method for identifying a suitable agent to treat a cancer, tumor-growing medium and / or organoid, t

专利摘要:
The present invention provides co-cultures of organoids and immune cells and methods of using these to identify agents to treat diseases.
公开号:BR112020012565A2
申请号:R112020012565-2
申请日:2018-12-21
公开日:2020-11-24
发明作者:Kai KRETZSCHMAR；Jotam Elazar Bar-Ephraim；Johannes Carolus Clevers；Sylvia Fernandez-Boj；Robert Gerhardus Jacob VRIES
申请人:Koninklijke Nederlandse Akademie Van Wetenschappen；Stichting Hubrecht Organoid Technology；
IPC主号:

专利说明:

[001] [001] All documents cited are hereby incorporated by reference in their entirety. TECHNICAL FIELD
[002] [002] The present invention relates to co-cultures of organoids and their use in the investigation of diseases. FUNDAMENTALS
[003] [003] Clinical research within the physiology of underlying diseases, such as cancer and immune diseases, remains a cornerstone of medical progress, although in vitro systems for carrying out such investigations remain basic. Likewise, modern regimens for the treatment of such diseases typically involve rigorous testing systems during development, to ensure the efficacy and safety of the regimens. Although recent advances in these fields have increased the effectiveness of investigative and therapeutic test systems, there is a need for improvements in terms of the systems' efficiency, accuracy, and cost effectiveness. An ideal test system would accurately replicate the physiology of a patient or patient population, at the biochemical, cellular, tissue, organ, and organism levels, without
[004] [004] In vitro models are needed to 'screen' candidate compounds to identify new regimes for investigating and treating cancer and immune diseases at the population level. In addition, there is growing interest in 'personalized medicine' in which in vitro models can be used to test (sometimes previously approved) regimes in patient subgroups with particular characteristics or even samples from a single patient, to determine the ideal regimen for this subgroup or patient in particular.
[005] [005] The field of organoid technology is revolutionizing our understanding of developmental biology. An organoid is a cell structure obtained by the expansion of epithelial cells and consisting of specific cell types of tissue that self-organize through cell classification and involvement of spatially restricted lineage (Clevers, Cell. June 16, 2016; 165 ( 7): 1586-1597). A limitation of organoid-based models in the prior art is that they contain only epithelial cells and thus are not fully representative of an in vivo tissue system that contains multiple cell types. In particular, human ‘cocultures’ of cancerous organoids (“tumoroids”) and immune cells have not been described, certainly not where the cancer and immune cells were obtained from the same patient. The immune cells improve the accuracy of the organoid as a test system, replicating the patient's physiology and ensuring that the immune system is represented in the test system.
[006] [006] Previous attempts have demonstrated the co-culture of murine intraepithelial lymphocytes (IELs) with murine intestinal epithelial organoids, for the purposes of understanding the spatial-temporal behaviors of IELs with intestinal epithelial cells - Nozaki et al. (J Gastroenterol.
[007] [007] There is a need for improved methods for preparing cocultures of organoids and tumoral cocultures and methods for using these cocultures in drug screening, particularly a system in which the interaction between diseased cells and immune cells can be stimulated to investigate an increased range of drugs with high yield capacity. SUMMARY OF THE INVENTION
[008] [008] The inventors have developed co-cultures of organoids useful for investigations related to diseases, such as cancer and immune diseases, including the identification of suitable treatments for such diseases. This involves in some embodiments preparing cocultures of organoids and immune cells, particularly disease organoids (such as tumoroids) and immune cells, which can be exposed to candidate agents to treat diseases and detect any changes to identify suitable candidate agents.
[009] [009] Consequently, the invention provides, among other things, a method for identifying a suitable agent to treat cancer, in which the method comprises: placing a tumoral coculture in contact with one or more candidate agents, in which the tumoroid coculture comprises cells immune and at least one tumoroid,
[0010] [0010] In some embodiments, the above method further comprises comparing the presence or absence of one or more changes in the tumor coculture with a reference organoid or reference tumor and in which the method further comprises: placing a reference coculture of reference or reference tumor coculture in contact with one or more candidate agents, where the reference organoid coculture or reference tumor tumor coculture comprises immune cells and at least one organoid or tumoroid, and detects the presence or absence of one or more further changes in the reference organoid coculture or reference tumor tumor coculture that is indicative of the suitability of a candidate agent to treat cancer.
[0011] [0011] The invention further provides a method for identifying a suitable agent for treating an immune disease, wherein the method comprises: bringing an organoid coculture into contact with one or more candidate agents, wherein the organoid coculture comprises diseased immune cells and at least one organoid, detect the presence or absence of one or more changes in the organoid co-culture that is indicative of the suitability of a candidate agent to treat the immune disease and identify a candidate agent as suitable to treat the immune disease if the presence or absence one or more of said changes
[0012] [0012] In some embodiments, the above method further comprises comparing the presence or absence of one or more changes in the organoid coculture with a reference immune cell (for example from a control patient lacking the immune disease) and in which the method additionally comprises: placing a reference organoid co-culture in contact with one or more candidate agents, in which the reference organoid co-culture comprises immune cells and at least one organoid, and detecting the presence or absence of one or more changes in the co-culture reference organoid that is indicative of suitability of candidate agent to treat immune disease.
[0013] [0013] A method of testing an immunotherapy of CAR-T, transgenic T cells in TCR, neoantigen or checkpoint inhibitor is also provided for efficacy and / or safety when used to treat epithelial cancer, the method comprising: optionally provide tumor epithelial cells, normal epithelial cells and immune cells from the same patient, expand tumor epithelial cells in tumoroid culture medium to form a tumoroid, and grow the tumoroid with immune cells in a tumoroid co-culture medium comprising interleukin to form an interleukin tumor tumor coculture, expand normal epithelial cells in organoid culture medium to form an organoid and grow the organoid with immune cells in an organoid coculture medium comprising interleukin to form a reference organoid coculture, place the tumor tumor coculture and the reference organoid co-culture in contact with the CAR-T, transgenic T cell immunotherapy in TCR, neoantigen or checkpoint inhibitor,
[0014] [0014] Also provided is a method of testing a candidate compound for efficacy and / or safety when used to treat epithelial cancer, the method comprising: optionally providing tumor epithelial cells, normal epithelial cells and immune cells, expanding cells tumor epithelial cells in tumor growth medium to form a tumor and grow the tumor with immune cells in a tumor cell culture medium comprising interleukin to form a tumor cell culture, expand normal epithelial cells in organoid culture medium to form a organoid and culturing the organoid with immune cells in an organoid co-culture medium comprising interleukin to form a reference organoid co-culture, placing the tumoroid co-culture and the reference organoid co-culture in contact with the candidate compound, detecting the presence or absence of one or more changes in the tumor coculture and organ coculture oide of reference, in which the presence or absence of one or more changes is indicative of the efficacy and / or safety of the candidate compound and to compare the coculture of tumoroid and the coculture of reference organoid.
[0015] [0015] A method is also provided for preparing a coculture of
[0016] [0016] A method is also provided for preparing a tumoroid cell-immune coculture, wherein the method comprises: optionally culturing tumor epithelial cells in contact with an extracellular matrix in a tumor culture medium to obtain an organoid; removing said extracellular matrix and tumoroid culture medium from said tumoroid; resuspending said tumoroid in the immune cell culture medium supplemented with interleukin; preparing an immune cell suspension comprising immune cells, immune cell culture medium supplemented with interleukin and collagen at a concentration of at least 5 to 10% in the suspension; and mixing the immune cell suspension comprising immune cells with the resuspended tumor.
[0017] [0017] A method is also provided for testing a therapeutic agent, in which the method comprises: placing an organoid co-culture in contact with one or more candidate agents, in which the organoid co-culture comprises immune cells and at least one organoid, detecting the presence or absence of one or more changes in the organoid coculture that is indicative of therapeutic efficacy, and to identify a candidate agent as a therapeutic agent if the presence or absence of one or more of said changes in the organoid coculture is detected.
[0018] [0018] An organoid coculture obtainable or obtained by the methods of the invention is also provided.
[0019] [0019] A tumor coculture obtainable or obtained by the methods of the invention is also provided.
[0020] [0020] An organoid population obtainable or obtained by the methods of the invention is also provided.
[0021] [0021] A tumoroid population obtainable or obtained by the methods of the invention is also provided.
[0022] [0022] An organoid co-culture medium suitable for use in the methods of the invention is also provided.
[0023] [0023] A tumoroid co-culture medium and organoid co-culture medium suitable for use in the methods of the invention are also provided. A tumoroid or organoid is also provided in a medium comprising an interleukin, optionally where the interleukin is selected from the group consisting of IL-2, IL-7 and IL-15
[0024] [0024] The invention also provides a kit comprising a tumoroid, organoid, tumoroid coculture or organoid coculture of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] [0025] Figure 1A. Schematic of the procedure. Biopsies of normal colonic mucosa and tumor tissue are collected from the resected colon and / or rectum of patients with colorectal cancer. Peripheral blood is also collected during surgery. The normal colonic mucosa is treated with EDTA to release the follicles for the derivation of normal and additionally digested colonic organoid to manufacture a single cell suspension containing intraepithelial lymphocytes (IELs) for T cell cultures. Tumor tissue is digested to make a suspension single cell cells containing epithelial tumor cells for tumor derivation as well as tumor infiltrating lymphocytes (TILs) for T cell cultures. Peripheral blood is processed to purify peripheral blood mononuclear cells enriched for peripheral blood lymphocytes (PBLs) and T cells. Primary analysis is performed by sequencing the T cell receptor (TCR) and immunophenotyping the T cells and sequencing the single cell messenger RNA (mRNA) of the cells present in the single cell suspensions of normal colonic epithelium and tumor epithelium. Organoid cultures are analyzed using whole genome sequencing, mRNA sequencing and peptidoma profiling.
[0026] [0026] Figure 1B. Bright field images representative of normal colonic organoids and tumors derived from patient samples. The colonic crypts were embedded in basement membrane extract (BME) and cultured with medium containing R-spondin-1, Noggin, medium conditioned with Wnt3A, supplement B27 without vitamin A, nicotinamide, N-acetylcysteine, EGF, TGF-inhibitor α A-83-01, gastrin, MAP38 SB202190 p38 inhibitor and prostaglandin E2. Normal colonic organoids developed within 1 week and were changed weekly thereafter (top panel). Single cell suspensions of
[0027] [0027] Figure 1C. Bright field images representative of clonal growth of intraepithelial lymphocytes (IELs) and tumor infiltrating lymphocytes (TILs) derived from patient samples (left panels). Flow cytometry analysis shows robust expansion of CD4 + helper T cells (Th) and CD8 + cytotoxic T cells (CTLs). Single cell suspensions of normal colonic mucosa or colorectal cancer tissue were maintained in T cell medium containing interleukin-2 (IL-2). Clonal T-cell growth was noticeable within 1 to 2 weeks (left panels).
[0028] [0028] Figure 1 is further described in Example 1. Figure 2. Proof of principle co-culture of normal colonic organoids and allogeneic CD3 + T cells in drops of basement membrane extract (BME).
[0029] [0029] Figure 2A. Schematic of the procedure. Normal colonic organoids were released from the BME drop using Cell Recovery Solution and washed in complete advanced DMEM / F12. The expanded CD3 + T cells were harvested from the culture and labeled with green dye (Vybrant CFDA SE Cell Tracer). The colonic organoids and labeled T cells were mixed in human colonic organoid medium and embedded in BME drops. The cocultures were maintained in human colonic organoid medium containing IL-2 for 60 hours. Cocultures were released from BME using Cell Recovery Solution and fixed in 4% paraformaldehyde. Fixed entire assemblies were dyed with phalloidin to manufacture polymerized actin and DAPI to label nuclei.
[0030] [0030] Figure 2B. Maximum projection of z-stack images of colonic organoid co-cultures. F-actin in organoids is labeled in dark gray and T cells are labeled in light gray. The insert in the right panel shows a T cell infiltrating the colonic epithelium.
[0031] [0031] Figure 2C. Three-dimensional reconstruction of a normal colonic organoid and T cells.
[0032] [0032] Figure 2 is further described in Example 8. Figure 3. Live imaging of tumoroid cocultures to assess optimal T-cell motility.
[0033] [0033] Figure 3A. Schematic of the procedure. The tumors were released from the BME drop using Cell Recovery Solution and washed in complete Advanced DMEM / F12. Allogeneic CD8 + T cells isolated from peripheral blood samples were labeled with green dye (Vybrant CFDA SE Cell Tracer). Tumor cells and T cells were mixed with human colonic organoid medium containing IL-2 and 10% BME or rat tail collagen I and imaged live for 80 hours in a Leica SP8X confocal microscope equipped with an imaging camera. live at 37 ° C and atmosphere at 5% CO2.
[0034] [0034] Figure 3B. Composite images representative of the tumoral coculture. The bright field channel and the green fluorescent channel have been merged to generate composite images. The T-cell pathways were plotted using Imaris software.
[0035] [0035] Figure 3C. Quantification of the T cell track length in both conditions shows significantly longer pathway of T cells co-cultured in 10% collagen compared with 10% BME.
[0036] [0036] Figure 3 is explained in more detail in Example 10. Figure 4. Generation of positive and negative clonal tumoroids for human leukocyte antigen (HLA) type A2.
[0037] [0037] Figure 4A. Schematic of the procedure. Tumoroids were dissociated in single cells using enzyme digestion TrypLE. Single cells were stained with anti-HLA-A2 antibody and purified based on anti-HLA-A2 immunoreactivity. The HLA-A2 + ve and HLA-A2-ve tumor cells were embedded and maintained to generate tumors.
[0038] [0038] Figure 4B. Flow cytometric analysis showing the establishment of pure HLA-A2 + ve or HLA-A2-ve tumor lines. Controls are the HLA-A2 + ve JY cell line as well as the normal colonic organoid lines derived from the same patient samples as the HLA-A2 + ve or HLA-A2-ve tumor lines.
[0039] [0039] Figure 4 is further explained in Example 11. Figure 5. Extermination assay for anti-tumor reactivity of T cells experienced in antigen.
[0040] [0040] Figure 5A. Schematic of the procedure. The HLA-A2 + ve or HLA-A2-ve tumors were pulsed for 2 hours with the HLA-A2 restricted Wilms tumor peptide 1 (WT1). The transgenic CD8 + T cells in TCR harboring a specific TCR of WT1 peptide were then co-cultured for 48 hours with HLA-A2 + ve or HLA-A2-ve tumors pulsed with the WT1 peptide.
[0041] [0041] Figure 5B. Bright field images representative of cocultures after 48 hours showing the significant death of HLA-A2 + ve tumors pulsed only with WT1 peptides. All other conditions, i.e., HLA-A2 + ve or HLA-A2-ve tumors not pulsed with WT1 peptides and HLA-A2-ve tumors pulsed with WT1 peptide, show normal growth.
[0042] [0042] Figure 5 is explained in more detail in Example 12.
[0043] [0043] Figure 6A. Schematic of the procedure. Co-culture was performed as described for Figure 5A but only for 12 hours and incubated with and without anti-PD1 checkpoint inhibitor. The cell viability test was performed using the CellTiter Glo Luminescent Cell Viability Assay kit (Promega) according to the manufacturer's instructions.
[0044] [0044] Figure 6B. Cell viability of tumors normalized to no peptide control.
[0045] [0045] Figure 6 is explained in more detail in Example 13. Figure 7. Assay to determine differential effect on T cell activation by organoid / tumoroid co-cultures.
