Compositions and methods for improving the treatment of cancer

Abstract

To provide a system and a method capable of providing a tumor model suitable for accurately predicting the success of therapy.SOLUTION: Systems, compositions, and methods are provided that utilize a three dimensional tumor model that incorporates a tissue barrier (such as a stromal barrier) around tumor cells in culture, allowing for the recapitulation of invivo immune cell responses to tumor cells. Such tumor models are used to accurately evaluate appropriate treatment modes and protocols that would be effective against the individual's tumor. Such tumor models can be used to selectively expand immune cells that respond to the tumor cells utilized in the model. The population of immune cells so expanded can then be utilized therapeutically.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 222,150, filed July 15, 2021. These and all other referenced external materials are incorporated herein by reference in their entirety. In the event that the definition or use of a term in a reference incorporated by reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein shall control.

[0002] FIELD OF THE INVENTION The field of the invention is cancer drug discovery and personalized medicine, in particular personalized cancer treatment. [Background technology]

[0003] The following background discussion contains information that may be useful in understanding the present invention, but is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Cancer is a highly heterogeneous condition, with neoplastic cells arising from various tissues and cell types, various presentations (e.g., solid, non-solid), and phenotypes (e.g., vascular, non-vascular, non-malignant, malignant, localized, metastatic, chemosensitive). Furthermore, individual immune responses to cancer vary widely (e.g., inflamed, excluded, cold, "immune desert") . While clinicians have a variety of therapeutic approaches at their disposal, including chemotherapy, radiation therapy, hyperthermia, antibody-based immunotherapy, and cell-based immunotherapy, they lack adequate tools to determine which therapeutic approach is likely to be most effective for a given individual. Furthermore, current tools available to drug discovery and preclinical researchers are severely limited in their ability to recapitulate the complex tumor microenvironment in vitro to advance top candidate compounds and therapies to the clinic.

[0005] For example, U.S. Patent No. 6,299,999 to Downing et al. and U.S. Patent No. 6,299,999 to Chalmers et al. provide methods for rapidly identifying specific mutations in tumor cells. While such mutations may provide some therapeutic insight, they are limited to genetic information and therefore do not provide direct data regarding effective in situ treatment of such tumors. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. If the definition or use of a term in an incorporated reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0006] U.S. Patent No. 6,269,999 describes a method for determining tumor sensitivity by exposing three-dimensional cell cultures containing neoplastic cells to anticancer drugs. Similarly, U.S. Patent No. 6,269,999 to Sikora and Pathak describes a test system based on xenografting of tumor cells onto the chick chorioallantoic membrane. However, such an approach fails to reproduce the structure of the tumor, the tissue surrounding the patient's tumor, and the patient's own immune response to the tumor. Therefore, it does not provide data that is directly useful for determining a therapeutic approach.

[0007] In US Patent Publication No. 2005 / 0129997, Presnell et al. used a three-dimensional cancer model containing tumor cells inserted into a bioprinted stromal microenvironment to determine the effect of candidate treatments on various aspects of tissue growth, including applications in personalized therapy. However, the described method does not allow for fixation of the implanted tumor cells, complicating visualization. Furthermore, the described method cannot account for tumor-associated pathological findings. Finally, relying on deposition to provide a stromal microenvironment necessarily limits the dimensions that can be achieved.

[0008] Therefore, there remains a need for systems and methods that can provide tumor models suitable for accurately predicting the success of treatments. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2012 / 0208706 [Patent Document 2] US Patent Application Publication No. 2018 / 0363066 [Patent Document 3] US Patent Application Publication No. 2018 / 0252703 [Patent Document 4] US Patent Application Publication No. 2019 / 0029235 [Patent Document 5] US Patent Application Publication No. 2019 / 309264 Summary of the Invention

[0010] The present subject matter provides systems, compositions, and methods for providing planar, three-dimensional constructs incorporating cells characteristic of cancer (e.g., tumor cells, fibroblasts, etc.) and other disease states, as well as therapeutic compounds and / or immune cells, to facilitate the treatment of cancer and other disease states. Such constructs can also be used for the selective clonal expansion of immune cells, which can then be utilized therapeutically.

[0011]

[0003] Embodiments of the present concepts include methods for diagnosing and predicting treatment for cancer patients by obtaining data regarding the pathology of the cancer patient's tumor, determining the distribution of cancer cells, stromal cells, or immune cells within or near the tumor, and generating multiple three-dimensional models of the tumor containing two or more of: (i) tumor cells obtained from the cancer patient, (ii) stromal cells, and / or (iii) immune cells. The cancer cells, immune cells, and / or stromal cells can be obtained from the cancer patient or from a non-patient source (i.e., an allogeneic source, tissue or cell culture, explanted tissue, etc.). In such methods, each of the three-dimensional models reflects the distribution of cancer cells, stromal cells, or immune cells indicated by the pathology, and each of the multiple three-dimensional models is deposited on a test surface, and tumor cells or an acellular layer of each of the multiple three-dimensional models is attached to the test surface. The multiple three-dimensional models are exposed to multiple anti-cancer treatments, and the effects of the anti-cancer treatments on the three-dimensional tumor models are characterized. The therapeutic effects of one or more anti-cancer treatments in the cancer patient can be predicted based on the effects on the three-dimensional tumor models.

