (e.g., astrocytomas, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngiomas, ependymomas, glioblastoma multiforme (GBM), etc.), bronchial tumors, carcinoid tumors (e.g., in children, gastrointestinal, etc.), cardiac tumors, central nervous system (e.g., atypical teratoid / rhabdoid tumors, embryonal tumors, germ cell tumors, lymphomas, etc.), embryonal tumors, extracranial germ cell tumors, extragonadal germ cell tumors, islet cell tumors (e.g., pancreatic neuroendocrine tumors, etc.), kidney cancers (e.g., renal cell, Wilms tumor, pediatric renal tumors, etc.), ovarian cancers (e.g., epithelial, germ cell tumors, low malignant potential tumors, etc.), pancreatic cancers, pancreatic neuroendocrine tumors (islet cell tumors), pituitary tumors, uterine tumors, esophageal tumors, breast tumors, skin tumors, liver tumors, etc. In some embodiments, as described herein, the tumor infiltrating lymphocytes (TILs) are reprogrammed TILs to have stem cell-like properties.

[0164] In some cases, the composition for administration depends on the desired formulation, a pharmaceutically acceptable, non-toxic carrier or diluent, which is defined as a medium commonly used for formulating pharmaceutical compositions for administration to animals or humans. In some cases, the choice of diluent does not affect the biological activity of the combination. Examples of diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, glucose solution, and Hank's solution. In some embodiments, the pharmaceutical composition or formulation may include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0165] In some embodiments, the composition further comprises an acceptable carrier, excipient, or stabilizer. The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosages and concentrations employed. In some embodiments, the acceptable carrier, excipient, or stabilizer includes buffering agents such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, 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 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG); and / or solvents or cryoprotectants such as dimethyl sulfoxide (DMSO).

[0166] In some cases, the composition is prepared as an injectable liquid solution or suspension. In some cases, solid forms of the composition suitable for dissolving or suspending in a liquid vehicle before injection can also be prepared. In some embodiments, the protein can be administered in the form of a depot injection or implant formulation, which can be formulated in a manner that permits sustained or pulsed release of the active ingredient. The pharmaceutical composition is formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

[0167] In some cases, once expanded, an effective dose of immune cells (e.g., TIL or rALITIL) can be administered to a patient, including but not limited to a patient from whom the PDO is derived. In some embodiments, the effective dose can be at least about 10 2 cells per administration, at least about 10 3 cells per administration, at least about 10 4 cells per administration, at least about 10 5 cells per administration, at least about 10 6 cells per administration, at least about 10 7 cells per administration, at least about 10 8 cells per administration, at least about 10 9 cells per administration, at least about 10 10 cells per administration, at least about 1011 cells, at least about 10 12 cells, at least about 10 13 cells or more. In some embodiments, the effective dose can be at least about 10 2 cells to about 10 13 cells, at least about 10 3 cells to about 10 13 cells, at least about 10 4 cells to about 10 13 cells, at least about 10 5 cells to about 10 13 cells, at least about 10 6 cells to about 10 13 cells, at least about 10 7 cells to about 10 13 cells, at least about 10 8 cells to about 10 13 cells, at least about 10 9 cells to about 10 13 cells, at least about 10 10 cells to about 10 13 cells, at least about 10 11 cells to about 10 13 cells or at least about 10 12 cells to about 10 13 cells. In some embodiments, the effective dose can be at least about 10 2 cells to about 10 11 cells, at least about 10 3 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 7 cells to about 10 11 cells, at least about 10 8 cells to about 10 11 cells, at least about 10 9 cells to about 10 11 cells or at least about 10 10 cells to about 10 11 cells. In some embodiments, the effective dose can be delivered systemically by intratumoral injection or other administration routes as described above.

[0168] In some cases, after administration, the enhanced immune response can be manifested as an enhanced cytolytic response of T cells to target cells present in the recipient.

[0169] In some embodiments, a patient receiving the expanded immune cells can also receive chemotherapy.

[0170] In some embodiments, the composition further comprises an acceptable carrier, excipient, or stabilizer. In some embodiments, the composition can be administered in combination with other therapeutic treatments. In some embodiments, the other therapeutic treatments include chemotherapeutic agents, immune checkpoint inhibitors, cancer therapeutic agents, targeted therapeutic agents, immunomodulators, cytokines, antibiotics, or antiviral agents.

[0171] E. Combination Therapies

[0172] In one aspect, the present disclosure provides a composition that can be administered in combination with other therapeutic treatments. In some embodiments, the other therapeutic treatments include chemotherapeutic agents, immune checkpoint inhibitors, cancer therapeutic agents, targeted therapeutic agents, immunomodulators, cytokines, antibiotics, or antiviral agents. In some cases, cancer cells prevent immune cells from attacking by exploiting the braking mechanism of the immune system or using signals from the tumor that weaken the immune response. In some cases, immune checkpoint inhibitors are used in combination with adoptive cell therapy. In some embodiments, the expanded immune cells or the reprogrammed immune cells are treated with an immune checkpoint inhibitor prior to administration to a patient.

[0173] In some embodiments, treating a subject or patient's condition with the compositions and / or cells of the present disclosure can be combined with one or more additional active agents. In some cases, the available additional active agents include active agents for treating cancer.

[0174] In some embodiments, the treatment can be combined with other active agents, including antibiotics, cytokines, and antiviral agents. In some embodiments, the antibiotics include penicillins, such as penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, and the like. In some embodiments, the penicillins are used in combination with: β-lactamase inhibitors, cephalosporins, such as cefaclor, cefazolin, cefuroxime, moxalactam, and the like; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin, and the like. In some embodiments, the cytokines include interferon γ, tumor necrosis factor α, interleukin 12, and the like. In some embodiments, the antiviral agents include acyclovir, gancyclovir, and the like.

[0175] In some cases, when the treatment is directed against cancer, chemotherapeutic agents can be administered in combination with expanded immune cells or reprogrammed immune cells. In some embodiments, the chemotherapeutic agents include abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, 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, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lanvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, mutamycin, myleran, mylosar,Navelbine, Nipent, Novantrone, Oncovin, Oxaliplatin, Paclitaxel, Paraplatin, Pentostatin, Platinol, Plicamycin, Procarbazine, Purinethol, Ralitrexed, Taxotere, Taxol, Teniposide, Thioguanine, Tomudex, Topotecan, Valrubicin, Velban, Vepesid, Vinblastine, Vindesine, Vincristine, Vinorelbine, VP-16 and Vumon.

[0176] In some embodiments, targeted therapeutic agents that can be co-administered with the amplified immune cells or the reprogrammed immune cells include tyrosine kinase inhibitors such as imatinib mesylate (Gleevec, also known as STI-571), gefitinib (Iressa, also known as ZD1839), erlotinib (sold under the name Tarceva), sorafenib (Nexavar), sunitinib (Sutent), dasatinib (Sprycel), lapatinib (Tykerb), nilotinib (Tasigna), and bortezomib (Velcade); Janus kinase inhibitors such as tofacitinib; ALK inhibitors such as crizotinib; Bcl-2 inhibitors such as obatoclax, venclexta, and gossypol; FLT3 inhibitors such as midostaurin (Rydapt), IDH inhibitors such as AG-221; PARP inhibitors such as iniparib and olaparib;

[0177] PI3K inhibitors such as perifosine; VEGF receptor 2 inhibitors such as apatinib; AN-152 (AEZS-108) doxorubicin conjugated to [D-Lys(6)]-LHRH; Braf inhibitors such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors such as trametinib; CDK inhibitors such as PD-0332991 and LEE011; Hsp90 inhibitors such as salinomycin; and / or small molecule drug conjugates such as vintafolide; serine / threonine kinase inhibitors such as temsirolimus (Torisel), everolimus (Afinitor), vemurafenib (Zelboraf), trametinib (Mekinist), and dabrafenib (Tafinlar).

[0178] In some embodiments, the amplified or reprogrammed immune cells can be administered in combination with an immunomodulator such as a cytokine, lymphokine, monokine, stem cell growth factor, lymphotoxin (LT), hematopoietic factor, colony stimulating factor (CSF), interferon (IFN), transforming growth factor (TGF) (such as TGF-α or TGF-β), insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF) (such as TNF-α or TNF-β), vascular endothelial growth factor, integrin, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon (such as interferon-α, interferon-β or interferon-γ), S1 factor, interleukin (IL) (such as IL-1, IL-1cc, 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, LIF, kit ligand, FLT-3, endostatin, and LT.