[0046] [0046] Figure 7A. Schematic of the procedure. The tumors were released from the Matrigel® drop using Dispase and subsequently passed over 70 µm and 20 µm filters. The organoids were recovered from the 20 µm filter, counted and plated. The tumors and T cells were mixed with human colonic organoid medium containing RPMI, IL-2 and 5% Matrigel® and incubated at 37 ° C and atmosphere with 5% CO2. After 24 hours of incubation the organoids were imaged using an inverted bright field microscope.
[0047] [0047] Figure 7B. Representative images of tumoroid co-cultures.
[0048] [0048] Figure 7C. Representative images of the organoid co-cultures.
[0049] [0049] Figure 7D. Quantification of IFN-γ levels in cocultures.
[0050] [0050] Figure 7 is further explained in Example 14. Figure 8. Live imaging of tumoroid cocultures to evaluate
[0051] [0051] Figure 8A. Schematic of the procedure. The tumors were released from the Matrigel® drop using Dispase and subsequently passed over 70 µm and 20 µm filters. The organoids were recovered from the 20 µm filter, counted and plated. The cultured T cells were labeled with dark red dye (CellVue Claret). Tumors and T cells were mixed with human colonic organoid medium containing RPMI, IL-2 and 5% Matrigel® and imaged live for 68 hours in a Leica SP8X confocal microscope equipped with a 37 ° C live imaging camera and atmosphere with 5% CO2.
[0052] [0052] Figure 8B. Composite images representative of the tumor and non-targeting T cell co-cultures. The bright field channel and the dark red fluorescence channel were generated fused composite images.
[0053] [0053] Figure 8C. Composite images representative of the tumor and T-cell targeting cultures. The bright field channel and dark red fluorescence channel were fused composite images generated.
[0054] [0054] Figure 8 is further described in Example 15.
[0055] [0055] Figure 9. CRC organoids express immunomodulatory molecules Normal colon and CRC organoid strains were generated in a patient-specific manner and RNA was extracted and analyzed using Affymetrix single transcript microarrays.
[0056] [0056] Figure 9A. Mean gene expression of different immunomodulators in normal colon and CRC organoid strains; n.s., not significant; *, p <0.05.
[0057] [0057] Figure 9B. Hierarchical grouping of individual normal colon organoid and CRC strains in the ‘living biobank’ showing the expression of selected immunomodulators gene. Color gradients represent z in the value of each row (gene transcripts).
[0058] [0058] Figure 9C. Human colonic organoid strains genetically engineered to carry one or more mutations found in CRCs. The expression levels of CD274 (PD-L1) in organoid strains (n = 2) in constant state (Ctrl) and in stimulation with 20 ng / mL of recombinant human IFN-γ evaluated by quantitative PCR. A, APCKO / KO; N.D., not detected; K, KRASG12D / +: P, P53KO / KO; S, SMAD4KO / KO, WT, wild type.
[0059] [0059] Figure 9D. Human colonic organoid strains genetically engineered to carry one or more mutations found in CRCs. Expression levels of CD274 (PD-L1) in organoid strains (n = 2) in constant state (Ctrl) and in stimulation with 20 ng / mL of recombinant human IFN-γ evaluated by flow cytometry. A, APCKO / KO; N.D., not detected; K, KRASG12D / +: P, P53KO / KO; S, SMAD4KO / KO, WT, wild type.
[0060] [0060] Figure 10. Expression of HLA-A2 in the CRC organoid strains HLA-A2 + and HLA-A2- clonally expanded. The Figure shows a representative plot of multiple repeated experiments. Flow cytometry analysis of HLA-A2 expression in normal strains (left panel), CRC HLA-A2 + (central panel) and CRC HLA-A2– (right panel) with and without stimulation with 20 ng / mL of IFN- recombinant human γ. Figure 11. CRC Organoids as tools for the evaluation of specific antigen extermination by CD8 + T cells
[0061] [0061] Figure 11A. Experimental scheme.
[0062] [0062] Figure 11B. Flow cytometry analysis of HLA-A2 expression in HLA-A2 + and HLA-A2– cloned strains.
[0063] [0063] Figure 11C. Bright field images of CRC organoids co-cultured with specific transgenic T cells from peptide specific T cell receptor WT1 for 48 hours; scale bars: 1 mm.
[0064] [0064] Figure 11D. Images showing HLA-A2 + pulsed peptide CRC organoids at the beginning and end of co-culture with indicated peptide specific T cells; scale bars: 70 µm.
[0065] [0065] Figure 11E. Production of IFN-γ by peptide-specific T cells WT1 (top) and EBV (bottom) as measured by the ELISA of supernatants collected after 18 hours co-culture with HLA-A2 + CRC organoids pulsed with the indicated peptides.
[0066] [0066] Figure 11F. Live cell photographs of an 18-hour coculture experiment with HLA-A2 + CRC organoids pulsed with EBV peptide co-cultured with an EBV-specific T cell clone.
[0067] [0067] Figure 11G. Quantification of CRC organoid extermination by specific T cells. The graphs are representative of multiple repeated experiments with co-cultures of EBV peptide and T cell EBV or peptide WT1 and T cell WT1.
[0068] [0068] Figure 11H. Representative projection image of T cells (blue) infiltrating a CRC organoid pulsed with peptide as recorded during live cell imaging experiments.
[0069] [0069] Figure 11I. Quantification of extermination of CRN organoids treated with IFN-γ by specific T cells in the presence or absence of a blocking antibody against PD-1. The graphs are representative of multiple repeated experiments with cocultures with EBV peptide and T cell EBV or peptide WT1 and T cell WT1.
[0070] [0070] Figure 11J. Quantification of cell viability after 18-hour cocultures of HLA-A2 + organoids with peptide-pulsed or non-pulsed antigen-specific T cells. The plots represent the ratio between peptide-pulsed and non-pulsed-peptide conditions.
[0071] [0071] “Allogeneic” refers to entities (for example, cells,
[0072] [0072] "Approximately" or "about", as used in this application, are equivalent. Any numerals used in this application with or without approximately / approximately are intended to cover any normal fluctuations assessed by the person skilled in the art. As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to an established reference value. In certain modalities, the term "approximately" or "about" refers to a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14% , 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in each direction (greater than or less than) the established reference value unless otherwise established or otherwise evident from the context (except where such number exceeds 100% of a possible value).
[0073] [0073] "Biologically active" refers to a characteristic of any agent that has activity in a biological system and particularly in an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
[0074] [0074] "Co-culture" refers to two or more types of cell maintained in conditions suitable for their mutual growth. In the context of the present description, an "organoid coculture" refers to an epithelial organoid, as defined elsewhere, in culture with a non-epithelial cell type, specifically an immune cell type. In some modalities, cell types in co-culture exhibit a structural, biochemical and / or phenomenological association that they do not exhibit in isolation. In some
[0075] [0075] "Understanding", "understands" and "understanding" will be understood to imply the inclusion of an established step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.
[0076] [0076] "Dose" refers to a specified amount of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose can be administered in two or more cakes, tablets or injections. For example, in certain modalities, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such modalities, two or more injections can be used to achieve the desired dose. In certain embodiments, a dose can be administered in two or more injections to minimize the reaction at the injection site in an individual. In certain embodiments, a dose is administered as a slow infusion.
[0077] [0077] "Immune disease" refers to any disorder of the immune system. Immune diseases typically have a genetic component and include autoimmune diseases (in which the immune system mistakenly acts on the "own" components) and immune-mediated diseases (in which the immune system exhibits excessive function).
[0078] [0078] "Immunotherapy" refers to any medical intervention that induces, suppresses or enhances a patient's immune system for the treatment of a disease. In some embodiments, immunotherapies activate a patient's innate and / or adaptive immune response (eg, T cells) to more effectively target and remove a pathogen or cure for a disease, such as cancer or an immune disease.
[0079] [0079] "Intestine" and "intestinal" refer to the gastrointestinal tract, including the mouth, oral cavity, esophagus, stomach, large intestine, intestine
[0080] [0080] "Organoid" refers to a cell structure obtained by the expansion of adult epithelial stem cells (post-embryonic), preferably distinguished by the expression of Lgr5 and consisting of specific tissue cell types that self-organize through classification of cell and compromise of spatially restricted lineage (for example, as described in Clevers, Cell. June 16, 2016; 165 (7): 1586-1597, see particularly the section called “Organoids derived from adult stem cells” on page 1590 onwards). In the present application, the term "organoid" can be used to refer to normal organoids (for example, non-tumor). Where organoids are described as “disease” organoids, this means that the organoid has a disease phenotype, for example, typically because the organoid was derived from one or more epithelial stem cells having a disease phenotype or in some modalities, because the organoid has been genetically modified to demonstrate particular characteristics of a disease phenotype.
[0081] [0081] "Population" refers to a group of entities sharing common features. In some modalities, “population” refers to patients sharing a set of relevant clinical features. Preferably, a "population" may refer to a group of patients sharing the same cancer and / or being treated with the same agent and / or susceptible to successful treatment with the same agent. A population can differ in one or more characteristics, including specific genotype and / or agent response characteristics during treatment. A population can also refer to a group of cells, organoids and / or co-cultures sharing one or more genotypic, phenotypic or biochemical traits. A “subpopulation” refers to a group of entities sharing a greater number of common traits or a smaller number of dissimilar traits, than a larger population in which
[0082] [0082] "Safe" refers to a treatment for a disease, which has no side effects or only has side effects within a tolerable level according to standard clinical practice.
[0083] [0083] "Side effect" or "deleterious effect" refers to a physiological response attributable to a treatment other than the desired effects.
[0084] [0084] "Subject" or "patient" or "individual" may refer to a human or any non-human animal (such as any mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate ). In preferred embodiments, the patient is a mammal, more preferably a human being. “Human being” can refer to pre- and / or post-natal forms. A subject can be a patient, which refers to a human being who comes to a doctor for diagnosis or treatment of a disease. The term "subject" is used interchangeably with "individual" or "patient." A patient may be afflicted with or is susceptible to a disease or disorder, but may or may not show symptoms of the disease or disorder.
[0085] [0085] "Suffering from" refers to a patient who has been diagnosed with or exhibits one or more symptoms of a disease, disorder and / or condition.
[0086] [0086] "Susceptible to" refers to a patient who has not been diagnosed with a disease, disorder and / or condition. In some embodiments, a patient who is susceptible to a disease, disorder and / or condition may not exhibit symptoms of the disease, disorder and / or condition. In some embodiments, a patient who is susceptible to a disease, disorder, condition or event can be distinguished by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder and / or condition; (3) increased and / or decreased expression and / or activity of a protein associated with
[0087] [0087] "Therapeutically effective amount" refers to an amount of a therapeutic agent that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder and / or condition, to treat, diagnose, prevent and / or delay the onset of symptom (s) of the disease, disorder and / or condition. It will be appreciated by the skilled person that a therapeutically effective amount is typically administered via a dosage regimen comprising at least one unit dose.
[0088] [0088] "Treating", "treating", "treatment" refers to any method used to partially or completely relieve, improve, mitigate, inhibit, prevent, delayed onset, reduce the severity of and / or reduce the incidence of a or more symptoms or traits of a particular disease, disorder and / or condition. Treatment can be administered to a subject who does not show signs of a disease and / or only shows initial signs of the disease for the purpose of decreasing the risk of developing the pathology associated with the disease.
[0089] [0089] "Tumoroid" refers to an organoid comprising cells that exhibit one or more genetic, phenotypic or biochemical traits classified as cancerous. In the present application, the term "tumoroid" encompasses "organoids" derived from cancerous tissue. The term "tumoroid" can also encompass tumor progression organoids (TPOs), which are engineered tumor organoid cultures where a normal organoid has been engineered to contain cancerous mutations, for example, using
[0090] [0090] General. The invention relates to cocultures of organoids and immune cells ('cocultures of organoids') and / or cocultures of disease organoids (such as tumors) and immune cells ('cocultures of disease organoids' or more specifically 'tumoroid cocultures') ) and its use to investigate the physiology of diseases and / or the suitability of candidate agents to treat diseases. The suitability to treat a disease may comprise the effectiveness to treat the disease and / or safety to treat the disease. Diseases of particular interest include cancer and immune diseases.
[0091] [0091] Consequently, the invention provides, among other things, a method for identifying a suitable agent to treat cancer, in which the method comprises: placing a tumoroid co-culture in contact with one or more candidate agents, in which the tumor tumor co-culture comprises immune cells and at least one tumoroid, detect the presence or absence of one or more changes in the tumoral coculture that is indicative of the suitability of a candidate agent to treat cancer, and identify a candidate agent as suitable to treat cancer if the presence or absence of one or more of said changes in the tumoral coculture is detected.
[0092] [0092] A method for testing a therapeutic agent is also provided, in which the method comprises: placing an organoid co-culture in contact with one or more candidate agents, in which the organoid co-culture comprises immune cells and at least one organoid, detecting the presence or absence of one or more changes in the organoid co-culture that is indicative of therapeutic efficacy, and
[0093] [0093] In some embodiments, the organoid is a disease organoid, for example, an organoid exhibiting an immune disease phenotype. Due to the presence of immune cells in the co-cultures of the invention, co-cultures are particularly suitable for investigating the suitability of candidate immunotherapy agents.
[0094] [0094] The methods of the invention have high throughput capacity (HTP). In some embodiments, the methods of the invention can be performed on 96-well plates and / or on 384-well plates.
[0095] [0095] Contact Step. This may involve exposing the organoid co-culture to the therapeutic levels of a known or unknown therapeutic product. Typically, an agent will be dissolved in solution at a therapeutically effective (predicted) concentration and administered to the coculture by injection (or other appropriate administration) into a vessel in which the coculture is maintained.
[0096] [0096] Detection step. In some embodiments, the invention comprises a step of detecting the presence or absence of one or more changes in the tumoral coculture that are indicative of the suitability of a candidate agent for treatment.
[0097] [0097] In principle, any biochemical, genetic, phenotypic or phenomenological change in co-culture can be detected. In some embodiments, the one or more changes may be in one or more disease biomarkers, such as cancer biomarkers. In some embodiments, one or more changes may include a reduction in cell viability, a reduction in cell proliferation, an increase in cell death, a change in cell or organoid size, a change in motility, dissociation or disruption of layer cell
[0098] [0098] In principle, detection can be performed using any suitable laboratory method known to the person skilled. In some embodiments, detecting one or more changes may comprise a cell proliferation assay, a viability assay, flow cytometric analysis, ELISA for IFN-γ (Interferon gamma) (as performed for example, in Figure 8D), expression analysis gene and / or cell imaging.
[0099] [0099] A reduction in cell viability can be detected by the CellTiter Glo Luminescent Cell Viability Assay kit (Promega), intracellular flow cytometric staining for active Caspase 3 (BD) or positive strain for dead cells. The positive strain for dead cells includes non-cellular membrane permeable DNA strains such as NucRed Dead 647 ReadyProb.
[00100] [00100] An increase in cell death can be detected by bright field imaging.
[00101] [00101] Identification step. Identification can comprise identifying a change of a particular magnitude and can be an automated and / or high-throughput process.
[00102] [00102] Comparison step. In some embodiments, the invention may comprise a step of comparing the organoid coculture or tumoroid coculture with a control, which may or may not be associated with the identification step. This may involve comparing the presence or absence or magnitude of one or more changes in the tumor coculture with a reference organoid or reference tumor and may additionally comprise: placing a reference organoid or tumor coculture