[0012] Each of the multiple three-dimensional tumor models can include a first compartment and a second compartment, the first compartment having a first side and a second side, the first side being in contact with the test surface and the second side being in contact with the second compartment, and one or both of the first and second compartments (optionally) including one or more of an extracellular matrix, a biomaterial, and a biopolymer scaffold. Coexistence of tumor cells and immune cells in the first compartment can provide a model of an immunoinflammatory tumor. Alternatively, to provide a model of an immune-exclusion tumor, tumor cells can be present in the first compartment and immune cells can be absent from the first compartment, with immune cells present in or on the surface of the second compartment. In some embodiments, stromal cells (e.g., fibroblasts, endothelial cells, mesenchymal stem cells (MSCs), adipocytes, and / or pericytes) are positioned near the first compartment such that the stromal cells are sandwiched between the tumor cells and the immune cells (e.g., stimulatory immune cells of the innate or acquired immune system, inhibitory immune cells of the innate or acquired immune system, peripheral blood mononuclear cells (PBMCs), T cells, NK cells, B cells, dendritic cells, mast cells, neutrophils, and / or macrophages). Alternatively, in some embodiments, the tumor cells and stromal cells can coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, the tumor cells and immune cells coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, the immune cells and stromal cells coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, the tumor cells, stromal cells, and immune cells coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, immune cells are absent from the tumor model to provide a model of an immune desert tumor.

[0013] Such methods can include providing a liquid medium to a portion of a three-dimensional model. The liquid medium can include immune cells and, optionally, stromal cells. The tumor cells, stromal cells, and / or immune cells can be obtained from a surgical tumor sample, a tumor biopsy, normal tissue from a cancer patient, from the circulation of a cancer patient, or from an established cell line. In some embodiments, the immune cells are activated T cells that are prevented from infiltrating three-dimensional tumors under controlled, untreated conditions. Alternatively, the immune cells can be activated T cells that can infiltrate three-dimensional tumors under controlled, untreated conditions.

[0014] The data utilized in such methods can be obtained by one or more of immunohistochemistry (IHC), flow cytometry, gene expression, and other methods that provide information about the tumor microenvironment or tumor composition of an individual patient. The IHC data so provided can relate to the relative orientation and location of tumor, immune, and stromal compartments in cancer patients. Such data can be collected by automated analysis, such as high-content imaging, cell sorting, flow cytometry, proteomic analysis, expression analysis, and / or genomic sequencing. In some embodiments, data collected by automated analysis can be evaluated to quantify one or more of immune cell infiltration, tumor cell death, and other tumor microenvironment changes within multiple three-dimensional tumor models.

[0015] The anti-cancer treatments evaluated in such methods can be targeted cancer therapies, immunomodulators, chemotherapy, repurposed drugs traditionally used to treat conditions other than cancer, radiation, or a combination of two or more of these. The three-dimensional tumor models utilized in such methods can be generated and automated using a liquid handling system, such as a bioprinter. Such liquid handling systems can include a photomask and a light source. Such liquid handling systems can deposit one or more of an extracellular matrix, a biomaterial, or a biopolymer scaffold onto the test surface.

[0016] Some embodiments of the inventive concepts are methods for optimizing cancer treatment for a cancer patient by generating a first dataset including predicted past treatment outcomes developed using the methods described above for a plurality of cancer patients with a history of the disease, generating a second dataset including treatment outcomes for the plurality of cancer patients with a history of the disease, and providing an artificial intelligence system with a learning algorithm configured to access the first and second datasets and generate a proposed treatment planning algorithm that correlates or otherwise relates the predicted treatment outcomes to the treatment outcomes, and applying the treatment planning algorithm to the predicted treatment outcomes to provide or suggest one or more treatment protocols believed to be effective for treating the cancer patient. Such an artificial intelligence system can be configured as a neural network. In some embodiments, the artificial intelligence system is accessed via an information network and may also be accessed via a subscription service.