[0179] In some embodiments, tumor-specific monoclonal antibodies that can be co-administered with the amplified immune cells or reprogrammed immune cells include ipilimumab (for treating melanoma, prostate cancer, RCC) that targets CTLA-4; tremelimumab (for treating CRC, gastric cancer, melanoma, NSCLC) that targets CTLA-4; nivolumab (for treating melanoma, NSCLC, RCC) that targets PD-1; MK-3475 (for treating melanoma) that targets PD-1; pidilizumab (for treating hematological malignancies) that targets PD-1; BMS-936559 (for treating melanoma, NSCLC, ovarian cancer, RCC) that targets PD-L1; MEDI4736 that targets PD-L1; MPDL33280A (for treating melanoma) that targets PD-L1; rituximab (for treating non-Hodgkin lymphoma) that targets CD20; ibritumomab tiuxetan and tositumomab (for treating lymphoma); brentuximab vedotin (for treating Hodgkin lymphoma) that targets CD30; gemtuzumab ozogamicin (for treating acute myeloid leukemia) that targets CD33; alemtuzumab (for treating chronic lymphocytic leukemia) that targets CD52; IGN101 and adecatumumab (for treating epithelial tumors (breast, colon, and lung)) that targets EpCAM; labetuzumab (for treating breast, colon, and lung tumors) that targets CEA; huA33 (for treating colorectal cancer) that targets gpA33; pemtumomab and oregovomab (for treating breast, colon, lung, and ovarian tumors) that targets mucin; CC49 (minretumomab) (for treating breast, colon, and lung tumors) that targets TAG-72; cG250 (for treating renal cell carcinoma) that targets CAIX; J591 (for treating prostate cancer) that targets PSMA; MOv18 and MORAb-003 (farletuzumab) (for treating ovarian tumors) that targets folate-binding protein; 3F8, ch14 that targets gangliosides such as GD2, GD3, and GM2.18 and KW-2871 (for treating neuroectodermal tumors and some epithelial tumors); hu3S193 and IgN311 targeting Le y (for treating breast, colon, lung, and prostate tumors); Bevacizumab targeting VEGF (for treating tumor vasculature); IM-2C6 and CDP791 targeting VEGFR (for treating solid tumors of epithelial origin); Etaracizumab targeting integrin αvβ3 (for treating tumor vasculature); Volociximab targeting integrin α5β1 (for treating tumor vasculature); Cetuximab, panitumumab, nimotuzumab, and 806 targeting EGFR (for treating gliomas, lung, breast, colon, and head and neck tumors); Trastuzumab and pertuzumab targeting ERBB2 (for treating breast, colon, lung, ovarian, and prostate tumors); MM-121 targeting ERBB3 (for treating breast, colon, lung, ovarian, and prostate tumors); AMG 102, METMAB, and SCH 900105 targeting MET (for treating breast, ovarian, and lung tumors); AVE1642, IMC-A12, MK-0646, R1507, and CP751871 targeting IGF1R (for treating gliomas, lung cancer, breast cancer, head and neck cancer, prostate cancer, and thyroid cancer); KB004 and IIIA4 targeting EPHA3 (for treating lung, kidney, and colon tumors, melanoma, gliomas, and hematological malignancies); Mapatumumab (HGS-ETR1) targeting TRAILR1 (for treating colon, lung, and pancreatic tumors and hematological malignancies); HGS-ETR2 and CS-1008 targeting TRAILR2; Denosumab targeting RANKL (for treating prostate cancer and bone metastases); Sibrotuzumab and F19 targeting FAP (for treating colon, breast, lung, pancreatic, and head and neck tumors); 81C6 targeting tenascin (for treating gliomas, breast, and prostate tumors); Blinatumomab (Blincyto; Amgen) targeting CD3 (for treating ALL); pembrolizumab targeting PD-1, for cancer immunotherapy; 9E10 antibody targeting c-Myc; and so on.

[0180] Other applications of expanded immune cells or reprogrammed immune cells

[0181] In one aspect, the expanded immune cells (e.g., TIL) or reprogrammed immune cells (e.g., rALI TIL) described in the present disclosure can be used in other applications besides immunotherapy. In some embodiments, the expanded immune cells (e.g., TIL) or reprogrammed immune cells (e.g., rALI TIL) can be used in functional in vitro assays for disease modeling or for determining a patient's specific reactivity to immunotherapy agents. In some embodiments, the expanded immune cells (e.g., TIL) or reprogrammed immune cells (e.g., rALI TIL) can be used in screening assays. In some embodiments, the expanded immune cells (e.g., TIL) or reprogrammed immune cells (e.g., rALI TIL) can be used to determine the preclinical efficacy of immunotherapy agents. In some embodiments, the expanded immune cells (e.g., TIL) or reprogrammed immune cells (e.g., rALI TIL) can be used in various applications / experiments modified according to methods known in the art.

[0182] Figure 1 Depicts the timeline of the pre-rapid expansion protocol (pre-REP) in the standard tumor-infiltrating lymphocyte (STD TIL) protocol and the air-liquid interface tumor-infiltrating lymphocyte (ALI TIL) protocol. Standard (“STD”) refers to immune cells obtained from a method that involves obtaining and culturing TIL from fresh tumor tissue and treating with one concentration of cytokine during the pre-REP step. Briefly, on day 0 of the STD pre-REP step, tumor tissue fragments are transferred to a G-Rex10 plate. 5 - 30 tumor tissue fragments are added to each well, and these fragments are in 10 to 40 mL of medium that contains RPMI, 10% FBS, gentamicin, and 6000 IU / mL of IL-2. The tumor tissue fragments are then cultured at 37 °C, 5% CO 2 2. The medium is removed and replaced with fresh medium and 6000 IU / mL of IL-2 on days 5 and 10 of the STD pre-REP step. After day 10, half of the medium is replaced every 2 to 3 days. At the end of day 11, the cells are collected and then treated with a mixture for the rapid expansion protocol (REP). Certain types of immune cells are sorted from the tumor tissue fragments for the REP step. The protocol for the REP step has been previously described in the present disclosure.

[0183] Contrary to the STD TIL protocol described in this disclosure, in the ALI TIL protocol, the starting sample is patient-derived organoids (PDOs) generated from tissue samples. These PDOs are cultured in an air-liquid interface (ALI) environment. These PDOs contain various immune populations, such as tumor-infiltrating immune cells, lymphocytes, T cells, etc., and they can then be used for expansion. In the pre-REP phase of the ALI TIL protocol, the PDOs or ALI organoids are treated with 50 IU / mL of IL-2 for 1-2 weeks and then with a higher concentration of IL-2 (6000 IU / mL) for 11 days. At the end of this, the cells are harvested for REP treatment.

[0184] Figure 2 It is shown that ALI tumor-infiltrating lymphocytes (ALITIL) exhibit better tumor-killing ability compared to standard TIL (STD TIL). ALI TIL and STD TIL were co-cultured with autologous tumor epithelial cells derived from submerged organoids for 48 hours. Single-cell tumor organoids alone were used as a control. After 48 hours of incubation, the cells were stained with fluorescent dye-conjugated antibodies against CD45, EPCAM, Annexin V, Zombie NIR live dead, and other T cell markers. Flow cytometry analysis was performed on the live tumor cell count after staining. To evaluate the tumor-killing ability of TIL, the absolute number of tumor cells under the condition of co-culturing autologous tumor organoids with TIL was divided by the absolute number of tumor cells under the condition of culturing autologous tumor organoids without TIL (control condition). The results of these analyses are expressed as a percentage of live cells. Data from samples from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non-parametric ANOVA was performed, corrected with Geisser-Greenhouse correction, for comparison between different conditions; *≤Pv = 0.05.

[0185] Figures 3A-3C It is shown that ALI tumor-infiltrating lymphocytes (ALITIL) are more reactive to tumor cells compared to standard TIL (STD TIL). Figure 3A The experimental protocol is shown. Briefly, ALI TIL and STD TIL were co-cultured with autologous tumor epithelial cells derived from submerged organoids for 16 hours. ALI TIL or STD TIL alone was used as a control. After 16 hours of incubation, the cells were stained with fluorescent dye-conjugated antibodies against IFNγ secretion, CD107a, CD45, EPCAM, Annexin V, Zombie NIR live dead, and other T cell markers for flow cytometry analysis. Next, flow cytometry analysis was performed on live CD3+ cells after staining. In Figure 3BAnd Figure 3C In, to evaluate the tumor reactivity of TILs, the percentage of CD3+ cells secreting IFNγ and the percentage of CD3+ cells expressing CD107a on their surface were evaluated after 16 hours of incubation. Data from samples from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non-parametric ANOVA was performed, corrected with Geisser-Greenhouse correction for comparison between different conditions; *≤Pv = 0.05.

[0186] Figures 4A-4D ALI tumor-infiltrating lymphocytes (ALITILs) were shown to express higher levels of HLA-DR compared to standard TILs (STD TILs). Figure 4A The experimental protocol is shown. Briefly, ALITILs 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 controls. After 48 hours of incubation, cells were stained with fluorescent dye-conjugated antibodies against HLA-DR, CD137, PD1, CD45, EPCAM, Annexin V, Zombie NIR live / dead, and other T cell markers for flow cytometry analysis. Next, flow cytometry analysis of live CD3+ cells was performed. In Figures 4B-4D In, to evaluate the tumor reactivity of TILs, the MFI (median fluorescence intensity) of PD1, HLA-DR, and CD137 was quantified and compared to the MFI of ALITILs or STD TILs not exposed to tumor cells. Results are shown as fold change. Data from samples from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non-parametric ANOVA was performed, corrected with Geisser-Greenhouse correction for comparison between different conditions; *≤Pv = 0.05.