[00103] [00103] In some embodiments, a candidate agent is identified as a suitable agent if the presence or absence of a change is detected in the tumor coculture, but not in the reference coculture.
[00104] [00104] In some embodiments, the reference organoid coculture or reference tumoroid coculture is used as a control, such as a negative control or a positive control.
[00105] [00105] Selection step. In some embodiments, the method of the invention comprises a step of selecting a candidate agent as suitable for treating cancer. Selecting is distinct from identifying, since selecting may involve considerations considering the presence or absence or magnitude of one or more changes in the method provided. For example, selecting may comprise additional considerations such as agent bioavailability, applicability to a subpopulation of patient, or agent release mechanisms, which may or may not be tested in the method.
[00106] [00106] In some embodiments, this step may be the final step of the method of the invention. In other modalities, additional steps are considered. For example, the methods of the invention may additionally comprise the step of using the selected candidate agent in the treatment.
[00107] [00107] Agents. Any agent can be tested according to the method of the invention. This includes any biological, chemical, physical or other agent or multiple agents administered concomitantly or in sequence.
[00108] [00108] The agents (or 'candidate agents') are tested for suitability to treat cancer, can be selected from one or more of the following therapeutic classes: immunotherapeutic, tumor-specific peptides, checkpoint inhibitors, alkylating agent, antimetabolite, metabolic agonist, metabolic antagonist, plant alkaloid, mitotic inhibitor, antitumor antibiotic, topoisomerase inhibitor, radiotherapeutic products, chemotherapeutic products, antibodies, photosensitizing agent, stem cell transplantation, vaccine, cytotoxic agent, cytostatic agent, tyrosine kinase inhibitor, proteasome inhibitor, cytokine, interferon, interleukin, intercalating agent, targeted therapy agent, small molecule drug, hormone, steroid, cellular therapeutic product, viral vector and nucleic acid therapeutic product.
[00109] [00109] Preferably, the agents are tumor-specific peptides, checkpoint inhibitors or immunotherapeutic products.
[00110] [00110] The agents are more preferably immunotherapeutic products, for example, therapeutic products of chimeric antigen (CAR) -cell receptor, therapeutic product of transgenic T cell in TCR or neoantigens. Other agents include agents associated with antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP).
[00111] [00111] Context. The methods of the claimed invention can be carried out in vivo, ex vivo, in vitro, in situ, ex situ or any combination thereof. Preferably the methods are carried out in vitro. Personalized medicine
[00112] [00112] General. A means of testing different regimens can be described as a ‘personalized medicine’ approach to testing. A personalized medicine approach may involve testing one or more candidate agents that are of known suitability for the treatment and / or identification of one or more candidate agents as suitable agents
[00113] [00113] Personalized medicine applications of the invention may require that both the tumor tumor coculture and the reference organoid coculture or reference tumor tumor coculture be derived from the particular patient for whom the suitability of candidate agents to treat cancer is being identified.
[00114] [00114] The inventors have shown that it is possible to derive immune cells, normal epithelial cells (for example, non-tumor) and tumor epithelial cells from a single tissue in a single patient and obtain immune organ-cell co-cultures and immune cell-tumor co-cultures from these cells. These co-cultures provide a particularly useful model for testing individual patient's response to candidate agents.
[00115] [00115] A patient for whom a candidate agent has been identified as being suitable for treating cancer, can subsequently be treated with the candidate agent so identified. Screening
[00116] [00116] General. Another means of testing different regimens can be described as a 'screening' approach to testing. A screening approach may involve testing one or more candidate agents that are of unknown suitability for treatment and / or identifying a subset of one or more candidate agents as suitable agents for treatment.
[00117] [00117] The screening applications of the invention may require that the one or more candidate agents are of known suitability to treat a first cancer and unknown suitability to treat a second cancer, with screening comprising identifying a subset of the one or more candidate agents as agents suitable to treat the second cancer.
[00118] [00118] In some modalities, the screening approach identifies
[00119] [00119] Species. Cells, cancers, organoids and / or co-cultures of the invention or suitable for use with the methods of the invention can be mainly of any multicellular organism, preferably a cancer-susceptible multicellular organism. In some embodiments, the cells, cancers, organoids and / or co-cultures of the invention are mammals (meaning derived from mammals), such as murine, primate or human cells, cancers, organoids and / or co-cultures. In a preferred embodiment, the cells, cancers, organoids and / or co-cultures of the invention are human (meaning derived from humans).
[00120] [00120] Epithelial cells. Organoids and / or organoid co-cultures of the invention are obtained from epithelial cells. Organoids and / or organoid co-cultures can be obtained from normal (i.e., non-diseased) skin cells or from diseased epithelial cells (sometimes specifically referred to as 'disease organoids' or 'disease co-cultures'). The tumors and / or tumoral coculture of the invention are obtained from tumor epithelial cells. Any epithelial cell from which an organoid or tumoroid can be generated is suitable for use in the invention. Preferred tumor epithelial cells and / or normal epithelial cells include lung cells, liver cells, breast cells, dermal cells, intestinal cells, crypt cells, rectal cells, pancreatic cells, endocrine cells, exocrine cells, duct cells, kidney cells, cells adrenal glands, thyroid cells, pituitary cells, cells of the
[00121] [00121] In some embodiments, tumor epithelial cells and / or normal epithelial cells are obtained from a sample from a cancer patient. In a particular embodiment, tumor epithelial cells and normal epithelial cells are obtained from samples from the same cancer patient, optionally from the same sample. Samples suitable for obtaining epithelial cells include tissue biopsy, such as ascites from a patient with colorectal or ovarian cancer; urine of a patient with kidney cancer; or biopsy of colon and / or resected rectum tissue from a patient with colorectal cancer.
[00122] [00122] Immune cells. Any immune cell that can be incorporated into a co-culture is suitable for use with the methods of the invention. Preferred immune cells include one or more cell types selected from the group consisting of intraepithelial lymphocytes (IELs), tumor infiltrating lymphocytes (TILs), peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), T cells, cytotoxic T lymphocytes (CTLs), B cells, NK cells, mononuclear phagocytes, α / β receptor T cells and γ / δ receptor T cells. Preferred immune cells also include myeloid-derived suppressor cells.
[00123] [00123] Immune cells can be obtained from established cell lines available in the art (for example, from ATCC or similar libraries of cell lines). Alternatively, immune cells can be purified from an impure sample from a subject. There are advantages associated with obtaining immune cells from the same patient as tumor epithelial cells to derive the tumor from the
[00124] [00124] An impure immune sample from which immune cells can be obtained, can include a tumor sample, normal colonic tissue (non-tumor) and / or peripheral blood. In some embodiments, immune cells are obtained from a sample from a cancer patient. In some embodiments, immune cells are obtained from a peripheral blood sample and / or a tissue biopsy. For example, peripheral blood lymphocytes (PBLs) and / or T cells can be obtained from a peripheral blood sample; or tumor infiltrating lymphocytes (TILs) and / or intraepithelial lymphocytes (IELs) are obtained from biopsy of tumor or healthy tissue, respectively.
[00125] [00125] Immune cells suitable for use in the methods of the invention can be allogeneic with the tumoroid and / or organoid. In some embodiments, the immune cells are matched in HLA with the tumor and / or organoid, that is, the immune cells can be antigenically compatible with the patient from which the tumor and / or organoid are derived (Shiina et al., (2016). MHC Genotyping in Human and Nonhuman Species by PCRbased Next-Generation Sequencing, Next Generation Sequencing - Advances, Applications and Challenges, Dr. Jerzy Kulski (Ed.), InTech, DOI: 10.5772 / 61842) (Choo, Yonsei Med J. February 28, 2007; 48 (1): 11-23).
[00126] [00126] T cell engineering An important aspect of the present invention is the use of engineered T cells, such as chimeric antigen receptor (CAR) T cells (Sadelain et al., Nature. May 24, 2017; 545 ( 7655): 423-431). The invention provides methods and co-cultures that can be used to test the suitability of different types of CAR T cells for different tumor phenotypes and tumor microenvironments. The present invention is an advantageous means of improving the selection process
[00127] [00127] Organoids and tumors. Organoids can be prepared by culturing normal epithelial cells in an organoid culture medium. Tumors can be prepared by culturing tumor epithelial cells in a tumoroid culture medium. Normal epithelial cells can be autologous with tumor epithelial cells (that is, from the same patient). The organoids / tumors of the invention can be distinguished by the expression of Lgr5. In some embodiments, an organoid / tumor is a three-dimensional cell structure. In some embodiments, an organoid / tumoroid comprises a lumen surrounded by epithelial cells. In some embodiments, the epithelial cells surrounding the lumen are polarized. Polarization can be disrupted in tumors. The epithelial cells from which organoids / tumors are obtained are preferably primary epithelial cells.
[00128] [00128] Types of cancer. The methods of the invention are applicable to any cancer. In some modalities, the cancer can be one or more of adenoma, adenomatous polyps, renal carcinoma, adrenal adenoma, thyroid adenoma, pituitary adenoma, parathyroid adenoma, hepatocellular adenoma, fibroadenoma, cystadenoma, bronchial adenoma, adenoma sebacic, prostate adenoma, prostatic adenoma, adenoma cholangiocarcinoma, squamous cell cancer, ductal carcinoma, lobular carcinoma, carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, spindle cell carcinoma, sarcomatoid carcinoma, carcinoma
[00129] [00129] Cancers for which the methods of the invention are particularly applicable include epithelial cancer, such as gastrointestinal or colorectal cancer, pancreatic cancer and breast cancer.
[00130] [00130] Cancer stages. The invention is applicable to cancer at any stage of progression. Cancer progression can be distinguished in several systems. The TNM system (Tumor, Nodule, Metastasis) comprises three categories, each designated with a numerical degree. T refers to the size of the cancer and how far it has spread in the surrounding tissue - it can be 1, 2, 3 or 4, with 1 being small and 4 being large. N refers to whether the cancer has spread to the lymph nodes - it can be between 0 (none of the lymph nodes containing cancer cells) and 3 (large amounts of lymph nodes containing cancer cells). M refers to whether the cancer has spread to another part of the body - it can be 0 (the cancer has not spread) or 1 (the cancer has spread). A second system is the Numerical Staging System, which comprises four stages. Stage 1 usually means that a cancer is relatively small and contained within the organ within which it started. Stage 2 usually means that the cancer has not started to spread within the surrounding tissue, but the tumor is larger than in stage 1. Sometimes stage 2 means that the cancer cells have spread in the lymph nodes near the tumor. This depends on the particular type of cancer. Stage 3 usually means that the cancer is bigger. It may have started to spread
[00131] [00131] Certain agents tested in the methods of the invention, such as immunotherapy, are more relevant in the later (metastatic) stages of cancers such as colorectal cancers, because frequent surgical resection is sufficient when no metastasis is present. Consequently, the invention is applicable to cancer at or below a Stage III, Grade III or T2 N1 M1.
[00132] [00132] For other cancers that are less easily resected surgically, immunotherapy may also be relevant in the early stages. In addition, the use of the invention in tumor progression organoids (TPOs) also allows investigation of treatments for cancers in the early stages. Consequently, the invention is applicable to cancer at or below a Stage II, Grade II or T2 N1 M0.
[00133] [00133] Immune diseases. In addition to cancers, immune cell diseases can also be investigated using the methods of the invention. In principle, any immune system disorder that affects immune cells can be investigated in coculture. Preferred immune diseases include immune diseases of the digestive and respiratory systems, especially the intestines and lungs. Exemplary immune diseases include irritable bowel disease (IBD), ulcerative colitis (UC), obstructive lung disease
[00134] [00134] When testing for immune disorders using the methods of the invention, organoids can be separately cultured with sick immune cells and immune cells from a healthy control patient.
[00135] [00135] Biopsies and sample acquisition. Organoid and / or tumoroid samples can be obtained during surgery of normal mucosal and tumor tissue, for example taken from resected colon, rectum, small intestine and / or ileum from patients with colorectal cancer and / or healthy control patients. Immune cells can be derived from peripheral blood collected during surgery. Organoids, tumors and cocultures
[00136] [00136] Preparation of coculture of tumoroid. In one aspect, the invention provides a method for preparing a tumoroid cell-immune coculture. The method comprises the step of mixing a tumoroid as described herein with immune cells in an in vitro culture. The mixture can comprise sequential layers of T cells and organoids in the same well in a multi-well plate or can comprise sequential pipetting of T cells and organoids in a gel. In a preferred embodiment, the tumoroid co-culture is maintained in a co-culture medium as described herein.
[00137] [00137] In some embodiments, the method for preparing the tumor-cell-immune co-culture further comprises one or more of the following preparation steps: preparing at least one tumoroid by culturing tumor epithelial cells in a tumoroid culture medium; and / or preparing immune cells by culturing the immune cells in an immune cell expansion medium.
[00138] [00138] In a preferred embodiment, the tumor culture medium (optionally including any extracellular matrix) is removed from the hair
[00139] [00139] In some embodiments, the method additionally comprises the step of obtaining the immune cells from an impure immune sample. Methods for isolating immune cells from impure immune samples are known in the art. Exemplary methods for isolating lymphocytes from single cell suspensions and T cell expansion cultures are described in Example 5.
[00140] [00140] The invention provides a coculture of tumoroid-immune cell obtained by the above method. The invention also provides uses of said immune tumor-cell co-culture in drug screening, toxicology screening, drug research and development.
[00141] [00141] The tumoral coculture can be ex situ, ex vivo and / or in vitro. It is preferably in vitro.
[00142] [00142] Preparation of coculture of organoids. In one aspect, the invention provides a method for preparing an organoid-immune cell co-culture. The method comprises the step of mixing an organoid as described herein with immune cells in an in vitro culture. In a preferred embodiment, the organoid co-culture is maintained in a co-culture medium as described herein.
[00143] [00143] In some embodiments, the method for preparing the immune cell-organoid co-culture comprises one or more of the following steps: preparing the at least one organoid by culturing normal epithelial cells in an organoid culture medium; and / or culturing immune cells in an immune cell expansion medium.
[00144] [00144] In a preferred embodiment, the organoid culture medium (optionally including any extracellular matrix, such as basal membrane matrix 'BME' or matrigel) is removed from at least one organoid before mixing at least one organoid with the immune cells. The extracellular matrix can be disrupted using commercially available kits, such as Cell Recovery Solution® (Corning). An alternative matrix, such as collagen, can be used in place of the removed matrix.
[00145] [00145] In some embodiments, the method additionally comprises the step of obtaining the immune cells from an impure immune sample. Methods for isolating immune cells from impure immune samples are known in the art. Exemplary methods for isolating lymphocytes from single cell suspensions and T cell expansion cultures are described in Example 5.
[00146] [00146] The invention also provides an immune cell-organ co-culture obtained by the above method. The invention also provides uses of said immune organ-cell co-culture in drug screening, toxicology screening, drug research and development.