[0017] Some embodiments of the inventive concepts are methods for optimizing cancer treatment for a cancer patient by generating a first dataset including predicted past treatment responses using the methods described above for a plurality of cancer patients with a history of the disease, generating a second dataset including recorded treatment outcomes for the plurality of cancer patients with a history of the disease, and providing the first and second datasets to an artificial intelligence system including a learning algorithm to generate a treatment planning algorithm that correlates the predicted treatment responses with the treatment outcomes, and applying the treatment planning algorithm to data related to tumor pathology of the cancer patient to report or propose a treatment plan for the cancer patient. Such an artificial intelligence system can be configured as a neural network. In some embodiments, the artificial intelligence system can be accessed via an information network and can also be accessed via a subscription service.

[0018] Embodiments of the inventive concept include methods for conducting three-dimensional cell-based assays by obtaining a planar three-dimensional construct comprising a planar or essentially planar first layer of extracellular matrix or hydrogel within a culture vessel, where first cells (e.g., immune cells, target cells, or a combination of immune and target cells) are provided below or intermixed within the first layer, and second cells (e.g., immune cells, target cells, and a combination of immune and target cells) are provided on top of the first layer, and measuring or otherwise characterizing interactions between the first and second cells over a period of time. The second cells can be tumor cells, non-tumor diseased mammalian cells, pathogens, bacteria, viruses; a source of chemokines, chemoattractants, stimuli that enable an immune response, and / or immune cells (e.g., T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and / or their derivatives). The second cells can be of human, non-human, or non-animal origin. Such constructs may further comprise stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and / or viruses.

[0019] Such constructs may include biomaterials such as polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and / or chemically modified derivatives thereof formulated for chemical or physical crosslinking. Such constructs may include at least one layer comprising a polymer polymerized using electromagnetic radiation, temperature, and / or time. Such constructs may include a planar or essentially planar second layer. The at least one layer may be selected for biocompatibility, modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers of the construct.

[0020] Constructs suitable for these methods can be fabricated using standard or customized molding methods, photolithography, bioprinting, and / or techniques to control the size, shape, and planarity of one or more layers of the construct. The first layer of such a construct can have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter, and a stiffness of between 50 Pa and 20 kPa.

[0021] An embodiment of the inventive concept includes a three-dimensional cell-based assay system comprising a first planar or essentially planar layer of extracellular matrix or hydrogel within a culture vessel; first cells (e.g., immune cells, target cells, or a combination of immune and target cells) below, mixed within, or above the first layer; a second layer of extracellular matrix or hydrogel above the first layer; and second cells (e.g., immune cells, target cells, a combination of immune and target cells, tumor cells, non-tumor diseased mammalian cells, pathogens, bacteria, viruses; a source of chemokines, a source of chemoattractants, a source of stimuli that enable an immune response) mixed in or disposed on the second layer. Such second cells can be of human, non-human animal, or non-animal origin. Suitable immune cells include stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and / or viruses. Such assay systems may further comprise stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and / or viruses. Such assay systems may include a planar or essentially planar second layer. Such assay systems may include immune cells such as T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and / or derivatives thereof.

[0022] Such assay systems may include biomaterials such as polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. Such assay systems may include at least one layer comprising a polymer produced by polymerization using electromagnetic radiation, temperature, or time.

[0023] At least one layer of such an assay system is selected for biocompatibility, modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. Such assay systems can be constructed using standard or customized molding methods, photolithography, bioprinting, or other techniques to control the size, shape, and planarity of the layers of the construct. The first layer of such an assay system can have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter, and a stiffness of between 50 Pa and 20 kPa.

[0024]

[0010] Embodiments of the present concepts include methods for assessing the effectiveness of a therapy by measuring at least one of cell viability, cell structure, and the presence of a marker of cell apoptosis in either a first or second cell in the manner described above. Such measurements may be obtained by at least one of live cell imaging, immunofluorescence, flow cytometry, proteomic analysis, and genomic analysis. Such methods may include obtaining measurements of immune cell infiltration into a layer of a construct or into a collection of target cells, analyzing protein secretion, characterizing cytokines, observable changes to the target or immune cells, characterizing apoptosis in the target or immune cells, characterizing proliferation in the target or immune cells, characterizing cell aggregate size in the target or immune cells, characterizing phenotypic changes in the target or immune cells, and / or characterizing genotypic changes in the target and immune cells. Such methods may include subjecting at least a portion of a layer of a three-dimensional construct or assay system to at least one of chemical digestion, enzymatic digestion, mechanical digestion, or disruption by addition of a chelating agent. Such steps may include robotic selection or aspiration to collect cells released by the digestion or disruption of the layer.

[0025] Embodiments of the inventive concepts include methods of providing personalized medicine by carrying out the above-described methods incorporating patient- or pathogen-derived target cells and autologous or allogeneic immune cells, subjecting the construct or assay system used to one or more therapies (e.g., radiation therapy, chemotherapy, targeted therapy, and / or immunotherapy), determining the efficacy of the one or more therapies with the construct or assay system, and providing efficacy information based on such determinations to medical professionals to aid in clinical decision-making for a particular patient or group of patients.