[0187] Figure 5 ALI tumor-infiltrating lymphocytes (ALITILs) were shown to express a higher percentage of HLA-DR+. The timing and conditions of the experiment were similar to those described in Figures 4A-4D Results are shown as percentages rather than Figure 4CFold changes in. Data from samples derived from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non-parametric ANOVA was performed, corrected by Geisser-Greenhouse correction for comparison between different conditions; *≤Pv = 0.05.

[0188] Figures 6A-6C An example of the establishment of ALI tumor organoids is shown. Figure 6A Representative bright-field images of ALI tumor organoids generated from tissues obtained from the kidney, lung, esophagus, and uterus are shown. Figure 6B Examples of ALI tumor organoids generated from tissues obtained from the colon and glioblastoma (GBM) are shown. The top panel shows samples grown as air-liquid interface (ALI) organoids, which can be used for TIL expansion and / or reprogramming. The bottom panel shows submerged organoids, which can be used for assays. Figure 6C Sequencing results of three colorectal cancer (CRC) organoid lines are shown, presented as CNV plots and mutated genes (APC, TP53, KRAS).

[0189] Figures 7A-7C Demonstration of the generation and phenotypic characterization of ALI TIL. Figure 7A A schematic diagram depicting the 2-step ALI TIL process and cryopreservation of the product containing ALI TIL is shown. Figure 7B The cell numbers of ALI TIL obtained from 14 preparations (left) and the fold expansion distribution of 14 products (right) are shown. Figure 7C Flow cytometry analysis results of T cell, lineage, and memory subsets of twelve ALI-TIL preparations detected using CD3, γδTCR, CD4, CD8, CD45RA, and CD62L are shown. The results are plotted as the percentage of the parent for each individual sample, with the mean and SEM. (TEM: effector memory T cell; TCM: central memory T cell; TN / TSCM: stem cell memory T cell; TEMRA: terminally differentiated effector memory cell)

[0190] Figure 8A and 8B Detection of ALI TIL tumor reactivity and cytotoxicity is shown. Figure 8AShows flow cytometry analysis of ALITIL from CRC co-cultured with autologous tumor organoids at a 1:1 effector:target ratio for 16 hours. Increased IFNγ, CD107a, and 4-1BB were observed. PMA / ionomycin was used as an activation control, and W2 / 36 blocked MHC I presentation and confirmed antigen specificity. Cells were stained for live / dead, EPCAM, CD3, CD8, and CD107a&IFNγ or 4-1BB using a Quanteon instrument (Agilent, Santa Clara, CA) and analyzed by flow cytometry. Data were processed using FlowJo (Ashland, OR), and the indicated markers were gated as shown. Figure 8B Shows results obtained from confocal imaging at 0 hours (0h) and 24 hours (24h) after a 24-hour co-culture experiment of ALITIL obtained from CRC with autologous tumor organoids at a 10:1 effector:target ratio. Tumor cell killing over time was observed.

[0191] Figure 9A and 9B Shows the results of single-cell RNA sequencing analysis of CRC and melanoma ALI-TIL. Figure 9A Shows the number of unique clonotypes (upper row) and the clonotype frequency distribution (lower row) relative to the number of sequenced cells. Unique paired αβ CDR3 sequences (assimilated to TCR clonotypes) were counted and sorted in frequency order. Figure 9B Shows the expression levels of 397 immune genes identified in 10 clusters using SeqGeq.

[0192] Example

[0193] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0194] Example 1: Air-liquid interface tumor-infiltrating lymphocytes (ALITIL) exhibit better tumor-killing ability compared to standard tumor-infiltrating lymphocytes (STD TIL)

[0195] To evaluate the tumor killing ability of tumor-infiltrating lymphocytes (TIL) generated by the air-liquid interface (ALI) as described in the present disclosure, an ALI culture and expansion method was performed.

[0196] Table 1. Materials and reagents for establishing PDO cultures in the ALI environment and pre-rapid expansion protocol TIL.

[0197]

[0198]

[0199] Patient-derived organoids (PDO) or ALI organoids are established from primary tumor tissues in a 3D ALI environment

[0200] Preparation of culture inserts and collagen before processing tissues

[0201] In this step, prepare the collagen liquid mixture according to the manufacturer's instructions. Prepare the collagen liquid mixture and keep it on ice. Next, add 1 mL of the collagen liquid mixture to the 0.4 μm insert. Then let the insert containing the collagen mixture stand at room temperature for at least 30 min to solidify it. Keep the remaining collagen liquid mixture on ice until it is subsequently used in the organoid culture step.

[0202] Prepare the number of inserts according to the size of the tissue sample. The total volume of each insert is 2 mL.

[0203] Preparation for organoid culture

[0204] Prepare the tissue sample for organoid culture by chopping the tissue with scissors until it becomes a pasty texture and nearly liquid form. There are no lumps in this step, and the tissue is kept hydrated with an appropriate volume of medium throughout the process. In this step, some of the chopped tissue can be frozen at -80 °C. Alternatively, all or the remaining tissue can be used for organoid culture.

[0205] Next, add 5 mL of F12 medium containing Normocin (F12-Normocin medium) to the culture dish to transfer the chopped tissue to a 15 mL conical tube. Then wash the culture dish with 5 - 10 mL of F12-Normocin and collect all the remaining chopped tissue into the 15 mL conical tube. Then spin the chopped tissue at 400 xg for 3 min using a centrifuge. Then aspirate the medium and wash the chopped tissue pellet with 10 mL of F12-Normocin medium. Then spin the chopped tissue at 400 xg for 3 min. Depending on the tissue mass obtained and the amount of adipose tissue in the sample, additional washing steps may be required.

[0206] After the washing step, resuspend and redissolve the chopped tissue in the previously prepared collagen liquid mixture. Then add 1 mL of the chopped tissue dissolved in the collagen liquid mixture to each insert, placing it directly on top of the previously prepared collagen matrix insert. Then incubate the insert containing the chopped tissue at 37 °C for 30 - 45 min to solidify the collagen liquid mixture.

[0207] Next, add 1.5 mL of medium outside the insert. Gently shake the plate or outer well containing the insert to allow the medium to cover the bottom of the insert.

[0208] Before establishing organoid cultures, a portion of the tissue sample can be reserved for later histological and / or sequencing experiments. To store the tissue sample, wash the tissue in a 10 cm dish with 5-fold (5x) volume of F12-Normocin. Optionally, the tissue can be transferred to a dish on ice and cut into smaller pieces for IHC experiments. The tissue can be fixed overnight at 4°C in 4% PFA or 10% formalin.

[0209] Passaging of ALI organoids

[0210] After culturing ALI organoids for 2 to 4 weeks, the ALI organoids can be passaged.

[0211] Prepare the collagen liquid mixture as described above and place it on ice. Add 1 mL of the collagen liquid mixture to each 0.4 μm insert and let it stand at room temperature for 30 min to solidify.

[0212] To passage ALI organoids, prepare a collagenase solution by adding 950 μL of F12 medium and 50 μL of collagenase stock solution (the collagenase stock solution is prepared by adding 5 mL of PBS to 1 vial of collagenase). Use a scraper to collect the tissue in the collagen gel insert, add 50 - 100 μL of the collagenase solution to each insert, so 300 U of collagenase is added to each insert. Then incubate the tissue in the collagen gel and collagenase solution at 37°C in a gentle shaker for 45 min.

[0213] During the first wash, add 10 mL of ADMEM / F12 medium. Next, centrifuge the tissue at 600 xg for 3 min. Discard the supernatant and add 1 mL of Matrigel Recovery Solution (MRS) to each tube. Use a P1000 pipette to break up the gel / precipitate by gently grinding.

[0214] Next, during the second wash, add 9 mL of ADMEM / F12 to 10 mL and then centrifuge the sample at 600 xg for 3 min. Then discard the supernatant.

[0215] During the third wash, add 1 mL of ADMEM / F12 and use a P1000 pipette to break up the gel / precipitate by gently grinding. Then centrifuge the sample at 600 xg for 3 min and then discard the supernatant.

[0216] Next, resuspend the pellet in the collagen liquid mixture. Then, add 1 mL of the pellet / collagen liquid mixture to each insert and place it on top of the collagen layer. Then let the insert stand at room temperature for 30 min to solidify.

[0217] Then, add 1.5 mL of culture medium to each outer well of the insert. Then place the solidified insert in the center of the outer well to ensure that the culture medium diffuses and covers the bottom of the insert.

[0218] Optionally, the ALI organoids can be cryopreserved. Before cryopreservation, prepare a cryopreservation medium containing 90% FBS and 10% DMSO. Aspirate the culture medium from the culture dish. Next, use a cell scraper or a P1000 pipette tip to collect the collagen gel containing the ALI organoids, and then transfer it to a 15 mL conical tube. Calculate the volume of collagenase IV according to the number of inserts; use approximately 50 - 100 μL of collagenase IV and 500 μL of culture medium for each insert. Then shake the ALI organoids at 37 °C for 30 - 60 min until the collagen gel dissolves. Next, add 3 volumes of culture medium to the tube. Then centrifuge the tube at 400 x g for 3 min. Discard the supernatant, add 3 mL of culture medium to each tube, and gently grind the ALI organoids using a P1000 pipette. Next, add 5 mL of culture medium to the test tube and centrifuge the tube at 400 x g for 3 min. Aspirate the supernatant, and then resuspend the ALI organoids in the cryopreservation medium. Gently mix the ALI organoids by pipetting. Then add 0.5 mL of each suspension to each cryotube. Place the cryotubes at -80 °C for 24 hours, and then transfer them to liquid nitrogen for long-term storage.