[00147] [00147] The organoid co-culture can be ex situ, ex vivo and / or in vitro. It is preferably in vitro.
[00148] [00148] Primary analysis. In some embodiments, the methods of the invention additionally comprise one or more stages of primary analysis. The primary analysis of tumors and / or organoids may comprise entire genomic sequencing, mRNA sequencing, peptidoma profiling and / or microscopy. Primary analysis can be used to ensure that tumors and / or organoids are uniform and / or meet expectations, in a form of information disclosure and / or information verification. For example, primary analysis can be used to determine differences in mRNA transcription between organoids and
[00149] [00149] Immune cells can be subjected to one or more stages of primary analysis. For example, primary analysis of immune cells can comprise immunophenotyping and / or T cell receptor sequencing. Primary analysis can be used to check whether CAR T cells express the receptor needed to recognize tumor cells. The overloading of specific receptors recognizing the tumor can also be investigated.
[00150] [00150] In a particular embodiment, the methods of the invention comprise a step of determining the HLA type of the cells, organoids or tumoroids.
[00151] [00151] Co-cultures can also be submitted to one or more stages of primary analysis. The primary analysis of the tumoral coculture and / or organoid coculture can comprise image analysis, flow cytometric analysis and / or analysis of cytokine secretion. Primary analysis can be used to ensure that co-cultures are uniform and / or meet expectations.
[00152] [00152] Source of tumors and organoids. The tumors and / or organoids of the invention may comprise or consist of autologous cells, i.e., cells obtained from the same patient. For example, the tumoroid can be obtained by culturing a tumor cell (for example, a colorectal cancer cell), whereas the organoid can be obtained by culturing a normal (non-tumor) cell from the same tissue in the same patient. (for example, a normal colon cell). This can be particularly useful in
[00153] [00153] The invention also provides tumoroids and / or organoids in a medium comprising an interleukin, such as IL-2, IL-7 or IL-15. In some embodiments, the at least one tumoroid or at least one organoid comprises or consists of mammalian cells, preferably human cells.
[00154] [00154] Tumor and organoid separation. In some modalities, tumors and / or organoids are separated into populations sharing one or more genotypes, phenotypes and / or epigenetic markers, before mixing with immune cells. Preferably, genotypes, phenotypes and / or epigenetic markers contribute to the interaction between (i) the tumoroid and / or organoid and (ii) the immune cells.
[00155] [00155] Separate tumor or organoid populations may share the presence or absence of an HLA haplotype, for example an HLA haplotype such as HLA-A2.
[00156] [00156] This separation step can allow relevant patient groups and subgroups to be determined. Means
[00157] [00157] Immune cell culture media. The immune cell culture medium can be used to prepare immune cells for coculture, for example, by facilitating the growth and division (expansion) and / or differentiation of immune cells to produce a population suitable for coculture.
[00158] [00158] In a preferred embodiment, the immune cell culture medium comprises an interleukin. In some embodiments, interleukin is selected from IL-2, IL-7 and IL-15. In a preferred embodiment, the interleukin in the immune cell culture medium is IL-2.
[00159] [00159] In some modalities the interleukin concentration is from 2000 to 6000 IU / mL. A preferred concentration of IL-2 in the culture medium
[00160] [00160] The immune cell culture medium may additionally comprise an RPMI medium (for example, RPMI 1640, Gibco), optionally supplemented with penicillin / streptomycin and / or hepes and / or glutaMAX® and / or sodium pyruvate and / or serum (eg 5% human AB serum, Sigma-Aldrich). In principle, any mammalian basal cell culture medium can be used in place of the RPMI medium, such as DMEM / 12.
[00161] [00161] Organoid and tumoroid media. Tumor growth media and organoid culture media can be used to prepare organoids and tumors for co-culture, for example, by facilitating growth, division (expansion), structural organization or other development to produce a suitable tumoroid and / or organoid for coculture.
[00162] [00162] Suitable tumor tumor culture media and organoid culture media for different tissues are known in the art (for example, Clevers, Cell. June 16, 2016; 165 (7): 1586-1597). Preferred organoid / tumoroid culture media comprise a Wnt agonist (for example, any of R-spondin 1-4), a mitogenic growth factor (for example, selected from EGF, FGF, HGF and BDNF) and a BMP inhibitor (for example, Noggin) (for example, as described in WO2010 / 090513). In some embodiments, the organoid / tumoroid culture medium further comprises a TGF-beta inhibitor (for example, A83-01, Tocris) (for example, as described in WO2012 / 168930). The addition of a TGF-beta inhibitor is particularly suitable for culturing human cells. The TGF-beta inhibitor preferably inhibits the ALK4 / 5/7 signaling pathway.
[00163] [00163] In some embodiments, certain components of culture medium are optional for the tumor culture medium, because certain
[00164] A preferred organoid culture medium, which is particularly suitable for colonic organoid culture, comprises one or more (or preferably all) of a basal medium (such as DMEM / F12 Advanced, Gibco medium) and a Wnt linker (such as such as Wnt-3a), a Wnt agonist (such as any of Respondina 1-4), a BMP inhibitor (such as Noggin), EGF and a TGF-α inhibitor (such as A83-01, Tocris) and optionally it further comprises one or more (or all) of a MAPK p38 inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or an antimicrobial (such as primocin).
[00165] [00165] A preferred tumor culture medium, which is particularly suitable for colon cancer tumor culture, comprises one or more (or preferably all) of a basal medium (such as DMEM / F12 Advanced, Gibco medium) an agonist Wnt (such as any of Respondina 1-4), a BMP inhibitor (such as Noggin), EGF and a TGF-α inhibitor (such as A83-01, Tocris) and optionally further comprises one or more (or all) of a MAPK p38 inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or an antimicrobial (such as primocin). The tumor culture medium can optionally comprise a Wnt ligand (such as Wnt-3a), which is especially useful for colorectal tumors most sensitive to immune therapy (for example, MSI tumors that normally lack Wnt path mutations) .
[00166] [00166] In some embodiments, tumors or organoids are grown in an immune cell expansion medium or a mixture of
[00167] [00167] The respondent is aware of the culture media specific to other types of organoids and tumors and can adapt the invention for use with other organoids and tumors accordingly.
[00168] [00168] Means of co-culture. The invention provides means (for example, as described in the examples) for the coculture of tumoroids and immune cells. The invention also provides means (for example, as described in the examples) for the coculture of organoids and immune cells. Any of the immune cell culture media or the tumoroid / organoid culture medium described above can be used as a co-culture medium for the culture of the immune-organoid / tumor cell cell culture.
[00169] [00169] The co-culture means of the invention advantageously allow the co-culture of immune and organoid / tumor cells. In the case of tumors, such co-culture is difficult or even impossible without using the adaptations of means used in the co-culture means of the invention. The inventors have observed for the first time that the media for co-culture between tumoroids and immune cells benefit from a reduced Wnt component (in relation to the organoid culture medium), to preserve the immune cell function. This can be achieved by co-culturing in 100% immune cell culture medium or in a mixture of immune cell culture medium and organoid / tumoroid culture medium. The same means can be used for the coculture of organoids and immune cells, although a reduced Wnt component is not so beneficial for the coculture of organoids.
[00170] [00170] Consequently, in some embodiments, the co-culture medium comprises part of the immune cell culture medium (for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% at least 60%, at least 70%, at least 80% or at least 90%) and part of the organoid / tumor cell culture medium (for example
[00171] [00171] In some embodiments, an immune cell culture medium (such as a T cell medium, for example, RPMI 1640 (Gibco)) is used for the co-culture medium. This culture medium is particularly useful for supporting the maintenance of immune cells in coculture, particularly for human immune cells. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the medium coculture culture consists of an immune cell culture medium.
[00172] [00172] Extracellular matrix. The cells are preferably grown in a microenvironment that mimics at least in part a cell niche in which said cells naturally reside. A cell niche is partly determined by cells and an extracellular matrix (ECM) that is secreted by cells in said niche. A cell niche can be imitated by cultivating said cells in the presence of biomaterials or synthetic materials that provide interaction with cell membrane proteins, such as integrins. An extracellular matrix as described herein is, therefore, any biomaterial or synthetic material or combination thereof that mimics the cell niche in vivo, for example, by interacting with cell matrix proteins, such as integrins. Any suitable extracellular matrix can be used.
[00173] [00173] In a preferred method of the invention, cells are
[00174] [00174] In other modalities, the ECM is in suspension, that is, the cells are in contact with the ECM in a suspension system. In some embodiments, ECM is in suspension at a concentration of at least 1%, at least 2% or at least 3%. In some embodiments, ECM is in suspension at a concentration of 1% to about 10% or 1% to about 5%. The suspension method can have advantages for sophisticated methods.
[00175] [00175] One type of ECM is secreted by epithelial cells, endothelial cells, parietal endoderm cells (for example, Englebreth Holm Swarm Parietal Endoderm Cells described in Hayashi et al. (2004) Matrix Biology 23: 47-62) and connective tissue cells. This ECM comprises of a variety of polysaccharides, water, elastin and glycoproteins, where the glycoproteins comprise collagen, entactin (nidogen), fibronectin and laminin. Therefore, in some embodiments, the ECM for use in the methods of the invention comprises one or more of the components selected from the list: polysaccharides, elastin and glycoproteins, for example, where the glycoproteins comprise collagen, entactin (nidogen), fibronectin and / or laminin. For example, in some modalities, collagen is used as the ECM. Different types of ECM are known, comprising different compositions including different types of glycoproteins and / or different combination of glycoproteins.
[00176] [00176] Examples of commercially available extracellular matrices include: extracellular matrix proteins (Invitrogen) and Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell-based membrane preparations (for example, Cultrex® basement membrane extract (Trevigen, Inc.) or Matrigel® (BD Biosciences)).
[00177] [00177] In some embodiments the ECM is an ECM containing laminin such as Matrigel® (BD Biosciences). In some modalities, ECM is Matrigel® (BD Biosciences), which comprises laminin, entactin and collagen IV. In some embodiments, ECM comprises laminin, entactin, collagen IV and proteoglycan heparin sulfate (for example, Basement membrane extract Cultrex® Type 2 (Trevigen, Inc.)). In some embodiments, ECM comprises at least one glycoprotein, such as collagen and / or laminin. Mixtures of naturally produced or synthetic ECM materials can be used, if desired. In some embodiments, ECM is BME (‘basement membrane extract’), which is a soluble form of base membrane purified from the Engelbreth-Holm-Swarm (EHS) tumor (for example, Cultrex® BME).
[00178] [00178] In another modality, the ECM can be a synthetic ECM. For example, a synthetic ECM, such as ProNectin (Sigma Z378666) can be used. In a further example, the ECM can be a plastic, for example, a polyester or a hydrogel. In some embodiments, a synthetic matrix can be coated with biomaterials, for example, one or more glycoproteins, such as collagen or laminin.
[00179] [00179] A three-dimensional ECM supports the cultivation of three-dimensional epithelial organoids. The extracellular matrix material will normally be a drop at the bottom of the plate in which the cells are suspended. Typically, when the matrix solidifies at 37 ° C, the medium is added and spread over the ECM. The cells in the medium stick to the ECM by interacting with its surface structure, for example interaction with integrins.
[00180] [00180] The culture medium and / or cells can be placed in, embedded in or mixed with ECM.
[00181] [00181] Preferred ECM's for growing tumor / organoids include BME and Matrigel.
[00182] [00182] A preferred ECM for growing co-cultures is collagen, such as rat tail collagen I. Rat tail collagen I has been shown to improve immune cell motility during co-culture - see Example
[00183] [00183] Interleukin. Co-culture media can comprise an interleukin (IL), optionally in which the interleukin or one or more of IL-2 (in a concentration of 100 to 200 IU / mL), IL-7 (in 10 to 100 ng / mL) and IL-15 (at a concentration of 10 to 100 ng / ml). A preferred concentration of interleukin used in co-culture media is 25 µM. These concentrations for coculture contrasts with the concentrations of IL used in expansion, which are higher (for example, IL-2 is used in a concentration of 2000 to 6000 IU / mL for immune cell expansion).
[00184] [00184] IL-2 is the preferred interleukin for use with tumor-associated immune cells. For other immune cells or diseases, such as irritable bowel syndrome (IBD) or ulcerative colitis (UC), IL-7 and / or IL-15 are preferred (Rabinowitz et al., Gastroenterology. Mar 2013; 144 (3) : 601-612.e1).
[00185] [00185] In some embodiments, the tumor-growing medium and / or organoid-growing medium comprises a mixture of (a) the immune cell expansion medium and (b) the tumor-growing medium or culture medium organoid, optionally in which the media are present in a ratio of 50:50 (v / v).
[00186] [00186] Motility and protein concentration. In some modalities, co-culture and / or co-culture medium advantageously confers
[00187] [00187] The inventors have shown that the greatest improvements in motility are seen using collagen, particularly rat tail collagen I. In particular, immune cells (eg, T cells) in BME-based media exhibit an average path length of 43.635 µm, while immune cells (eg, T-cells) in media based on tail I collagen. rat exhibit an average trail length of 135.08 µm. This is a 3-fold increase in motility. The co-culture medium can comprise a protein concentration of at least 0.15 mg / (ml of Matrigel®) to 0.95 mg / (ml of Matrigel®) for a medium comprising 2% to 10% Matrigel®.
[00188] [00188] In some embodiments, at least 20%, at least 30%, at least 40% or at least 50% of the immune cells in a coculture are capable of moving a distance of at least 200 µM, at least 250 µM, at least least 300 µM, at least 350 µM or at least 400 µM in 80 hours, as determined using the test in Figure 3 and Example 10.
[00189] [00189] Persistence and duration of activity. In some embodiments, the means of the invention allows immune cells to persist in the immune cell expansion medium for at least 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours or 240 hours.
[00190] [00190] In some embodiments, the means of the invention allow the immune cells to remain active for at least 4 hours, 8 hours, 12 hours, 24 hours, 48 hours or 72 hours after the formation of coculture (that is, after the point of mixing the immune cells with organoid / tumoroid cells).
[00191] [00191] In some embodiments, the means of the invention allows the tumoral cocultures to persist in the tumor coculture medium or the reference organoid coculture or the reference tumoroid coculture to persist in the organoid coculture medium for at least 4 hours , 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours or 240 hours. In some modalities, co-cultures can persist for 10 days or more or for as many days as the co-culture can remain in culture without being exchanged.
[00192] [00192] The activity of immune cells can be detected according to cell morphology (for example, the absence of a round shape and the presence of cell projections indicate that the cells remain active).
[00193] [00193] Legal notice. In some embodiments, IL-2 is not used in any medium of the claimed invention. Additional Methods and Products of the Invention
[00194] [00194] Kits. The invention provides kits comprising any organoid, tumoroid or co-culture of the invention.
[00195] [00195] In some embodiments, the kit comprises one or more of the following: syringe, alcohol swab, cotton ball, gauze pad, instructions for carrying out the methods of the invention. EXAMPLES
[00196] [00196] Other features, objectives and advantages of the present invention are evident in the examples that follow. It should be understood, however, that the examples, while indicating the modalities of the present invention, are given by way of illustration only, not by limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples. The invention is exemplified using tumoroids as disease organoids, but it is expected that other disease organoids, particularly disease organoids referring to immune diseases, would be used in the same way.
[00197] [00197] The following media are used in the Examples: Human colonic organoid media.