[0219] Pre-rapid expansion protocol (pre-REP) of ALI TIL in ALI organoids

[0220] To prepare tissues and cells for rapid expansion of ALI-derived TIL, after establishing organoids from tumor tissues, 50 IU / mL of IL-2 was added to the culture medium. Then the ALI organoids were cultured for 7 to 14 days. Between day 7 and day 14, the ALI organoids were collected using a cell scraper. Collagenase IV was used to collect the ALI organoids in this step. Next, to prepare for TIL priming between day 7 and day 14, the organoid pellet was resuspended into one well of a G-Rex 24-well plate, and 6000 IU / mL IL-2 in RPMI medium was added to each well. The total volume per well was approximately 6 mL. 6000 IU / mL IL-2 was added to the culture every three days. On day 11 after TIL priming (or day 18 or day 24 after starting 50 IU / mL IL-2 treatment), the cells were harvested and filtered through a 100 μm filter. Next, the cells were spun at 600 xg for 3 min. Then the cells were ready for the REP step. For comparison, standard TIL (STD TIL) from a standard pre-REP protocol was harvested by culturing with 6000 IU / mL IL-2 for 11 days. Alternatively, these cells could be counted and frozen using freezing medium as described previously. Rapid Expansion Protocol (REP) for ALI TIL

[0221] Table 2: Materials and Reagents for the REP Step of ALI-Derived TIL

[0222]

[0223] Table 3: Complete Medium (CM) Reagents

[0224]

[0225] Table 4: 50 / 50 Medium Reagents

[0226] Final concentration 100 mL Complete medium (CM) 50％ 50 mL AIM-V 50％ 50 mL IL-2 1000 IU / μL 3000 IU / mL 0.3 mL Anti-CD3 1 mg / mL 30 ng / mL 3 μL

[0227] In this REP step, 5 million ALI TIL cells were obtained from the pre-REP step and the cells were suspended in 100 mL of 50 / 50 medium. Next, 500,000 ALI TIL were counted and mixed with 50 million irradiated PBMC, and then plated into each well of a G-Rex 6M well plate. This was day 0 of the REP. 3000 IU / mL IL-2 was added to the ALI TIL every three days. On day 11 of the REP, the ALI TIL were collected and filtered through a 100 μm cell filter. The ALI TIL could be frozen or used for experiments / treatments. For comparison, in this REP step, the ALI TIL were replaced with STD TIL.

[0228] ALI TIL or STD TIL are co-cultured with autologous tumor epithelial cells

[0229] ALI TIL and STD TIL were co - cultured with autologous tumor epithelial cells derived from submerged organoids for 48 hours. Single - cell tumor organoids were used alone as a control. After 48 hours of incubation, the cells were collected for flow cytometry analysis.

[0230] Flow cytometry is used to evaluate the tumor-killing ability of ALI TIL and STD TIL

[0231] Cells were stained with antibodies conjugated to fluorescent dyes against CD45, EPCAM, Annexin V, Zombie NIR live - dead, and other T - cell markers. After staining, flow cytometry analysis was performed for viable tumor cell counting.

[0232] Results

[0233] Figure 1 The timeline and processing of the steps of the pre - rapid expansion protocol (pre - REP) of ALITIL and STD TIL are depicted. In the present disclosure, compared with the pre - REP of STD TIL, the pre - REP of ALITIL has an additional 1 - 2 weeks of low - IL - 2 treatment.

[0234] To evaluate the tumor - killing ability of TIL, the absolute number of tumor cells under the condition of co - culturing autologous tumor organoids with TIL was divided by the absolute number of tumor cells under the condition where autologous tumor organoids were not co - cultured with TIL (control condition). As Figure 2 shown, compared with the co - culture of standard TIL (or STD TIL) with autologous tumor epithelial cells, the co - culture of ALI tumor - infiltrating lymphocytes (ALITIL) with autologous tumor epithelial cells showed better tumor - killing ability. The results of these analyses are presented as the percentage of viable tumor epithelial cells. Data from samples derived from 3 different patients (CRC - 1 = colorectal cancer patient 1, CRC - 2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non - parametric ANOVA was performed, corrected by Geisser - Greenhouse correction for comparison between different conditions; *≤Pv = 0.05.

[0235] Example 2: ALI tumor-infiltrating lymphocytes (TIL) are more reactive to tumor cells compared to standard TIL

[0236] In this example, the reactivity of ALI TIL and STD TIL was evaluated.

[0237] PDO organoids were established from primary tumors as described in the previous example.

[0238] ALI TIL or STD TIL are co-cultured with autologous tumor epithelial cells

[0239] As described in the foregoing embodiments, ALI TIL and STD TIL were co-cultured with autologous tumor epithelial cells derived from submerged organoids for 16 hours. ALI TIL or STD TIL alone was used as a control. In this experiment, after 16 hours of incubation, cells were collected for flow cytometry analysis to examine the expression of IFNγ and CD107a.

[0240] Flow cytometry is used to evaluate the reactivity of TIL to tumor cells

[0241] Cells were stained for IFNγ secretion, CD107a, CD45, EPCAM, Annexin V, Zombie NIR live / dead, and other T cell markers using fluorescent dye-conjugated antibodies. After staining, flow cytometry analysis was performed on live CD3+ cells.

[0242] Results

[0243] To evaluate the tumor reactivity of TIL, the percentage of CD3+ cells secreting IFNγ and the percentage of CD3+ cells expressing CD107a on their surface were evaluated. Figure 3A The co-culture experiment timeline is depicted. As Figures 3B-3C shown, ALI TIL showed a higher percentage of IFNγ and CD107a compared to STD TIL, indicating that ALI TIL is more reactive to tumor cells compared to STD TIL. Data from samples from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non-parametric ANOVA was performed, corrected by Geisser-Greenhouse correction, for comparison between different conditions; *≤Pv = 0.05.

[0244] Example 3: ALI tumor-infiltrating lymphocytes (TIL) express higher levels of HLA-DR compared to standard TIL

[0245] In this embodiment, the levels of PD1, HLA-DR, and CD137 were evaluated.

[0246] PDO organoids were established from primary tumors as described in the previous embodiments.

[0247] ALI TIL or STD TIL are co-cultured with autologous tumor epithelial cells

[0248] ALI TIL and STD TIL were co-cultured with autologous tumor epithelial cells derived from submerged organoids for 48 hours. ALI TIL or STD TIL alone was used as a control. After 48 hours of incubation, cells were collected for flow cytometry analysis.

[0249] Flow cytometry is used to measure the levels of PD1, HLA-DR, and CD137

[0250] Cells were stained for HLA-DR, CD137, PD1, CD45, EPCAM, Annexin V, Zombie NIR live / dead, and other T cell markers using antibodies conjugated with fluorescent dyes. After staining, flow cytometry analysis was performed on live CD3+ cells.

[0251] Results

[0252] To evaluate the tumor reactivity of TILs, the MFI (median fluorescence intensity) of PD1, HLA-DR, and CD137 was quantified and compared to the MFI of ALI TILs or STD TILs not exposed to tumor cells. Figure 4A The experimental timeline of the co-culture experiment is depicted. Figures 4B-4D Flow cytometry results for PD1, HLA-DR, and CD137 are shown. As Figure 4C shown, in all samples, ALI TILs showed higher levels of HLA-DR compared to STD TILs. Results are presented as fold change. Additionally, as Figure 5 shown, in CRC-1, CRC-2, and melanoma (MLN), AIL TILs showed a higher percentage of HLA-DR+ compared to STD TILs, at least about 50%, 23%, and 35% respectively. Data from samples derived from 3 different patients (CRC-1 = colorectal cancer patient 1, CRC-2 = colorectal cancer patient 2, MLN = melanoma patient) are shown. Each experiment was repeated 3 times. Non-parametric ANOVA was performed, corrected with Geisser-Greenhouse correction, for comparison between different conditions; *≤Pv = 0.05.

[0253] Example 4: Air-liquid interface tumor-infiltrating lymphocytes

[0254] A method example is provided herein that combines the ALI and TIL processes to selectively reactivate and expand tumor rejection antigen-recognizing T cells and produces a TIL product with higher anti-tumor activity than standard TILs. Prior to the expansion phase, infiltrating lymphocytes were initially cultured with their tumor organoids, producing a TIL preparation with expected characteristics compared to the standard formulation. The method was carried out as described in Example 1.