[00198] [00198] Complete DMEM / F12 Advanced Medium (Gibco®) supplemented with 50% WNT3A conditioned medium (internal), 20% R-spondin-1 conditioned medium (internal), 10% Noggin conditioned medium (internal), 1 × B27 supplement (Gibco®), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 50 ng / mL human epidermal growth factor (EGF; Peprotech), 10 nM gastrin (Sigma-Aldrich), 500 nM TGF-β A-83-01 inhibitor (Tocris), 3 μM MAPK SB202190 p38 inhibitor (Sigma-Aldrich), 10 nM prostaglandin E2 (Tocris) and 100 mg / mL of Primocin (InvivoGen). Tumor medium of human colorectal cancer.
[00199] [00199] Complete DMEM / F12 Advanced medium supplemented with 20% R-spondin-1 conditioned medium, 10% Noggin conditioned medium, 1 × B27 supplement without vitamin A (Gibco®), 1.25 mM N-acetylcysteine , 10 mM nicotinamide, 50 ng / mL human EGF, 10 nM gastrin, 500 nM TGF-β A-83-01 inhibitor, 3 μM MAP38 SB202190 p38 inhibitor, 10 nM prostaglandin E2 and 100 mg / 100 mg / mL of Primocin. Human T cell medium.
[00200] [00200] RPMI1640 (Gibco®) supplemented with penicillin / streptomycin, 5% human AB serum (Sigma-Aldrich). Ijssel middle.
[00201] [00201] IMDM supplemented with penicillin / streptomycin, 1% human AB serum (Sigma-Aldrich), bovine serum albumin, insulin, oleic acid, linoleic acid, transferrin and ethanolamine (all Sigma-Aldrich).
[00202] [00202] In the following examples, the generation and characterization of
[00203] [00203] This example shows the isolation of cell samples, which are used for the preparation of organoid, tumoroid and immune cell samples in subsequent examples.
[00204] [00204] Biopsies of normal colonic mucosa and tumor tissue are collected from the resected colon and / or rectum of patients with colorectal cancer. Peripheral blood is also collected during surgery.
[00205] [00205] Specifically, biopsies of human colorectal cancer tissue as well as normal human (adult) colon mucosal epithelium were collected in 50 mL of canonical tubes containing 10 to 15 mL of frozen DMEM / F12 Advanced medium supplemented with Penicillin / Streptomycin (100 × stock 10,000 U / mL Penicillin and 10K μM / mL Streptomycin), HEPES (100 × 1 M stock), GlutaMAX (100 × stock; all from Gibco®) and Rho kinase Y inhibitor -27632 (Sigma-Aldrich). Biopsies kept on ice and immediately processed or can be stored for up to 24 hours at 4 ° C until isolation begins.
[00206] [00206] The process is shown schematically in Figure 1A. Example 2. Isolation of crypts from normal colon tissue and derivation of normal colonic organoids; isolation of intraepithelial T cells from normal colonic tissue for T cell culture
[00207] [00207] This example shows the processing of normal colon samples, for the development of organoid cultures, as well as for the isolation of immune cells from normal colon samples.
[00208] [00208] The normal colonic mucosa is treated with EDTA to release the follicles for the derivation of normal colonic organoid, after
[00209] [00209] The muscle layer and fat using surgical scissors and forceps are removed under a dissecting microscope. The clean tissue is cut into thin strips approximately 1 to 2 mm. One strip is fixed in 4% formaldehyde (Sigma-Aldrich) for histological analysis and one strip is quickly frozen (in dry ice or liquid nitrogen) and stored at - 80 ° C for gene and / or protein analysis. The remaining strips were washed 3 times with fresh chelation solution (5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM sucrose and 54, 9 mM D-sorbitol dissolved in sterile water; all from Sigma-Aldrich). The washed strips were incubated in a chelation solution supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA; internal) and 0.5 mM DL-dithiothreitol (DTT; Sigma-Aldrich) for 30 minutes at 4 ° C on a rotating wheel (room cold). The tubes were vigorously shaken to release the colonic crypts outside the mesenchyme. If no crypt was visible, the incubation was repeated with fresh, complete chelation solution. Tissue fragments were allowed to settle for 1 to 2 minutes and the supernatant containing the follicles was transferred to a new tube. 5 to 10 ml of fetal calf serum (FCS; Sigma-Aldrich) was added and the follicles were centrifuged at 300 × g for 5 minutes at 4 ° C. The remaining tissue fragments were kept on ice for the isolation of intraepithelial T cells. The crypts were washed 3 times in complete DMEM / F12 Advanced. The crypts were resuspended in a basement membrane extract (BME; Cultrex®) and plated at different densities and placed for 30 minutes in a humidified incubator at 37 ° C and 5% CO2. On solidification of BME inhibitor Rho kinase Y-27632
[00210] [00210] Subsequently, the organoid culture underwent primary analysis using whole genome sequencing, mRNA sequencing and peptidoma profiling. Isolation of intraepithelial T cells from normal colon tissue for T cell culture.
[00211] [00211] The tissue fragments kept from the isolation of the colonic crypt were placed inside a Petri dish and cut into very thin pieces (<1 mm) using forceps, scissors and scalpel. The tissue fragments were transferred into a 50 ml canonical tube and washed 3 times in 20 ml of RPMI 1640 medium (Gibco®) supplemented with 10% FCS and Penicillin / Streptomycin to remove any remaining EDTA and inhibit. The medium was removed with a pipette after the pieces of tissue had settled to the bottom of the beaker. The tissue pieces were then incubated in 10 ml of RPMI 1640 medium containing 1 mg / ml of collagenase 1A, 10 U / ml of DNase I (all from Sigma-Aldrich) and Rho kinase Y-27632 inhibitor for 1 hour at 37 ° C ° C under agitation. 2 mL of FCS was added to the cell suspension and the entire suspension was filtered through a 100 μm cell sieve. The single cell suspension was centrifuged at 300 × g for 5 minutes at 4 ° C. The supernatant was removed and the cell pellet was washed twice in complete RPMI 1640 medium. The single cell suspension was cryopreserved in liquid nitrogen in a freezing medium (Recovery® Cell Culture Freezing Medium or 10% DMSO in a 1: 1 mixture of FCS and DMEM / F12 Advanced, all from Gibco®) or additionally processed for T cell culture. Example 3. Digestion of colorectal cancer tissue for organoid tumor and T cell cultures; derivation of colorectal cancer tumors.
[00212] [00212] This example shows the processing of cancerous colon samples, for the development of tumoral cultures, as well as the isolation of immune cells from cancerous colon samples.
[00213] [00213] Tumor tissue is digested to make a single cell suspension containing epithelial tumor cells for tumor derivation as well as tumor infiltrating lymphocytes (TILs) for T cell cultures. Colorectal cancer tissue digestion for tumor cultures and T cell.
[00214] [00214] Tumor biopsies were cut into thin strips approximately 1 to 2 mm. One strip is fixed in 4% formaldehyde for histological analysis and each strip is quickly frozen (in dry ice or liquid nitrogen) and stored at –80 ° C for gene and / or protein analysis. The remaining strips were further cut using forceps until the tumor mass appeared viscous. The tumor mass was incubated and 10 ml of complete DMEM / F12 Advanced medium containing 1 mg / ml of Collagenase II, 10 μg / ml of hyaluronidase and Rho kinase Y-27632 inhibitor for 1 hour at 37 ° C under agitation. After incubation, 2 ml of FCS was added to the tumor mass slurry and the cell suspension was filtered through a 100 μm cell sieve and centrifuged at 300 × g for 5 minutes at 4 ° C. The supernatant was removed and the cell pellet was washed twice in complete DMEM / F12 Advanced medium. The single cell suspension was cryopreserved in liquid nitrogen in a freezing medium (Gibco® Recovery® Cell Culture Freezing Medium or 10% DMSO in a 1: 1 mixture of FCS and DMEM / F12 Advanced) or further processed to the derivation of colorectal cancer tumors and T cell culture. Derivation of colorectal cancer tumors.
[00215] [00215] A fraction of the single cell tumor suspension was resuspended in BME and plated at different dilutions. BME
[00216] [00216] Bright field microscopy was performed for analysis and confirmed successful single cell suspension of organoid and tumoroid samples. The bright-field images representative of normal organoids and colonic tumors derived from patient samples are shown in Figure 1B.
[00217] [00217] As described above, the colonic crypts were embedded within normal colonic organoid medium (basement membrane extract (BME) and cultured with medium containing R-spondin-1, Noggin, conditioned medium Wnt3A, supplement B27 without vitamin A, nicotinamide , N-acetylcysteine, EGF, TGF-β A-83-01 inhibitor, gastrin, MAP38 SB202190 p38 inhibitor and prostaglandin E2). The normal colonic organoids developed within 1 week and were changed weekly thereafter (top panel).
[00218] [00218] Single cell suspensions of colorectal cancer samples were embedded within basement membrane extract (BME) and cultured with medium containing tumor medium (R-spondin-1, Noggin conditioned medium, B27 supplement without vitamin A, nicotinamide , N-acetylcysteine, EGF, TGF-β A-83-01 inhibitor, gastrin, MAPK SB202190 p38 inhibitor and prostaglandin E2). The tumors formed within 1 week and were changed weekly thereafter (background panel).
[00219] [00219] As can be seen in each panel of Figure 1B, the single cell suspension of organoid, tumoroid and immune cells well resolved is obtained.
[00220] [00220] This example shows the further processing of immune cells, followed by the generation of immune cell expansion cultures.
[00221] [00221] 5 mL of pure Ficoll-Paque PLUS (GE Healthcare) was added to the 15 mL canonical tubes. Single cell suspensions obtained from digestions of normal colon or colorectal cancer tissue were resuspended in 5 mL of complete RPMI 1640 medium and carefully placed on top of the clear Ficoll-Paque PLUS layer. The samples were centrifuged at 800 × g for 20 minutes at room temperature. The cells from the layer above the clear Ficoll-Paque PLUS layer containing T cells were collected, resuspended in 10 mL of complete RPMI 1640 medium and centrifuged at 300 × g for 5 minutes. The cell pellet was resuspended in complete RPMI 1640 medium and counted. The single cell suspension was cryopreserved in liquid nitrogen in a freezing medium (Gibco® Recovery® Cell Culture Freezing Medium or 10% DMSO in a 1: 1 mixture of FCS and DMEM / F12 Advanced) or immediately used for expansion cultures . For T cell expansion cultures, lymphocytes were grown on cell culture plastic coated with anti-CD28 (Miltenyi) in a concentration of 1 × 106 total viable cells in 1 mL of RPMI 1640 medium supplemented with Penicillin / Streptomycin, 5 % human AB serum and 6000 IU of recombinant human IL-2 (Miltenyi) in a humidified incubator at 37 ° C and 5% CO2. The medium was renewed after 1 week.
[00222] [00222] In addition or alternatively, peripheral blood is processed to purify peripheral blood mononuclear cells enriched for peripheral blood lymphocytes (PBLs) and T cells.
[00223] [00223] Primary analysis is performed by sequencing the T cell receptor (TCR) and immunophenotyping the T cells (according to Figure 1C and
[00224] [00224] Figure 1C shows bright field images representative of clonal growth of intraepithelial lymphocytes (IELs) and tumor infiltrating lymphocytes (TILs) derived from patient samples (left panels).
[00225] [00225] Flow cytometry analysis shows robust expansion of CD4 + helper T cells (Th) and CD8 + cytotoxic T cells (CTLs). Single cell suspensions of normal colonic mucosa or colorectal cancer tissue were maintained in T cell medium containing interleukin-2 (IL-2). Clonal growth of T cells was noticeable within 1 to 2 weeks (left panels).
[00226] [00226] Consequently, analysis of isolated immune cells reveals that the immune cells remain functional and biologically representative. Example 7. Passage of organoids and epithelial tumors.
[00227] [00227] This example demonstrates the maintenance of organoid and tumoroid cultures.
[00228] [00228] Organoid cultures were disrupted ('divided') by pipetting BME drops up and down the growth medium using a 1 ml volume micropipette (ie P1000 Gilson). The disrupted organoids were centrifuged at 500 × g for 5 minutes. The pelleted organoids were resuspended in TrypLE (Gibco®) and incubated for 5 to 15 minutes at 37 ° C in a water bath. The organoids were dissociated into single cells using pre-moistened flame-polished glass Pasteur pipettes.
[00229] [00229] The dissociated organoids were collected in an excess of complete DMEM / F12 Advanced and centrifuged at 500 × g for 5 minutes. Single epithelial cells were replanted in BME at a density
[00230] [00230] Primary analysis is performed by sequencing single cell messenger RNA (mRNA) from cells present in single cell suspensions of normal colonic epithelium and tumor epithelium. Example 8. Generation of organoid cocultures and tumoroid cocultures.
[00231] [00231] This example demonstrates the coculture of organoids and tumors of Example 5 with immune cell cultures of Example 4.
[00232] [00232] In the division, (as in Example 7 above), 5000 cells were plated in BME and cultured for 3 to 4 days in human colon medium or human colorectal cancer tumor medium. In culture, the medium was removed and drops of BME / Matrigel® were ruptured using Cell Recovery Solution® (Corning) after 25 minutes of incubation on ice. The cells are subsequently centrifuged (5 minutes at 500 × g) and resuspended in T cell medium supplemented with 100 IU / ml of recombinant human IL-2 before mixing with T cells.
[00233] [00233] T cells were counted and brought to a concentration of 100,000 cells / ml in complete T cell medium supplemented with 100 IU / ml of recombinant human IL-2. 100 μL of epithelial cancer tumor suspension was mixed with 100 μL of T cell suspension in a 96-well plate. 22 μL of rat tail collagen (Gibco®) was dissolved in the mixture to achieve a concentration of 10% collagen in the suspension. The cells were rested at 37 ° C and 5% CO2 for 30 minutes to allow the cells and collagen to settle before analysis.
[00234] [00234] A co-culture of proof of principle of normal colonic organoids and allogeneic CD3 + T cells in drops of basement membrane extract (BME) is shown in Figure 2.
[00235] [00235] Figure 2A shows a schematic of the procedure. As described above, normal colonic organoids were released from the BME drop using Cell Recovery Solution and washed in completed DMEM / F12 Advanced. The expanded CD3 + T cells were harvested from the culture and labeled with green dye (Vybrant CFDA SE Cell Tracer). The colonic organoids and labeled T cells were mixed in human colonic organoid medium and embedded in the drop and BME. The cocultures were kept in human colonic organoid medium containing IL-2 for 60 h. Cocultures were released from BME using Cell Recovery Solution and fixed in 4% paraformaldehyde. The entire fixed assemblies were dyed with Phalloidin to mark the polymerized actin and DAPI to label the nuclei. Whole mounts were mounted on a slide in ProLong Gold antismaking mounting medium and imaged under a Leica SP8X confocal microscope.
[00236] [00236] The maximum projection of z-cell images of colonic organoid co-cultures is shown in Figure 2B. Organoid F-actin is labeled in dark gray and T cells are labeled in light gray. The insert in the right panel shows a T cell infiltrating the colonic epithelium.
[00237] [00237] A three-dimensional reconstruction of a normal colonic organoid and T cells is shown in Figure 2C.
[00238] [00238] As seen in the Figure, the organoid shows the expected level of structural organization and interacts with immune cells with noticeable similarity to an in vivo system. Example 9. Analysis of cocultures by imaging, flow cytometry and cytokine secretion.
[00239] [00239] This example analyzes co-cultures of organoids and co-cultures of
[00240] [00240] Image analysis is used to determine the percentage of cell staining in cocultures.
[00241] [00241] Prior to culture, T cells were labeled with cell tracer dye (eg CFSE, Molecular Probes®). The organoids were labeled with directly conjugated anti-mouse EPCAM antibodies (BD Bioscience) or cell tracer dye (different from that for T cell labeling). The cells were imaged overnight (12 to 18 hours) at 37 ° C and 5% CO2 using a confocal laser scanning microscope (for example, Leica SP8X; or any type of fluorescence microscope with time-lapse imaging) living cell) in the presence of a dye to mark apoptotic cells (for example, NucRed Dead®, Molecular Probes). Subsequently, the time-lapse images were analyzed using Imaris software (Bitplane) and the percentage of stained organoids was calculated by evaluating the percentage of voxels where EPCAM colocalization and dead cell marker can be viewed. Flow cytometric analysis
[00242] [00242] Cytometric flow analysis is used to evaluate surface markers present in immune cells present in cocultures.
[00243] [00243] In the division (as in Example 7 above), 5000 cells were plated in BME and cultured for 3, 4 days in human colon medium or human colorectal cancer tumor medium. In culture, the medium was removed and drops of BME / Matrigel® were ruptured using Cell Recovery Solution® (Corning) after 25 minutes of incubation on ice. The cells are subsequently centrifuged (5 minutes at 500 × g) and
[00244] [00244] T cells were counted and brought to a concentration of 500000 / ml in complete T cell medium supplemented with 100 IU / ml of recombinant human IL-2. 100 μL of epithelial cancer tumor suspension was mixed with 100 μL of T cell suspension in a 96-well plate. The cells were co-cultured overnight, harvested and single cell suspensions were made using TripLE (Gibco®). The single cell suspensions were fixed with 4% paraformaldehyde (Sigma-Aldrich) and permeabilized using a buffer containing 0.5% saponin (BD Bioscience). Alternatively, commercially available kits (for example, BD Cytofix / Cytoperm Plus Fixation / Permeabilization Kit, BD Bioscience) were used. The cells were subsequently incubated with antibodies for flow cytometry against CD3, EPCAM, interferon (IFN) γ and / or tumor necrosis factor (TNF) α, along with an antibody recognizing active Caspase-3 (all from BD Bioscience) followed flow cytometric analysis. Analysis of cytokine secretion.
[00245] [00245] The organoids were divided, plated, cultivated and prepared for coculture as described above. T cells were counted and brought to a concentration of 500000 / ml in complete T cell medium supplemented with 100 IU / ml of recombinant human IL-2. 100 μL of epithelial cancer tumor suspension was mixed with 100 μL of T cell suspension in a 96-well plate. 72h after the start of the culture, the supernatant was collected for evaluation of cytokine production by the T cell (for example, IFNγ, TNFα) by ELISA. The culture supernatant was stored at -20 ° C until analysis. Example 10. Live imaging of tumoral coculture shows increased T-cell motility when cocultures are performed with
[00246] [00246] This example tests the effect of different structural components used in the development of co-cultures, on the motility of the resulting immune cells.
[00247] [00247] A schematic of the procedure is shown in Figure 3A. As described above, tumors were released from the BME drop using Cell Recovery Solution and washed in complete DMEM / F12 Advanced. Allogeneic CD8 + T cells isolated from peripheral blood samples were labeled with green dye (Vybrant CFDA SE Cell Tracer).
[00248] [00248] Tumor cells and T cells were mixed with human colonic organoid medium containing IL-2 and 10% BME or rat tail collagen I and imaged live for 80 h in a Leica SP8X confocal microscope equipped with a live imaging at 37 ° C and 5% CO2 atmosphere.
[00249] [00249] Figure 3B shows composite images representative of tumoroid co-cultures. The bright field channel and the green fluorescent channel were generated fused composite images. The path of the T cell was traced using Imaris software.
[00250] [00250] The quantification of the T cell track length in both conditions, as graphically represented in Figure 3C, shows significantly longer path path of T cells co-cultured in 10% collagen compared with 10% BME.
[00251] [00251] The results suggest that a more in vivo type system can be developed using mouse tail collagen I in the coculture, which produces longer trails and thus preserves the motility of the immune cell. Example 11: Generation of clonal coculture of tumors.
[00252] [00252] This example illustrates the generation of positive and negative clonal tumoroids for human leukocyte antigen (HLA) type A2.
[00253] [00253] A schematic of the procedure is shown in Figure 4A. Tumors were dissociated into single cells using enzyme digestion TrypLE. Single cells were stained with anti-HLA-A2 antibody and purified based on anti-HLA-A2 immunoreactivity. The HLA-A2 + ve and HLA-A2-ve tumor cells were embedded and maintained to generate tumors.
[00254] [00254] Flow cytometric analysis in Figure 4B showed the establishment of pure HLA-A2 + ve or HLA-A2-ve tumor strains. Controls are the HLA-A2 + ve JY cell line as well as the normal colonic organoid lines derived from the same patient samples as the HLA-A2 + ve or HLA-A2-ve tumor lines. Example 12. Assay for specific antigen extermination mediated by cytotoxic T cell of epithelial cancer tumors.
[00255] [00255] This example involves performing a ‘cell extermination assay’ on a tumoroid coculture. This is an example of the method of the invention applied to αβ T cells experienced with neoantigen to treat cancer.
[00256] [00256] Colorectal cancer tumors or normal tissue organoids were divided and maintained as single cells as described above. 10,000 to 50,000 T cells (TILs or derived from PBMC) were co-cultured with 50,000 unique cells derived from tumoroid / organoid in the presence of αCD28 antibody stimulation for 2 weeks in human T cell medium and 200 IU / ml of recombinant human IL-2 . The medium was renewed every 2 to 3 days. The expanded cells were subsequently clonally expanded in the presence of irradiated feeder cells (1 × 106 / ml, mixture of PBMCs from 3 different donors and 1 × 105 / ml of JY cells and / or LAZ509) in complete Ijssel medium supplemented with 200 IU / ml of recombinant human IL-2. Alternatively, T cells were classified by FACS directly from the single cell TIL or IEL prep plates on the plates
[00257] [00257] The putative tumor neoantigens identified were loaded onto the epithelial cancer organoids as follows. Drops of BME / Matrigel® in which organoids were cultured were broken by resuspending the medium on the plates. The relevant peptides were added to the organoids and the organoids were cultured for 2 h at 37 ° C and 5% CO2. The clonally expanded T cells were then cultured with autologous organoids for imaging, flow cytometric analysis and / or analysis of cytokine secretion as described above.
[00258] [00258] An extermination assay for anti-tumor reactivity of T cells experienced in antigen is shown in Figure 5 and a schematic of the procedure is shown in Figure 5A. The HLA-A2 + ve or HLA-A2-ve tumors were pulsed for 2 h with the HLA-A2-restricted Wilms tumor peptide (WT) 1. The transgenic CD8 + T cells in TCR harboring a specific WT1 peptide TCR were then co-cultured for 48h with HLA-A2 + ve or HLA-A2-ve tumors pulsed with WT1 peptide.
[00259] [00259] Bright field images representative of co-cultures after 48 hours are shown in Figure 5B.
[00260] [00260] Significant death is observed for HLA-A2 + ve tumors pulsed only with WT1 peptides. All other conditions, i.e., HLA-A2 + ve or HLA-A2-ve tumors not pulsed with WT1 peptides and HLA-A2-ve tumors pulsed with WT1 peptide, showed normal growth.
[00261] [00261] The results suggest that the neoantigen peptide WT1 is effective in killing tumoroids (and possibly treating cancers) with
[00262] [00262] A cell viability assay for anti-tumor reactivity of αβ T cells experienced with antigen with and without checkpoint inhibition is shown in Figure 6. This is an example of the method of the invention applied to a chemical agent to treat cancer.
[00263] [00263] A Schematic of the procedure is shown in Figure 6A. Co-culture was performed as described in Figure 5A but only for 12 hours and incubated with and without anti-PD1 checkpoint inhibitor. The cell viability test was performed using the CellTiter Glo Luminescent Cell Viability Assay kit (Promega) according to the manufacturer's instructions.
[00264] [00264] Figure 5B shows normalized tumor cell viability for peptide controls. Cocultures were therefore successfully used to show that tumor cell viability was lower when a combination of HLA-A2, IL-2 and anti-PD1 checkpoint inhibitor were present, that is, that treatment with the inhibitor anti-PD1 checkpoint may be more potent when applied to patient subpopulations displaying IL-2 and HLA-A2 types of cancer. Example 14. Assay to determine differential effect on T cell activation by organoid / tumoroid co-cultures.
[00265] [00265] This example illustrates that the presence of γδ T cells activates coculture tumors in a non-specific antigen manner, where they do not activate coculture organoids beyond a T cell baseline. IFN-γ was used for determine activation.
[00266] [00266] A schematic of the procedure is shown in Figure 7A. Tumors were released from the Matrigel® drop using Dispase and
[00267] [00267] Bright-field images representative of tumoroid co-cultures and organoid co-cultures are shown in Figures 7B and 7C (respectively).
[00268] [00268] The levels of IFN-γ quantification of cocultures is shown in Figure 7D. Example 15. Live imaging of coculture of tumors to assess the association and ability of cell extermination.
[00269] [00269] T cells were investigated for their ability to exterminate cells and their variation with different T cell subtypes and for different T cell / tumor antigen combinations.
[00270] [00270] A schematic of the procedure is shown in Figure 8A. The tumors were released from the Matrigel® drop using Dispase and subsequently passed through 70 µm and 20 µm filters. The organoids were recovered from the 20 µm filter, counted and plated. Cultured T cells labeled with dark red dye (CellVue Claret). The tumors and T cells were mixed with human colonic organoid medium containing RPMI, IL-2 and 5% Matrigel® and imaged live for 68h in a Leocal SP8X confocal microscope equipped with a 37 ° C live imaging camera and 5% CO2 atmosphere.
[00271] [00271] The composite images representative of the tumoral cocultures containing non-targeting T cells are shown in Figure 8B. The bright field channel and dark red fluorescence channel have been merged to generate composite images.
[00272] [00272] Composite images representative of the tumoral co-cultures containing targeting T cells are shown in Figure 8C. The bright field channel and the dark red fluorescence channel have been merged to generate composite images. Example 16. Modeling of cancer immunomodulation using epithelial organoid culture.
[00273] [00273] Here we use organoid technology to study immune-cancer interactions and evaluate colorectal cancer (CRC) immunomodulation. The formation of transcriptional profile and flow cytometry revealed that organoids maintain differential expression of immunomodulatory molecules present in primary tumors. Finally, we have established a method to model the extermination of antigen-specific epithelial cells and cancer immunomodulation in vitro using CRC organoids co-cultured with cytotoxic T cells (CTLs).
[00274] [00274] CRC is among the most common cancers worldwide. Although early stages of CRC are highly treatable by surgical removal, advanced stages are usually incurable. CRC arises through a multi-step process from small lesions of the epithelium of the large intestine. These lesions grow in adenomas with a low degree of dysplasia that progress into high-grade dysplasia, eventually giving rise to infiltrating carcinomas. Genetic mutations in signaling pathways such as the canonical Wnt signaling pathway are the molecular basis of CRC4. However, the interaction of the tumor with its microenvironment is another critical mark. Cancer cells reshape their microenvironment (for example, fibroblasts, the vasculature and immune cells) to support tumor growth. Infiltrating immune cells (ICs) such as CTLs or macrophages plays a crucial role in generating different immune responses such as anti-tumor cytotoxicity (the former) or chronic inflammation in tumor promotion (the latter). As such, escaping the
[00275] [00275] The culture of organoids grown from different epithelial tissues serves as an excellent tool for studying tissue homeostasis and disease. In addition, organoid biobanks from multiple epithelial organ systems have been established and tumor-derived organoids have been used successfully as platforms for different drug screens to predict the patient's response. Here, we describe the establishment of a method to model the extermination of antigen-specific epithelial cell and cancer immunomodulation in vitro using tumoroids co-cultured with immune cells (specifically, CRC organoids co-cultured with CTLs).
[00276] [00276] We first evaluated whether CRC organoids expressed immunomodulatory molecules in long-established expanded cultures. For this purpose, we compared the gene expression of specific T cell immunomodulators in CRC organoids to the expression levels found in normal colonic organoids using a set of transcriptome data generated using our CRC patients' live organoid biobank (van de Wetering, M. et al.
[00277] [00277] Four of the most commonly mutated genes in CRC are APC, P53, KRAS and SMAD4, reflecting the staggered progression of the normal intestinal epithelium in a metastatic carcinoma. The introduction of these cancerous mutations into human intestinal organoid cultures
[00278] [00278] Next, we aim to establish a co-culture system for CRC and CTL organoids to model the specific antigen extermination of tumor cells in vitro. For this purpose, we use αβ T cells, carrying a transgenic T cell receptor (TCR) recognizing a HLA-A2 restricted Wilms tumor derived peptide (WT) 1. We first screened CRC organoids from the 'living biobank' as well as newly generated CRC organoids for HLA-A2 expression using flow cytometry. We verified three strains of CRC organoid that showed partial HLA-
[00279] [00279] Colonic tissues (both normal colon and tumor tissue) were obtained from the Departments of Surgery and Pathology of the Diakonessenhuis Hospital, Utrecht, Netherlands. All patients included in this study were diagnosed with CRC. Informed consent was signed by all included patients. The tissue collection was approved by the medical ethics committee (METC) of Diakonessenhuis Hospital, in accordance with the Declaration of Helsinki and in accordance with Dutch and European Union legislation. Organoid generation and cultures
[00280] [00280] Epithelial organoid strains were derived from healthy colon or tumor tissue (van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi: 10.1016 / j. cell.2015.03.053 (2015)). In summary, healthy colonic crypts were isolated by digesting the colonic mucosa in chelating solution (5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM of Sucrose and 54.9 mM of D-Sorbitol, Sigma) supplemented with dithiothreitol (0.5 mM, Sigma) and EDTA (2 mM, internal), for 30 minutes at 4 ° C. The colonic crypts were subsequently plated in basement membrane extract (BME; Cultrex PC BME RGF type 2, Amsbio) and the organoids were grown in human intestinal stem cell (HISC) medium, which is composed of Dulbecco Advanced's modified Eagle medium / F12 supplemented with penicillin / streptomycin, 10 mM HEPES and Glutamax (all from Gibco, Thermo Fisher Scientific) with
[00281] [00281] Tumoroids (specifically, CRC organoids) were dissociated into small pieces using TrypLE and then transduced with H2B-mNeonGreen (pLV-H2B-mNeonGreen-ires-Puro). T cells
[00282] [00282] The generation of αβ T cells carrying a transgenic TCR recognizing an HLA-A2 restricted WT1-derived peptide has been described in Kuball, J. et al. Facilitating matched pairing and expression of TCR chains introduced into humans cells T. Blood 109, 2331-2338, doi: 10,1182 / blood-2006-05-023069 (2007). In summary, the TCRα and β chains were cloned from high tetramer positive T cell clones. Subsequently, CD8 + αβ TCR cells were transduced using retroviral supernatant from the Phoenix-Ampho packaging cells that
[00283] [00283] Tumoroids stably transfected with H2B-mNeonGreen were divided and digested 5 to 7 days before co-culture and seeded at a density of 5000 cells per 10 μL BME (25,000 cells per well in a 12-well cell culture plate ). Two days before coculture, T cells were deprived of IL-2. The day before the coculture, the tumors were stimulated with IFN-γ at the indicated concentrations.
[00284] [00284] Before cocultivation, T cells were stained with Cell Proliferation Dye eFluor 450 (eBioscience) according to the manufacturer's instructions. The tumors were pulsed with a specific TCR peptide (ProImune) for 2 hours at 37 ° C before coculture. Tumor and T cells were harvested and harvested in T cell medium, supplemented with 10% BME, 100 IU / mL IL-2 and NucRed Dead 647 (Thermo Fischer). Where indicated, anti-PD1 blocking antibodies (2 μg / mL) were added to the coculture. The cells were plated on 96-well plates with a glass bottom and the cocultures were imaged using an SP8X confocal microscope (Leica). Flow cytometry
[00285] [00285] APC-labeled pentamers for the EBV-derived peptide, restricted with HLA-2: 02 FLYALALLL (ProImmune) were used to classify CD8 + CD3 + pentameric T cells from PBMCs isolated from the buffy coat layers of healthy individuals. The cells were classified as single cells in 96-well plates using a BD FACS Aria cytometer (BD Biosciences). For flow cytometry, the following antibodies were used (all anti-human): CD8 – PE (clone RPA-T8), CD45 – PerCP-Cy5,5 (2D1), CD274 (PD-L1) –APC (MIH1 ) (all
[00286] [00286] For qPCR analysis, RNA was isolated from organoids / tumors using the RNAeasy kit (QIAGEN) according to the manufacturer's protocol. PCR analysis was performed using the SYBR Green Reagent (Biorad). PCR reactions were performed in duplicate with a standard curve for each primer. The initiators were planned using the NCBI initiator planning tool. The initiators used in this study: Advanced GAPDH (GTC GGA GTC AAC GGA TT), reverse GAPDH (AAG CTT CCC GTT CTC AG), advanced HPRT (GGC GTC GTG ATT AGT GAT), reverse HPRT (AGG GCT ACA ATG TGA TGG), Advanced CD274 (TGC AGG GCA TTC CAG AAA GAT), reverse CD274 (CCG TGA CAG TAA ATG CGT TCAG). Transcriptional profile formation
[00287] [00287] The microarray analysis of biobank organoids was performed as described in van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi: 10.1016 / j.cell.2015.03.053 (2015). Enzyme-linked immunosorbent assays (ELISA)
[00288] [00288] Culture supernatants were kept at –20 ° C and ELISA was performed for the indicated cytokines using ELISA MA Standard (Biolegend) according to the manufacturer's protocol. Cell viability assay
[00289] [00289] Cell viability after cocultures was evaluated using CellTiter-Glo luminescent cell viability assay (Promega), according to the manufacturer's protocol. Image analysis
[00290] [00290] Image analysis was done using the software package
[00291] [00291] Bioinformatics analysis of normalized gene expression data from microarray experiments (van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi: 10.1016 /j.cell.2015.03.053 (2015)) was performed using standard packages (ie, gplots) in version R 3.4.0 (R Foundation, https://www.r-project.org) and version RStudio 1.0. 143 (https://www.rstudio.com). Statistical analysis
[00292] [00292] All experiments were repeated at least three times unless otherwise indicated. All data were shown as mean ± SEM. Statistical significance was analyzed by ANOVA or two-tailed Student t test using Graphpad Prism 6 or Microsoft Excel
2010.