[0255] ALI tumor organoid technology is successfully achieved

[0256] As Figure 6A and Figure 6B shown, using the air-liquid interface (ALI), organoids were successfully grown from fresh tumor tissues (e.g., kidney, lung, esophagus, uterus, and glioblastoma multiforme (GBM)). Figure 6AShows representative brightfield images of ALI tumor organoids from kidney, lung, esophageal, and uterine cancers. For samples used to generate ALI TIL, organoids were ultimately maintained under submerged conditions, which provides a robust stem cell-based cancer model to allow for extensive passaging and cryopreservation for subsequent in vitro and in vivo evaluation( Figure 6B ). Figure 6B Shows colorectal cancer (CRC) tissue (left) and GBM samples (right) grown as ALI organoids for TIL expansion (top) and as submerged organoids for assays (bottom). The cancer origin of CRC organoids was confirmed by whole-genome / exome sequencing, which matched the clinical sequencing of the original tumor( Figure 6C ). As Figure 6C shown, the sequencing results of 3 CRC organoid lines are shown as CNV plots and mutated genes (APC, TP53, KRAS).

[0257] The process based on ALI tumor organoids generates clinically relevant TIL

[0258] A research-scale process was developed that integrates an initial ALI tumor organoid and infiltrating lymphocyte co-culture phase (representing the first time period of pre-REP, during which cells are treated with low-dose IL-2), followed by a T cell outgrowth phase (representing the second time period of the pre-REP step) in the presence of high-dose IL-2, and a rapid expansion phase (during the REP step) in the presence of IL-2, anti-CD3, and irradiated PBMC( Figure 7A ). Figure 7A Shows a schematic of the 2-step ALI-TIL process and product cryopreservation. The process has been successfully performed using different samples, including CRC, gastric cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and melanoma. Cells were counted and cryopreserved after harvest to simulate product formulation. The extrapolated full-scale yield averaged 11x 10 9 cells (75x 10 7 to 64x10 9 ), which are within the 1x 10 9 to 150x 10 9 cell dose range for clinical administration( Figure 7B ). As Figure 7BAs shown, ALI-TIL obtained from 14 preparations were counted and the values were corrected according to tissue sampling and pre-REP dilution. The estimated full-scale yields were plotted for each individual preparation with mean and SEM (left). The box plot illustrates the fold amplification distribution of 14 REP products (right). To characterize the ALI-TIL products, thawed cells were analyzed by FACS. Consistent with the TIL phenotype, ALI-TIL were predominantly composed of αβ T cells of the CD4 and CD8 lineages, each consisting of approximately 83% effector memory T cells (TEM) and approximately 16.5% central memory T cells (TCM). Two additional subsets, naive / TSCM and effector memory CD45RA+ T cells (TEMRA), were present on average at <1% ( Figure 7C ). Figure 7C Twelve ALI-TIL preparations (14 original minus 2 CRCs with insufficient material) were analyzed by flow cytometry on an Agilent Quanteon analyzer (Santa Clara, CA) using CD3, γδ TCR, CD4, CD8, CD45RA, and CD62L to detect T cells, lineage, and memory subsets. Results were analyzed using FlowJo (Ashland, OR) and plotted as the parental percentage for each individual sample with mean and SEM. This advanced analysis demonstrated that TIL generated by the ALI process had the expected phenotypic characteristics of TIL products.

[0259] An in vitro cell-based assay for TIL functional characterization is established

[0260] Tumor reactivity was tested by co-culturing ALI-TIL from CRC organoids with autologous tumor cells established in parallel organoid cultures, which grew as conventional organoids with only tumor cells and no immune cells. As Figure 8A shown, IFNγ, CD107a, and 4-1BB in T cells were detected by flow cytometry and increased by 0.12% (6.3-fold relative to MHC blockade control induction), 0.8% (3.8-fold), and 0.4% (1.6-fold), respectively. In Figure 8AAmong them, ALI-TIL from 1 CRC was co-cultured with autologous tumor organoids at an effector:target ratio of 1:1 for 16 hours. PMA / ionomycin was used as an activation control, and an antibody generated from clone W6 / 32 was used to block major histocompatibility complex (MHC) class I (MHC-I) presentation and confirm antigen specificity. Cells were stained for live / dead, EPCAM, CD3, CD8, CD107a, and IFN-γ or 4-1BB and analyzed by flow cytometry using a Quanteon instrument (Agilent, Santa Clara, CA). Data were processed using FlowJo (Ashland, OR), and gating was performed on the indicated markers as shown. The results showed that the level of reactive T cells could be used for clinically active TIL products. In addition, tumor cell killing was evaluated by longitudinal high-content confocal imaging ( Figure 8B ). ALI-TIL from 1 CRC was co-cultured with autologous tumor organoids at an effector:target ratio of 10:1 for 24 hours. Tumor cell killing was monitored at 0 and 24 hours after co-culture using a Molecular Devices ImageXpress (San Jose, CA). As Figure 8B shown, TIL / autologous organoid co-culture, staining, and detection conditions were established. A decrease in tumor organoids and an increase in cell death were observed over time.

[0261] A preliminary single-cell RNA sequencing experiment confirms the polyclonality and diversity of T cell subsets of the ALI-TIL product ity

[0262] Cell indexing of transcriptomes and epitopes by sequencing (CITE-seq) was used to generate TCR repertoires and gene expression profiles for 4 ALI-TIL preparations (3 CRCs and 1 melanoma). As Figure 9A shown, the analysis revealed an average of 2334 unique TCR clonotypes per preparation, with individual frequencies following a "head-to-tail" distribution. Unique paired αβ CDR3 sequences (assimilated into TCR clonotypes) were counted and sorted by frequency order. Figure 9A The number of unique clonotypes relative to the number of sequenced cells (upper row) and a pie chart of the clonotype frequency distribution (decreasing from red to black, where the fraction is occupied by the sum of the top 20 frequencies) (lower row) are shown. Cell surface marker data were consistent with FACS analysis, confirming product purity and T cell subsets. As Figure 9B shown, unsupervised clustering of gene expression data generated 10 T cell subsets that appeared to be differentially expressed in CD4 (5 clusters), CD8 (3 clusters), and mixed (2 clusters) lineages for differentiation (LEF1, CD27), activation (HLA-DR, CD25), effector (GZMA, PRF1, KLRC1), and exhaustion (LAG3, HAVCR2) markers.Figure 9B The expression levels of 397 immune genes identified using SeqGeq (FlowJo, Ashland, OR) in 10 clusters are shown as a UMAP, where different colors represent each cluster. These preliminary data indicate that (1) there is significant clonal diversity in the in vitro expanded REP products, and (2) CITE-seq can robustly analyze the proposed GBM rALI-TIL candidates and determine how they differ from non-reprogrammed, non-ALI TIL preparations from the same samples.

[0263] Example 5: Reprogrammed ALI-TIL (rALI-TIL)

[0264] To generate the required number of T cells, TILs were isolated by mechanical dissociation of tumor tissue and subjected to two in vitro culture steps. These steps included a pre-rapid expansion protocol (pre-REP), followed by the REP phase. TILs were cultured in the presence of high-dose interleukin-2 (IL-2) to promote expansion, resulting in a several-fold increase in cell number (e.g., at least a 10-fold increase). The expanded TILs were cryopreserved and can be used for adoptive cell therapy (ACT). A therapeutic product consisting of autologous brain TILs reprogrammed to a stem cell-like state (termed stem cell memory T cells (TSCM)) was developed for ACT against glioblastoma (GBM). To achieve this, immunocompetent GBM organoids were used and signaling pathway modifications were induced during the in vitro TIL expansion process to promote differentiation into the TSCM phenotype.

[0265] 1. Determine the reagents and culture conditions for reprogramming TIL into a stem cell state

[0266] The interferon gamma (IFN-γ) signaling pathway is known to inhibit the maintenance and diversity of stem cell-like T cells, while Notch signaling has been identified as a potent regulator of T cell activation that can convert activated T cells into stem cell memory T cells (TSCM), which have enhanced self-renewal capacity and are crucial for long-term immune memory and effective immunotherapy. By blocking the interferon gamma (IFN-γ) signaling pathway and activating the Notch signaling pathway during the REP phase of the TIL preparation process, it is hypothesized that a portion of terminally differentiated effector memory T cells (TEM) are reprogrammed into TSCM, thereby enhancing the anti-tumor activity of TILs.

[0267] IFN-γ neutralizing antibodies prevent IFN-γ from binding to its receptor, thereby inhibiting downstream signaling pathways, while agonist antibodies targeting the Notch receptor induce Notch signal activation. Commercially available monoclonal antibodies (mAbs) can be used to modulate the IFNγ and Notch pathways in TILs.

[0268] Cryopreserved pre-REP TILs were used to culture pre-REP TILs in media supplemented with various concentrations of IFNγ-inhibiting or Notch-activating antibodies to identify the most effective mAb mixture capable of blocking the IFN-γ signaling pathway and activating the Notch signaling pathway. To verify successful pathway modulation, the expression levels of Myc, Deltex1, and Hes1 were measured to characterize Notch activation levels, and the IFNγ-induced genes Gbp5, Irf1, and Ccl2 were measured to evaluate IFNγ signaling levels by qPCR at 8 hours, 24 hours, and 48 hours after mAb administration.

[0269] To optimize the TSCM reprogramming conditions during the REP phase, which typically takes 11 - 14 days, TILs during REP culture were exposed to the most effective mAb mixture that targets both IFNγ inhibition and Notch activation for the duration of the entire REP TIL phase or the last 7 days of the REP TIL phase. To potentially further enhance the TSCM reprogramming conditions, a portion of the IL-2 in the culture was replaced with IL-7 and / or IL-15 to further improve the TIL reprogramming process through IFN-γ inhibition and Notch activation. Each condition was tested on at least 10 pre-REP TIL preparations (including melanoma, colon cancer, lung cancer, and GBM pre-REP TIL samples).

[0270] To verify TSCM reprogramming, the expression levels of the following markers in cultured cells were evaluated: including CCR7, CD45RA, CD62L, CD69, CD95, and CD103, which contribute to the determination of different T cell memory subtypes. These subtypes are (i) central memory T cells (TCM), which are characterized by CD45RA-CCR7+CD62L+ expression, (ii) tissue-resident memory T cells (TRM), which are identified by CD69+CD103+ expression, (iii) effector memory T cells (TEM), which are identified by CD45RA-CCR7-CD62L- expression, (iv) stem cell memory T cells (TSCM), which are characterized by CD45RA+CCR7+CD62L+CD95+ expression, (v) naive T cells, which are characterized by CD45RA+CCR7+CD62L+ expression, and (vi) terminally differentiated effector memory cells (TEMRA), which are identified by CD45RO- / CCR7- expression. 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) were evaluated, which are typically associated with impaired T cell function and increased sensitivity to apoptosis during cancer progression (REF). This system generates at least 5% detectable TSCM subsets and at least 20% TCM subsets. 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 can be evaluated and areas for further optimization to increase TSCM yield can be determined.

[0271] Alternative methods to ensure that the antibody does not affect other pathways or have any unexpected off-target effects on cells include using small molecule inhibitors that can target specific molecules within the IFN-γ pathway or administering exogenous Delta-like 1 ligand to activate Notch signaling.

[0272] 2. Generate a development of a reprogrammed TIL (rALI-TIL) based on the ALI organoid process from at least 4 primary GBM samples of

[0273] To enhance the therapeutic potential of TILs against GBM, donor- or patient-derived air-liquid interface (ALI) immunocompetent tumor organoids were cultured. By using the ALI culture system, a physiologically relevant microenvironment was established, which better mimics the in vivo tumor microenvironment than traditional cell or tissue culture and has lower immunosuppression than the in vivo tumor microenvironment (TME). The ALI-TIL process facilitates the selection of T cells that recognize tumor neoantigens and results in a shift in the T cell receptor (TCR) repertoire of the TIL preparation towards enhanced neoantigen recognition ability. Therefore, these conditions enrich the specificity and function of the TIL product against GBM and achieve better results in resisting GBM.

[0274] To generate GBM ALI TIL, ALI GBM organoids containing TIL were cultured in GMB medium supplemented with low-dose IL-2 to promote the growth of GBM ALI TIL. After 1 - 7 days, GMB ALI organoids were isolated by digesting the collagen chunks with collagenase IV. The isolated organoids containing TIL were transferred to 24-well G-Rex plates and then subjected to the pre-REP culture process. During this 11 - 14-day process, TIL were cultured in T cell medium enriched with high-dose IL-2. After 11 - 14 days, ALI pre-REP TIL were transferred to 6M G-Rex wells to initiate the REP phase. This protocol involves stimulating TIL in vitro with anti-CD3 antibody and high-dose IL-2 for 11 - 14 days. The ALI-TIL process for GBM has been successfully run twice, generating at least 250 million cells each time. rALI-TIL (reprogrammed ALI-TIL) is expected to exhibit a T cell-dominant phenotype, with at least 90% of the T cells represented by the CD4 and CD8 lineages. This high level of T cell representation is crucial for effective targeting of cancer cells.

[0275] To determine the most effective conditions for increasing the percentage of TSCM in GBM ALI-TIL preparations, the composition and concentration of supplemented mAb and other growth factors (including IL-2, IL-7, and IL-15) were further optimized. This approach helps direct GBM TIL towards an immunophenotype, which is expected to enhance their anti-tumor persistence. To study the efficacy of the optimized stem cell reprogramming protocol on GBM rALI-TIL, rALI-TIL were derived from at least 4 different GBM samples and their T cell memory phenotypes were compared with matched control TIL preparations (including ALI, CTRL, and CTRL TIL). After generating the TIL preparations, the yields were determined. The full-scale TIL yields were extrapolated from this, providing insights into the potential of the TIL generation process for clinical applications. To ensure long-term preservation of the samples, TIL preparations of 20 million cells per vial were aliquoted and cryopreserved. This allows the samples to be evaluated at a later time and the study of the efficacy of the optimized reprogramming protocol on GBM rALI-TIL to continue.

[0276] Detailed immunoprofiling was performed on autologous TIL preparations (including ALI, rALI, autologous control (CTRL), and rCTRL) from 4 GBM samples.

[0277] Flow cytometry was used to comprehensively analyze individual TIL preparations by evaluating T cell subsets, T cell memory, and function. The panel of markers included CCR7, CD45RA, CD62L, CD69, CD95, CD103, PD-1, Tim-3, LAG-3, TIGIT, and CTLA-4, which were used to distinguish T cell memory subsets, activation status, and functional potential, as well as to assess the level of T cell exhaustion. This analysis provided a comprehensive immunoprofile of GBM ALI-TIL and its autologous controls and identified differences in T cell subsets and activation status. The selection of the rALI-TIL process was based on a yield of at least 1x10 8 cells, with at least 90% being T cells, and at least 20% being TCM and 5% being TSCM, indicating successful reprogramming. To further ensure the purity of the TIL preparations, a flow cytometry panel was established to characterize the expression levels of several cell surface markers associated with glioblastoma (including CD133, CD44, CD45, and CD90). The goal was to identify cell surface markers that were uniquely expressed in autologous GBM cells but absent in their respective TIL preparations.

[0278] To evaluate the T cell fitness of the TIL preparations, an IFNγ release assay was performed on all 16 GBM TIL samples. This assay is a widely used method for evaluating T cell activation and cytokine production in response to pan-TCR stimulation. The assay was performed by incubating individual TIL preparations with a pan-TCR stimulator consisting of anti-CD3 and anti-CD28 antibodies. After 1 to 3 days of incubation, the supernatant was collected, and the amount of IFNγ released by the cells was measured using ELISA. Using this method, the reactivity of the TIL samples to pan-TCR stimulation could be evaluated and their overall T cell fitness could be assessed. Secretion of more than 200 pg / ml IFNγ could be used as an indicator of strong T cell fitness levels.

[0279] Co-culture of GMB TIL with submerged glioblastoma organoids (GBO) was used as an important tool to study the cytotoxic potential and cytokine secretion profile of TIL against autologous tumor cells in various in vitro assays described below. GBO is a three-dimensional (3D) culture that mimics the complex structure and cellular heterogeneity of GBM in vivo. By culturing 1 mm in suspension 3Patient-derived GBO tissue fragments were used to establish GBO models. Organoids were maintained in serum-free medium and propagated for several months. To avoid necrotic cell death in the inner core cells, the GBOs were propagated by cutting them into pieces with a diameter of 0.5 mm every 1-2 weeks. These organoids retained the genomic and transcriptomic characteristics of the original tumor and could be used to evaluate drug responses and gene expression changes. The goal was to establish and expand this GBO culture system from fresh glioblastoma tissue and cryopreserve it for future experiments. This ensured a continuous supply of GBOs that could be used to characterize the anti-tumor responses of matched TIL preparations. To ensure the establishment of high-quality GBO lines, specific criteria were developed. A GBO line was established if it could be passaged indefinitely, cryopreserved for future use, and exhibited antigen presentation in response to IFNγ exposure. In addition, by performing whole-genome sequencing, the genotype of the established GBO line was matched to the genotype of the original GBM sample. Meeting these criteria ensured the establishment of reliable and consistent GBO lines for future experiments.

[0280] 3. In vitro functional comparison of GBM reprogrammed ALI TIL with non-reprogrammed and control TIL

[0281] Mature in vitro assays were used to characterize and compare the anti-tumor activities of rALI TIL preparations with autologous controls. These assays were used to evaluate the cytotoxicity and cytokine secretion profiles of TILs against autologous patient-derived glioblastoma organoids in co-culture experiments. The results of these assays provided a comprehensive understanding of the functional differences between different TIL preparations and helped to determine the optimal culture methods for generating TILs with enhanced anti-tumor activity.

[0282] All GBM samples were amplified for the following functional TIL test assays.

[0283] To determine the most suitable GBO / TIL co-culture conditions that do not compromise cell viability and function, the focus was on finding a culture medium composition and co-culture system that both GBOs and TILs could tolerate well. By achieving this, in vitro functional TIL test assays, such as tumor reactivity assays (TRA) and GBO killing assays, could be performed to evaluate the anti-tumor potential of all 16 GBM TIL preparations.