权利要求:
Claims (78)
[1]
1. Method to identify a suitable agent to treat a cancer, characterized by the fact that the method comprises: putting a tumor co-culture in contact with one or more candidate agents, in which the tumor co-culture comprises immune cells and at least one tumor , detect the presence or absence of one or more changes in the tumoral coculture that is indicative of the suitability of a candidate agent to treat cancer, and identify a candidate agent as suitable to treat cancer if the presence or absence of one or more of said changes in the tumor coculture is detected.
[2]
2. Method according to claim 1, characterized by the fact that the suitability to treat cancer comprises efficacy to treat cancer and / or safety to treat cancer.
[3]
3. Method according to any of the previous claims, characterized by the fact that the one or more changes are a change in one or more cancer biomarkers.
[4]
4. Method according to any of the preceding claims, characterized by the fact that the one or more changes are selected from a reduction in cell viability, a reduction in cell proliferation, an increase in cell death, a change in cell size or organoid, a change in cell motility, change in the production of cytokines and cytotoxic molecules by cocultivated immune cells, dissociation or disruption of the intact / compact epithelial cell layer and a change in the expression of one or more genes.
[5]
5. Method according to any one of the preceding claims, characterized by the fact that the detection comprises a cell proliferation assay, a viability assay, flow cytometric analysis,
2/16 ELISA for IFN-γ, analysis of gene expression and / or cell imaging.
[6]
6. Method according to any of the preceding claims, characterized by the fact that one or more changes is a reduction in cell viability, for example as detected by the CellTiter Glo Luminescent Cell Viability Assay kit (Promega), cytometric dyeing of intracellular flow for active Caspase 3 (BD) or positive strain for dead cells.
[7]
7. Method according to any of the preceding claims, characterized by the fact that the one or more changes is an increase in cell death, for example as detected by the bright field imaging.
[8]
8. Method according to any one of the preceding claims, characterized in that the method is preceded by one or more of the following steps: preparing at least one tumoroid by culturing tumor epithelial cells in a tumoroid culture medium; preparing the immune cells by separating immune cells from an impure immune sample and culturing the immune cells in an immune cell expansion medium; and / or preparing the tumoral coculture, preferably by removing the tumoroid culture medium from the at least one tumoroid and mixing the at least one tumoroid with the immune cells in a tumoroid coculture medium.
[9]
9. Method according to any of the preceding claims, characterized by the fact that the method comprises comparing the presence or absence of one or more changes in the tumor coculture with a reference organoid or reference tumor and in which the method additionally comprises : placing a reference organoid co-culture or co-culture
3/16 of reference tumor tumor in contact with one or more candidate agents, in which the reference organoid coculture or reference tumor tumor coculture comprises immune cells and at least one organoid or tumoroid, and to detect the presence or absence of a or more changes in the reference organoid coculture or reference tumor tumor coculture that is indicative of the suitability of a candidate agent to treat cancer.
[10]
10. Method according to claim 9, characterized in that a candidate agent is identified as a suitable agent if the presence or absence of a change is detected in the tumoroid coculture, but not in the reference organoid or coculture culture. reference tumor.
[11]
11. Method according to claim 9 or 10, characterized in that the method is preceded by one or more of the following steps: preparing the at least one organoid by culturing normal epithelial cells in an organoid culture medium; preparing the immune cells by separating the immune cells from an impure immune sample and culturing the immune cells in an immune cell expansion medium; and / or preparing the reference organoid coculture or reference tumoroid coculture, preferably by removing the tumoroid culture medium from the at least one tumoroid or at least one organoid culture medium and subsequently mixing the at least one organoid from reference or at least one reference organoid with immune cells in an organoid co-culture medium or a tumoroid co-culture medium, optionally wherein the impure immune sample is a tumor sample, normal colon tissue and / or peripheral blood.
4/16
[12]
Method according to any one of claims 8 to 11, characterized in that the normal epithelial cells are autologous with the tumor epithelial cells.
[13]
Method according to any of claims 9 to 12, characterized in that the reference organoid coculture or reference tumoroid coculture is used as a control, preferably in which it is used as a negative control.
[14]
Method according to any one of claims 8 to 13, characterized by the fact that (i) the reference tumor co-culture medium and / or (ii) the reference organoid co-culture medium or reference tumor co-culture medium , comprises extracellular matrix, preferably selected from collagen or any animal-derived or synthetic basal membrane matrix.
[15]
15. Method according to claim 14, characterized in that the collagen is rat tail collagen I.
[16]
16. Method according to any one of the preceding claims, characterized by the fact that the coculture comprises at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% of collagen, preferably, where the coculture comprises about 10% (v / v) of collagen.
[17]
17. Method according to any of claims 8 to 16, characterized by the fact that (i) the reference tumor co-culture medium and / or (ii) the reference organoid co-culture medium or the reference tumor co-culture medium has a protein concentration of 0.15 mg / (ml of Matrigel®) to 0.95 mg / (ml of Matrigel®) for a Matrigel® concentration of 2% to 10%.
[18]
18. Tumor co-culture medium and / or organoid co-culture medium, characterized in that it is in accordance with any one of claims 8, 11 or 14 to 17.
5/16
[19]
19. Method according to any one of the preceding claims, characterized by the fact that the immune cells of the reference tumor cell and / or reference organoid cell and / or reference tumor cell coculture have a motility of at least 40 µm / day , 60 µm / day, 80 µm / day, 100 µm / day, 120 µm / day or 140 µm / day.
[20]
20. Method according to any of the preceding claims, characterized by the fact that at least 20%, at least 30%, at least 40% or at least 50% of the immune cells in the tumor and / or organoid coculture of reference and / or reference tumor tumor coculture are able to move a distance of at least 200 µm, at least 250 µm, at least 300 µm, at least 350 µm or at least 400 µm in 80 hours.
[21]
21. Method according to any one of the preceding claims, characterized by the fact that the immune cells remain active for at least 4h, 8h, 12h, 24h, 48h or 72h.
[22]
22. Method according to any one of the preceding claims, characterized in that the one or more candidate agents are of known suitability for treating cancer and the method further comprises identifying the one or more candidate agents as suitable agents for treating cancer in one particular patient.
[23]
23. Method according to any one of claims 9 to 22, characterized in that both the tumor tumor coculture and the reference organoid coculture or reference tumor tumor coculture are derived from the particular patient.
[24]
24. The method of claim 22 or 23, characterized in that the method further comprises treating the patient with the candidate agent identified as suitable for treating cancer in the particular patient.
[25]
25. Method according to any of the claims
6/16 above, characterized by the fact that the one or more candidate agents are selected from one or more of the following therapeutic classes: immunotherapeutic, tumor-specific peptides, checkpoint inhibitors, alkylating agent, antimetabolite, metabolic agonist, metabolic antagonist , plant alkaloid, mitotic inhibitor, antitumor antibiotic, topoisomerase inhibitor, radiotherapeutic products, chemotherapeutic products, antibodies, photosensitizing agent, stem cell transplantation, vaccine, cytotoxic agent, cytostatic agent, tyrosine kinase inhibitor, proteasome inhibitor, cytokine, interferon , interleukin, intercalating agent, targeted therapy agent, small molecule drug, hormone, steroid, cellular therapeutic product, viral vector and nucleic acid therapeutic product.
[26]
26. Method according to any one of the preceding claims, characterized in that the one or more candidate agents are selected from one or more of the following therapeutic classes: tumor-specific peptides, checkpoint inhibitors, T-cell therapeutic product of the chimeric antigen receptor (CAR), transgenic T cells in therapeutic TCR and neoantigen.
[27]
27. Method according to any one of the preceding claims, characterized by the fact that the one or more candidate agents are an immunotherapeutic product.
[28]
28. The method of claim 27, characterized by the fact that the immunotherapeutic product is a therapeutic product of chimeric antigen receptor (CAR), transgenic T cell in therapeutic TCR or a neoantigen.
[29]
29. Method according to any of claims 1 to 21, characterized in that the one or more candidate agents is of unknown suitability to treat cancer and the method further comprises identifying a subset of the one or more candidate agents
7/16 as suitable agents to treat cancer.
[30]
30. Method according to any one of claims 1 to 21 or 29, characterized in that the one or more candidate agents is of known suitability for treating a first cancer and unknown suitability for treating a second cancer and the method further comprising identifying a subset of the one or more candidate agents as suitable agents to treat the second cancer.
[31]
31. Method according to any of the preceding claims, characterized by the fact that cancer is an epithelial cancer.
[32]
32. Method according to any of the preceding claims, characterized by the fact that cancer is gastrointestinal cancer.
[33]
33. Method according to any of the preceding claims, characterized by the fact that the cancer is colorectal cancer.
[34]
34. Method according to any of the preceding claims, characterized by the fact that the cancer comprises cancer at or below a Stage II, Grade II or T2 N1 M0.
[35]
35. Method according to any one of claims 8 to 34, characterized in that the tumor epithelial cell and / or normal epithelial cell are obtained from a sample from a cancer patient.
[36]
36. Method according to any of claims 8 to 35, characterized in that the tumor epithelial cell and the normal epithelial cell are obtained from the same cancer patient, optionally from the same sample.
[37]
37. Method according to any one of the preceding claims, characterized by the fact that the sample is a tissue biopsy.
[38]
38. Method according to any one of the preceding claims, characterized by the fact that the tissue biopsy is collected from the resected colon and / or rectum of patients with colorectal cancer, from ascites of
8/16 patients with colorectal or ovarian cancer and / or urine from patients with kidney cancer.
[39]
39. Method according to any one of the preceding claims, characterized in that the tumor epithelial cells and / or normal epithelial cells are selected from the group consisting of lung cells, liver cells, breast cells, dermal cells, intestinal cells, crypt cells, rectal cells, pancreatic cells, endocrine cells, exocrine cells, duct cells, kidney cells, adrenal cells, thyroid cells, pituitary cells, parathyroid cells, prostate cells, stomach cells, esophageal cells, ovarian cells, tube cells fallopian tubes or vaginal cells.
[40]
40. Method according to any one of the preceding claims, characterized in that the tumor epithelial cells and / or normal epithelial cells are intestinal cells, for example, colorectal cells.
[41]
41. Method according to any one of the preceding claims, characterized in that the tumor epithelial cells and / or normal epithelial cells are epithelial stem cells, preferably distinguished by the expression of Lgr5.
[42]
42. Method according to any one of the preceding claims, characterized in that the immune cells comprise one or more cell types selected from the group consisting of intraepithelial lymphocytes (IELs), tumor infiltrating lymphocytes (TILs), blood mononuclear cells peripheral (PBMCs), peripheral blood lymphocytes (PBLs), cytotoxic T cells and T lymphocytes (CTLs), αβ T cells, γδ T cells, B cells, NK cells and mononuclear phagocytes.
[43]
43. Method according to any of the preceding claims, characterized by the fact that the immune cells are obtained from a sample from a cancer patient.
9/16
[44]
44. Method according to any one of the preceding claims, characterized in that the immune cells are obtained from a peripheral blood sample and / or a tissue biopsy.
[45]
45. Method according to any one of the preceding claims, characterized by the fact that peripheral blood lymphocytes (PBLs) and / or T cells are obtained from the peripheral blood sample.
[46]
46. Method according to any one of the preceding claims, characterized by the fact that tumor infiltrating lymphocytes (TILs) and / or intraepithelial lymphocytes (IELs) are obtained from tumor biopsy or normal tissue, respectively.
[47]
47. Method according to any of the preceding claims, characterized in that the immune cells are obtained from the same patient as the tumor epithelial cell and / or normal epithelial cell.
[48]
48. Method according to any one of the preceding claims, characterized by the fact that the immune cells are allogeneic) with the tumoroid and / or organoid, optionally in which the immune and tumoroid and / or organoid cells are derived from peripheral blood or biopsy tissue from a different patient or healthy control.
[49]
49. Method according to any one of the preceding claims, characterized by the fact that the immune cells are matched in HLA with the tumoroid and / or organoid.
[50]