[0284] To evaluate the reactivity of various GBM TIL preparations against tumor cells, the TRA, an in vitro assay, was used, in which different TIL preparations were co-cultured with patient-matched GBO. The production of IFN-γ, CD107a, and 4-1BB was used as a direct readout of tumor antigen-specific T cell activation and as a strong predictor of T cell cytotoxicity. IFN-γ is a cytokine produced by activated T cells and is an important marker of T cell function. The presence of IFN-γ in this assay indicates that T cells are activated and produce this cytokine in response to tumor antigens. CD107a is a lysosome-associated membrane protein expressed on the surface of activated T cells. The presence of cell surface CD107a indicates that T cells degranulate and release cytotoxic molecules responsible for killing tumor cells, such as perforin and granzyme B. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. CD137 signaling enhances T cell proliferation and survival and promotes the production of cytokines such as IFN-γ. The expression of CD137 on T cells in the TRA serves as an additional marker of T cell activation and function. In this experiment, TIL and GBO-derived tumor cells were co-cultured at a ratio of 1:1 for 4 - 24 hours. To ensure the functionality of the assay, a PMA / ionomycin T cell stimulation mixture was used as a positive control. To ensure the specificity of the assay, negative controls were used, including the addition of an MHC blocking antibody mixture, which can block T cell recognition of tumor antigens and thus prevent T cell activation. In addition, TIL cultured in the absence of the corresponding tumor cells can also serve as a negative control. After co-culture, all samples were subjected to flow cytometry analysis to evaluate the expression levels of IFN-γ, CD107a, and CD137 on T cells. This analysis provides a quantitative assessment of T cell reactivity against tumor cells and helps to identify any differences in tumor reactivity between GBM TIL preparations generated by different methods.

[0285] To evaluate the tumor cell killing ability of all GBM TIL samples, GMB TIL / GBO was set up to be co-cultured at a ratio of 10:1 for 24 h, 48 h, and 72 h. Negative controls (including MHC blocking antibodies) were used to ensure the specificity of the assay. After co-culture, the level of TIL-mediated tumor cell killing was evaluated by flow cytometry analysis. This analysis involved the measurement of various parameters, including the expression levels of CD3 and GBO-specific surface markers, which were used to label T cells and GBM tumor cells, respectively. In addition, the levels of apoptosis and necrosis in tumor cells were measured using Dapi, annexin V, or propidium iodide staining, enabling the differentiation of live cells, apoptotic cells, and necrotic cells.

[0286] To verify the tumor cell killing data obtained from flow cytometry analysis, live cell confocal imaging was performed to monitor TIL-mediated GBO cell death over a 72-hour period. TIL and GBO were co-cultured at a ratio of 10:1, and GBM TIL was labeled with CellTrace Violet while GBO was labeled with Cell Tracker Orange. To visualize cell death in the orange-labeled GBO, Sytox Green, a nucleic acid stain that cannot penetrate the cell membrane of live cells but can enter dead cells and bind to nucleic acids, resulting in green fluorescent labeling of dead cells, was added to the co-culture medium. Negative controls, including MHC blocking antibodies, were used to ensure the specificity of the assay.

[0287] A strong correlation between flow cytometry data and live cell imaging results is expected in the evaluation of TIL-mediated GBM cell death. This proposed method can comprehensively analyze the cytotoxic potential of TIL against autologous GBM tumor cells and identify the most effective TIL generation process for GBM immunotherapy.

[0288] 4. Test at least 2 reprogrammed in an orthotopic xenograft (ODX) mouse model derived from autologous GBM organoids Study on the efficacy and preliminary mechanism of action (MOA) of ALI-TIL.

[0289] The efficacy of rALI-TIL in controlling tumor growth in vivo was evaluated by monitoring its ability to control autologous ODX tumor growth. To achieve this, an organoid platform was used to generate an autologous tumor xenograft model. In addition, the in vivo persistence was characterized by detecting metastatic T cells in tumor deposits and circulation over time.

[0290] To study the tumor-forming potential and growth kinetics, an SQ xenograft model was used in immunodeficient NOG mice, and at least four donor-derived GBO lines were used. The GBO was dissociated into single cells and small cell aggregates and then resuspended in a mixture of 50% GBO medium and 50% Matrigel before transplantation. Each GBO line was implanted into five mice at doses ranging from 1x10 5 to 1x10 6 varying, and tumor growth was monitored weekly using caliper measurements until the tumor reached 2000 mm 3At this size, the mice were sacrificed. Tumor tissues were isolated from the mice, stored in the repository, and subjected to histological and molecular characterization. The purpose of this analysis was to verify that the genotype and phenotype of the ODX tumor tissues were similar to those of the corresponding primary GBM, thus providing a reliable model for subsequent experiments. In addition to the SQ xenograft model, an orthotopic GBM xenograft model was established, which was more clinically relevant than the subcutaneous model because it more closely replicated the tumor microenvironment of the brain. These models were used to study tumor invasion and better understand the behavior of GBM in its native environment. To establish the orthotopic GBO xenograft model, a stereotactic injection system was used to directly inject GBO-derived tumor cells through a small hole in the skull into the brains of immunodeficient mice to ensure precise targeting of the injection site. As with subcutaneous implantation, the GBO tumor cells were resuspended in a mixture of 50% GBO medium and 50% Matrigel prior to transplantation. After implantation, the neurological symptoms of the mice were monitored, and tumor growth was measured using MRI. To minimize variability, the GBO dissociation protocol and transplantation method were standardized. This involved using optimized concentrations and digestion times of digestive enzymes for each GBO line and employing a stereotactic injection system for orthotopic transplantation to minimize the risk of damage to the brain tissue.

[0291] To evaluate the cytotoxic anti-tumor activity of GBM rALI TIL in vivo, an ideal xenograft model was adopted and the optimal number of GBO cells with the best tumor formation and growth kinetics in each GBO line was utilized. For this set of experiments, immunodeficient NOG mice from Taconic were used, which expressed human IL-2 in the blood in the range of 0.5 - 2.0 ng / mL. The expression of human IL-2 in the mouse model was crucial for supporting the survival and function of the infused human TIL. When the tumor size reached 150 mm 3 36 female mice of each GBO ODX line were randomly divided into three groups. The first and second groups were infused with 20 million GMB patient-matched rALI TIL and CTRL TIL via the tail vein, respectively. The third group served as a control. The tumor size was monitored weekly until the tumor size of one mouse in the group reached 2000 mm 3 At this time, the mice were sacrificed. At the end of this GBO tumor xenograft experiment, all the remaining tumors from the three groups were harvested and evaluated. Based on the hypothesis that rALITIL had significantly enhanced cytotoxic anti-tumor activity, the administration of these cells could significantly reduce the tumor volume compared to the control cohort.

[0292] To study the in vivo persistence and tumor infiltration of metastatic cells, TIL invasion in the tumor microenvironment and T cell persistence in mouse blood samples were evaluated. Blood samples were collected at different time points after TIL infusion and analyzed using flow cytometry to determine the percentage of human T cells in mouse blood. These data enabled the evaluation of the in vivo persistence of GMB rALI TIL and autologous controls. Then, tumor samples were collected at the end of the GBO tumor xenograft experiment and subjected to flow cytometry and immunohistochemical staining to evaluate the degree of TIL infiltration in the tumor microenvironment. This provided important information on the localization of intratumoral TIL and its potential impact on tumor growth.

[0293] To verify the mechanism of action (MOA) mediated by tumor antigen-specific cytotoxic killing of rALI-TIL, the anti-tumor activity against an allogeneic ODX model was evaluated, and the effect of CD8+ cell depletion in the TIL preparation before infusion was studied. First, mismatched rALI-TIL was infused into mice bearing 150 mm 3 sized ODX tumors, and tumor growth was monitored using the above methods. This determined whether rALI-TIL exhibited non-specific or antigen-specific cytotoxic killing against ODX tumors. Second, autologous rALI-TIL was infused into mice bearing ODX tumors, and one group received a TIL product depleted of cytotoxic CD8+ T cells. A significant reduction in anti-tumor activity was expected in the CD8+ depletion group, thus verifying cytotoxic tumor cell killing by neoantigen-specific CD8+ cells in an in vivo experiment.

[0294] Characterize the TCR repertoire and gene expression profiles of reprogrammed and non-reprogrammed TILs at the single-cell level.

[0295] The T cell receptor (TCR) repertoire of the TIL preparation represents the diversity and composition of TCRs expressed by T cells in the sample. Each T cell expresses a TCR that recognizes a specific antigen presented by major histocompatibility complex (MHC) molecules, thus reflecting the specificity and diversity of T cell responses. To confirm that the ALI-TIL process may favor the selection of T cells that recognize tumor neoantigens and lead to a shift in the TCR repertoire therein, the TCR repertoire of GBM-derived TIL was analyzed.