50. Method according to any one of the preceding claims, characterized in that the immune cells persist in the immune cell expansion medium for at least 4h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h , 216h and 240h.
[51]
51. Method according to any one of the preceding claims, characterized in that the at least one tumoroid and / or at least one organoid comprises or consists of autologous cells.
[52]
52. Method according to any of the claims
Previous 10/16, characterized by the fact that the at least one tumoroid and / or at least one organoid are separated into populations sharing one or more genotypes, phenotypes and / or epigenetic markers, before mixing with the immune cells.
[53]
53. Method according to any of the preceding claims, characterized by the fact that genotypes, phenotypes and / or epigenetic markers contribute to the interaction between (i) the at least one tumoroid and / or at least one organoid and (ii) immune cells.
[54]
54. Method according to any one of the preceding claims, characterized by the fact that populations share the presence or absence of an HLA haplotype, optionally in which the HLA haplotype is HLA-A2.
[55]
55. Method according to any one of the preceding claims, characterized in that the at least one tumoroid or at least one organoid comprises or consists of mammalian cells, preferably human cells.
[56]
56. Method according to any one of the preceding claims, characterized by the fact that at least one tumoroid coculture or at least one organoid coculture is grown in an immune cell expansion medium or in a 50:50 (v / v) immune cell expansion medium and organoid culture medium or tumoroid culture medium (respectively).
[57]
57. Method according to any of the preceding claims, characterized by the fact that the tumor coculture persists in the tumor coculture medium or in which the reference organoid coculture or reference tumor tumor coculture persists in the organoid coculture medium , for at least 4h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h and 240h.
[58]
58. Tumoroid or organoid as defined in any one
11/16 of the preceding claims, characterized by the fact that the tumoroid or organoid is in a medium comprising an interleukin, optionally in which the interleukin is selected from the group consisting of IL-2, IL-7 and IL-15.
[59]
59. Tumor or organoid population, characterized by the fact that it is prepared according to the method as defined in any of the preceding claims.
[60]
60. Coculture of tumoroid and / or coculture of reference organoid, characterized by the fact that it is as defined in any of the preceding claims.
[61]
61. Method according to any one of the preceding claims, characterized in that the organoid culture medium comprises one or more (or preferably all) of a basal medium (such as DMEM / F12 Advanced, Gibco medium) a Wnt linker (such as Wnt-3a), a Wnt agonist (such as anyone from Respondina 1-4), a BMP inhibitor (such as Noggin), EGF and a TGF-β inhibitor (such as A83-01, Tocris ) and optionally further comprises one or more (or all) of a MAP38 p38 inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or an antimicrobial (such as primocin).
[62]
62. Method according to any of the preceding claims, characterized in that the tumor culture medium comprises one or more (or preferably all) of a basal medium (such as DMEM / F12 Advanced, Gibco medium) an agonist of Wnt (such as any of Respondina 1-4), a BMP inhibitor (such as Noggin), EGF and a TGF-β inhibitor (such as A83-01, Tocris) and optionally further comprises one or more (or all ) of a MAP38 p38 inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or an antimicrobial (such as primocin), optionally wherein the tumor culture medium additionally comprises a Wnt ligand (such as
12/16 Wnt-3a).
[63]
63. Method according to any one of the preceding claims, characterized in that the immune cell expansion medium comprises IL-2, optionally in a concentration of 2000 to 6000 IU / mL and optionally additionally comprising IL-7 and / or IL-15.
[64]
64. Method according to any one of the preceding claims, characterized in that the immune cell expansion medium additionally comprises an RPMI medium (for example, RPMI 1640, Gibco), optionally supplemented with penicillin / streptomycin and / or serum ( for example, 5% human AB serum, Sigma-Aldrich).
[65]
65. Method according to any one of the preceding claims, characterized in that the tumor-growing medium and / or organoid-growing medium comprises IL-2, optionally in a concentration of 100 to 200 IU / mL.
[66]
66. Method of testing a chimeric T cell antigen receptor (CAR-T), transgenic T cell to T cell receptor (TCR), neoantigen or checkpoint inhibitor, for efficacy and / or safety when used for treat epithelial cancer, the method characterized by the fact that it comprises: optionally providing tumor epithelial cells, normal epithelial cells and immune cells, expanding tumor epithelial cells in tumor growth medium to form a tumor and growing the tumor with the same immune cells in a tumoroid co-culture medium comprising interleukin to form a tumoroid co-culture, expand normal epithelial cells in organoid culture medium to form an organoid and grow the organoid with immune cells in an organoid co-culture medium comprising interleukin to form a reference organoid co-culture,
13/16 place the tumor tumor coculture and the reference organoid coculture in contact with the immunotherapy of CAR-T, transgenic T cells in TCR, neoantigen or checkpoint inhibitor, detect the presence or absence of one or more changes in tumor coculture and reference organoid coculture, in which the presence or absence of one or more changes is indicative of the efficacy and / or safety of the immunotherapy of CAR-T, transgenic T cells in TCR, neoantigen or checkpoint inhibitor , and to compare the tumor tumor coculture and the reference organoid coculture.
[67]
67. Method of testing a candidate compound for efficacy and / or safety when used to treat epithelial cancer, characterized by the fact that the method comprises: optionally providing tumor epithelial cells, normal epithelial cells and immune cells, expanding cells tumor epithelial cells in tumor growth medium to form a tumor and grow the tumor with immune cells in a tumor cell culture medium comprising interleukin to form a tumor cell culture, expand normal epithelial cells in organoid culture medium to form a organoid and culturing the organoid with immune cells in an organoid co-culture medium comprising interleukin to form a reference organoid co-culture, placing the tumoroid co-culture and the reference organoid co-culture in contact with the candidate compound, detecting the presence or absence of one or more changes in the tumor coculture and in the coculture of reference organoid, in which the presence or absence of one or more changes is indicative of the efficacy and / or safety of the candidate compound, and
14/16 to compare the tumor tumor coculture and the reference organoid coculture.
[68]
68. Method for preparing an organoid-immune cell co-culture, characterized by the fact that the method comprises: optionally growing epithelial cells in contact with an extracellular matrix in an organoid culture medium to obtain an organoid; removing said extracellular matrix and organoid culture medium from said organoid; resuspending said organoid in the immune cell culture medium supplemented with interleukin; preparing an immune cell suspension comprising immune cells, immune cell culture medium supplemented with interleukin and collagen at a concentration of at least 5 to 10% in the suspension; and mixing the immune cell suspension comprising immune cells with the resuspended organoid.
[69]
69. Method for preparing a tumoroid cell-immune coculture, characterized by the fact that the method comprises: optionally culturing tumor epithelial cells in contact with an extracellular matrix in a tumoroid culture medium to obtain an organoid; removing said extracellular matrix and tumoroid culture medium from said tumoroid; resuspending said tumoroid in the immune cell culture medium supplemented with interleukin; preparing an immune cell suspension comprising immune cells, immune cell culture medium supplemented with interleukin and collagen at a concentration of at least 5 to 10% in the suspension;
15/16 and mix the immune cell suspension comprising immune cells with the tumoroid resuspended.
[70]
70. The method of claim 68 or 69, characterized in that the collagen is rat tail collagen.
[71]
71. Method according to any one of claims 68 to 70, characterized in that the immune cell medium is RPMI1640 (Gibco).
[72]
72. Method according to any of claims 68 to 71, characterized in that the immune cells are T cells.
[73]
73. Method according to any one of claims 68 to 72, characterized in that the epithelial cells and immune cells are obtained from the same subject, optionally from the same sample.
[74]
74. Method according to any one of claims 68 to 72, characterized in that the epithelial cells and immune cells are human cells.
[75]
75. Coculture of an organoid-immune cell, characterized by the fact that it is obtained by the method as defined in any of claims 68 or 70 to 74.
[76]
76. Coculture of tumor-immune cell, characterized by the fact that it is obtained by the method as defined in any one of claims 69 to 74.
[77]
77. Method for testing a therapeutic agent, characterized by the fact that the method comprises: placing an organoid co-culture in contact with one or more candidate agents, in which the organoid co-culture comprises immune cells and at least one organoid, detecting the presence or absence of one or more changes in the organoid co-culture that is indicative of therapeutic efficacy, and
16/16 identify a candidate agent as a therapeutic agent if the presence or absence of one or more of said changes in the organoid coculture is detected.
[78]
78. The method of claim 77, characterized in that the therapeutic agent is suitable for the treatment of an immune disease, optionally in which the immune disease affects tissues of the lung and / or intestine, for example, in which the disease immune is selected from the group consisting of irritable bowel disease (IBD), ulcerative colitis (UC), chronic obstructive pulmonary disease (COPD) and asthma.
Figure 1
Petition 870200076768, of 19/06/2020, p. 104/125 patients surgical resection organoids and T cells analysis colorectal cancer tumoroid TCR sequencing normal colon peptidoma 1/20 organoid peripheral blood DNA sequencing (single cell) mRNA sequencing
Figure 1 organoid cultures T cell cultures flow analysis
Petition 870200076768, of 19/06/2020, p. 105/125 2/20 organoids (N18)
tumors (T18)
Figure 2 Halogenic IELs 60h coculture fixation & confocal assembly microscope
Figure 3 in 10% BME in 10% collagen I
tumors coculture live imaging 80h collagen track length I
Figure 4
Petition 870200076768, of 19/06/2020, p. 108/125 tumoroid staining with HLA-A2 antibody and 5/20 flow classification seeding of clones HLA-A2 + ve and HLA-A2-ve clonal tumoroid
Figure 4
Petition 870200076768, of 19/06/2020, p. 109/125 HLA-A2 cell line + control ve (JY)
HLA-A2 + ve organoids (N40) HLA-A2-ve tumoroid 6/20
(T40- clone) HLA-A2 + ve tumoroid (T40 + clone)
HLA-A2 + ve (N41) organoids HLA-A2-ve (clone T41-) tumors HLA-A2 + ve (clones T41 +)
Figure 5 Tumor cells pulsed coculture 48h transgenic peptide imaging in TCR HLA-A2-ve tumoroid HLA-A2 + ve tumoroid no peptide peptide
Figure 6
Petition 870200076768, of 19/06/2020, p. 111/125 cell viability pulsed tumors transgenic T cells in peptide in TCR 8/20 12h standardized coculture for no peptide cell viability assay
Figure 7 24-hour coculture T cell organoid images of bright field
-T cells + T cells
-T cells + T cells
Organoid
Tumoroid-T cells
+ T cells
Figure 8 Organoid T cells coculture live imaging
Figure 9
Petition 870200076768, of 19/06/2020, p. 114/125 colon colon colon cancer 11/20 row z colon colon colon cancer
Figure 9
Petition 870200076768, of 19/06/2020, p. 115/125 control control control control control 12/20 control control control control
Figure 10
Petition 870200076768, of 19/06/2020, p. 116/125 13/20 control (no stimulation) IFN-γ stimulation
Figure 11
Petition 870200076768, of 19/06/2020, p. 117/125 14/20 CRC organoids CRC organoids T cells that pulsed with peptide recognize coculture peptide
Figure 11
Petition 870200076768, of 19/06/2020, p. 118/125 HLA-A2 organoids - HLA-A2 + 15/20 organoids without peptide with HLA-A2 organoids - HLA-A2 organoids + EBV peptide Figure 11 WT1 organoid peptides / T cells T cell WT1 EBV no T cells T cells
Figure 11
Petition 870200076768, of 19/06/2020, p. 120/125 WT1 T cells without peptide 17/20 EBV T cells with peptide no peptide EBV peptide WT1 organoid peptide / T cells / apoptotic cells
Figure 11
Petition 870200076768, of 19/06/2020, p. 121/125 18/20 colocalization of 488+ no peptide no peptide / no T cell EBV peptide EBV peptide / no T cell
Figure 11
Petition 870200076768, of 19/06/2020, p. 122/125 EBV peptide / EBV T cells / anti-PD1 EBV peptide / EBV T cells / no antibody 19/20 no peptide / EBV T cells / anti-PD1 colocalization of 488+ organoids / T cells no peptide / EBV T cells / no antibody
Figure 11 ratio with peptide vs. without control peptide

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公开号 | 公开日

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US20210208131A1|2021-07-08|

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SG11202005891TA|2020-07-29|

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JP2021508249A|2021-03-04|

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AU2018390960A1|2020-07-09|

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CA3086290A1|2019-06-27|

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EP3729084A1|2020-10-28|

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WO2019122388A1|2019-06-27|

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GB201721615D0|2018-02-07|

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KR20200105852A|2020-09-09|

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CN111989569A|2020-11-24|

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法律状态:
2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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GBGB1721615.1A|GB201721615D0|2017-12-21|2017-12-21|Immune cell organoid co-cultures|

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