[0296] To evaluate the effect of different ex vivo TIL generation and expansion processes on TCR clone diversity, 1X10 in each GBM-derived TIL preparation 4Single cells were subjected to TCR sequencing (TCRseq). At least 4 GBM TIL groups were analyzed for each GBM sample, including autologous CTRL TIL, rCTRL TIL (reprogrammed CTRL TIL), ALI TIL, and rALI TIL preparations. Different TCR clonotype compositions between ALI and CTRL autologous samples could indicate that the ALI-TIL process selected T cells that recognize tumor antigens. Alterations in clonotype frequencies in reprogrammed TIL compared to non-reprogrammed TIL could reflect the impact of pathway regulation during TIL reprogramming. By understanding these differences, the methods for TIL production and selection for effective GBM immunotherapy could be further optimized. Gene expression profiling of all GBM-derived TIL preparations at the single-cell level using single-cell RNA sequencing (scRNA-seq) and CITE-seq was able to thoroughly validate the proposed reprogrammed stem cell-like state in TILs that were exposed to pathway regulation.

[0297] Sequencing data analysis using commercial (SeqGeq), academic (GLIPH258), and in-house (R scripts) pipelines was used to analyze the sequencing data. The commercial pipeline named SeqGeq was used for comprehensive analysis of the sequencing data. This pipeline could be used to characterize TCR clonotypes and RNAseq-derived immune profiling data to validate the upregulation of TSCM and TCM markers and the downregulation of exhaustion markers in reprogrammed TILs compared to the control group. Secondly, the academic pipeline named GLIPH258 was used to identify TCR clonotypes based on sequence homology. This pipeline could be used to identify clonotypes with common features and that could recognize similar antigens. Finally, the in-house R script pipeline was used for customized in-depth analysis of the sequencing data. This pipeline helped to determine the TCR clonotype overlap and diversity between autologous TIL groups and the altered clonotype frequencies.

[0298] Although the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for generating immune cells derived from a subject, the method comprising: (a) obtaining one or more tumor samples from the 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 a culture medium, and wherein one or more agents are added to the culture medium; and (c) collecting the immune cells from the one or more tumor samples, thereby generating the immune cells derived from the subject.

2. The method according to claim 1, wherein the incubation in (b) comprises a first time period and a second time period.

3. The method according to claim 2, wherein the first time period is at least about 1 day.

4. The method according to claim 2 or claim 3, wherein the first time period is about 7 to about 14 days.

5. The method according to any one of claims 2 to 4, wherein the second time period is at least about 1 day.

6. The method according to any one of claims 2 to 5, wherein the second time period is about 11 days.

7. The method according to any one of claims 2 to 6, wherein the one or more agents are added to the culture medium at a first concentration during the first time period.

8. The method according to any one of claims 2 to 7, wherein the one or more agents are added to the culture medium at a second concentration during the second time period.

9. The method according to claim 7 or claim 8, wherein the first concentration is lower than the second concentration.

10. The method according to any one of claims 1 to 9, wherein the in vitro culture process comprises a gas-liquid interface setting.

11. The method according to any one of claims 1 to 10, further comprising cryopreserving the immune cells from (c).

12. The method according to any one of claims 1 to 10, further comprising (d) expanding the immune cells obtained from (c) with one or more agents for a period of time.

13. The method according to claim 12, further comprising: (e) collecting the immune cells from (d).

14. The method according to claim 13, further comprising, after (e), (f) cryopreserving the immune cells.

15. The method according to any one of claims 1 to 14, wherein the immune cells comprise tumor-infiltrating lymphocytes.

16. The method according to claim 15, wherein the tumor-infiltrating lymphocytes comprise T cells.

17. The method according to claim 16, wherein the T cells include activated T cells; naive CD8+ T cells; cytotoxic CD8+ T cells; naive CD4+ T cells; helper T cells, such as T H 1, T H 2, T H 9, T H 17, T H 22, T FH ; memory T cells, such as central memory T cells, T stem cell memory cells (TSCM), effector memory T cells, NKT cells or γδ T cells.

18. The method according to any one of claims 1 to 17, wherein the one or more agents in (b) comprise cytokines.

19. The method according to claim 18, wherein the cytokines 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 a combination thereof.

20. The method according to claim 18 or claim 19, wherein the concentration of the cytokine added during the first time period in (b) is at least about 10 IU / mL.

21. The method according to any one of claims 18 to 20, wherein the concentration of the cytokine added during the first time period in (b) is about 50 IU / mL.

22. The method according to any one of claims 18 to 21, wherein the concentration of the cytokine added during the second time period in (b) is at least about 4000 IU / mL.

23. The method according to any one of claims 18 to 22, wherein the concentration of the cytokine added during the second time period in (b) is about 6000 IU / mL.

24. The method according to any one of claims 18 to 23, wherein the expansion of the immune cells in (d) does not include an air-liquid interface setting.

25. The method according to claim 12, wherein the period of time in (d) is at least 1 day.

26. The method according to claim 12 or claim 25, wherein the period of time in (d) is about 14 days.

27. The method according to any one of claims 12 to 26, wherein the one or more agents in (d) include cytokines, antibodies, modulators, or any combination thereof.

28. The method according to claim 27, wherein the one or more agents further include irradiated feeder cells.

29. The method according to claim 28, wherein the irradiated feeder cells are feeder cells derived from irradiated allogeneic PBMC.

30. The method according to claim 28 or claim 29, wherein the ratio of the immune cells to the irradiated feeder cells is about 1:

100.

31. The method according to any one of claims 27 to 30, wherein the cytokines include IL-2, IL-2 variants, IL-7, IL-7 variants, IL-15, IL-15 variants, IL-18, IL-18 variants, IL-21, IL-21 variants, or any combination thereof.

32. The method according to claim 31, wherein the cytokine is added at a concentration of at least about 2000 IU / mL.

33. The method according to claim 31 or claim 32, wherein the cytokine is added at a concentration of about 3000 IU / mL.

34. The method according to claim 27, wherein the antibody includes an anti-CD3 antibody.

35. The method according to claim 34, wherein the concentration of the anti-CD3 antibody is at least about 10 ng / mL.

36. The method according to claim 34 or claim 35, wherein the concentration of the anti-CD3 antibody is about 10 ng / mL.

37. The method according to claim 34 or claim 35, wherein the concentration of the anti-CD3 antibody is about 30 ng / mL.

38. The method according to claim 27, wherein the modulator comprises a Notch signaling pathway modulator, an interferon gamma (IFNγ) modulator, or a combination thereof.

39. The method according to claim 38, wherein the Notch signaling pathway modulator comprises a Notch activator.

40. The method according to claim 39, wherein the Notch activator comprises an antibody, a small molecule, or a combination thereof.

41. The method according to claim 38, wherein the IFNγ modulator comprises an IFNγ inhibitor.

42. The method according to claim 41, wherein the IFNγ inhibitor comprises an antibody, a small molecule, or a combination thereof.

43. The method according to any one of claims 2 to 42, wherein the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises one or more of PD-1, CD39, 4-1BB positive T cells, or a CXCR3-binding chemokine.

44. The method according to claim 43, wherein the CXCR3-binding chemokine comprises CXCL9 or CXCL10.

45. The method according to any one of claims 2 to 42, wherein the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises an interferon.

46. The method according to any one of claims 2 to 42, wherein the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises a checkpoint inhibitor.

47. The method according to any one of claims 2 to 42, wherein the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises TLR3, TLR7, TLR9, or other TLR agonists.

48. The method according to any one of claims 2 to 42, wherein the method further comprises providing an additional reagent during the first time period or the second time period, wherein the additional reagent comprises a modulator of RIG-I-like receptors, a modulator of NOD-like receptors, a modulator of C-type lectin receptors, a modulator of STING, or a combination thereof.

49. The method according to any one of claims 2 to 42, wherein the method further comprises combining the tumor organoids with immune cells obtained from other sources.

50. The method according to claim 49, wherein the other sources comprise peripheral blood cells or organoids grown from lymphoid tissue.

51. The method according to any one of claims 1 to 50, wherein the method further comprises stimulating antigen presentation.

52. The method according to any one of claims 1 to 51, wherein the method further comprises depletion of immunosuppressive cell types.

53. The method according to claim 52, wherein the immunosuppressive cell types comprise Tregs, myeloid-derived suppressor cells, TAMs, vascular endothelial cells, or CAFs.

54. The method according to any one of claims 1 to 53, wherein the method further comprises negative selection of bystander tumor-reactive immune cells.

55. The method according to any one of claims 1 to 53, wherein the method further comprises knocking down exhaustion regulators.

56. The method according to claim 55, wherein the knockdown exhaustion regulator is TOX.

57. The method according to any one of claims 1 to 56, wherein the method further comprises reprogramming the immune cells.

58. The method according to claim 57, wherein the immune cells are reprogrammed by activating the Notch signaling pathway during (d), inhibiting the interferon gamma (IFNγ) signaling pathway during (d), or both.

59. The method according to any one of claims 1 to 58, wherein the method further comprises identifying a T cell receptor (TCR) to identify TCRs enriched in the air-liquid interface culture.

60. The method according to claim 59, wherein the identification of the TCR is performed using sequencing techniques.

61. The method according to any one of claims 1 to 60, wherein the method comprises providing one or more tumor antigens during the first time period or the second time period.

62. The method according to claim 61, wherein the providing of one or more tumor antigens comprises providing cells expressing the tumor antigen.

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