[0026] Embodiments of the inventive concept include expanding an immune cell population by performing the above-described method, wherein the three-dimensional construct or assay system comprises immune cells cultured near target cells in a first or second layer, providing an incubation period sufficient for the immune cells to proliferate and generate an expanded immune cell population, and recovering the expanded immune cell population from the three-dimensional construct or assay system. The expanded immune cell population is modified after recovery, for example, by genetic modification, further expansion, or a combination thereof. Such immune cells may be autologous or allogeneic.

[0027] Embodiments of the inventive concept include methods of producing antibodies by performing the above-described clonal expansion method, where the three-dimensional construct or assay system includes immune cells, which are cultured B cells or hybridoma cells, to generate an expanded immune cell population, selecting a cell subpopulation from the expanded immune cell population, and harvesting antibodies from the selected cell subpopulation. Such selection can be performed using live imaging, manual picking, and / or robotic picking.

[0028] Embodiments of the inventive concept include methods of endogenous cell therapy by performing the clonal expansion method described above, where the cells to be expanded are immune cells derived from peripheral blood, spleen, or a disease microenvironment, expanding the immune cells to generate an expanded immune cell population, and infusing at least a portion of the expanded immune cell population into an individual in need of treatment. In some embodiments, a subpopulation of immune cells (which can be evaluated for efficacy in a patient) is separated from the expanded immune cell population and infused into a patient in need of treatment. Evaluation of patient efficacy can be performed after or before infusion.

[0029] Embodiments of the inventive concept include methods of providing immune cells having a particular phenotype (e.g., peripheral blood mononuclear cells or tumor-infiltrating lymphocytes) by generating a three-dimensional culture including a layer of antigen-producing cells (e.g., tumor cells or pathogen cells), contacting the three-dimensional culture with immune cells to generate a co-culture, and incubating the co-culture for a period of time sufficient to generate immune cells having the particular phenotype. Suitable phenotypes include cytolytic effector T cells, NK cells, macrophages, memory T cells, e.g., stem cell memory T cells, central memory T cells, effector memory T cells, and transitional memory T cells. The period of time can be selected to produce a stimulatory effect on the immune cells, such as antigen-specific stimulation, CD3 / CD28 stimulation, CD3 / CD28 stimulation with CD137, and costimulation with one or more cytokines (e.g., IL2, IL7, and IL5). Such three-dimensional cultures may include a stromal cell layer (which may include fibroblasts) interposed between the antigen-producing cell layer and the immune cells. In some embodiments, the three-dimensional cultures may include cytokine-producing cells (e.g., stromal cells, fibroblasts, endothelial cells, cytokine-producing immune cells, and / or dendritic cells).

[0030] Such three-dimensional cultures may include hydrogel or extracellular matrix components. The three-dimensional cultures may include dextran, gelatin, collagen, hyaluronic acid, and polyethylene glycol, and at least a portion of the three-dimensional culture may be degradable by metalloproteinases. The three-dimensional cultures may have a thickness of 20 μm to 2 mm and a stiffness of 50 Pa to 20 kPa. The three-dimensional cultures may be patterned into distinct regions and / or directions, for example, within a container or well or on a surface. In some embodiments, the three-dimensional cultures may include a porous membrane.

[0031] Embodiments of the inventive concept include methods of providing immunotherapy by isolating immune cells having a particular phenotype produced by the methods described above to provide isolated immune cells, contacting the isolated immune cells with a pharmaceutically acceptable carrier to form an immunotherapeutic composition, and administering the immunotherapeutic composition (e.g., by infusion) to an individual in need of treatment. Suitable pharmaceutically acceptable carriers include liquid media suitable for injection or infusion. In some embodiments, the pharmaceutically acceptable carrier is a biocompatible hydrogel or tissue scaffold, which can be administered by topical application to a portion of the individual to be treated.

[0032] Embodiments of the present concepts include systems for providing immune cells with specific phenotypes, including three-dimensional cultures comprising an antigen-producing cell layer (e.g., a pathogenic cell layer, a tumor cell layer, etc.) and immune cells (e.g., peripheral blood mononuclear cells or tumor-infiltrating lymphocytes) co-cultured in the three-dimensional culture, the three-dimensional culture being configured to enhance clonal expansion of the immune cells with the specific phenotype. The three-dimensional cultures in such systems may include a stromal cell layer interposed between the antigen-producing or tumor cell layer and the immune cells. The three-dimensional cultures may have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter, and may have a stiffness of between 50 Pa and 20 kPa. In such systems, the three-dimensional cultures may be patterned into distinct regions and / or directions.

[0033] In such systems, the three-dimensional culture can include a stromal cell layer interposed between the antigen-producing or tumor cell layer and the immune cells. Such a stromal cell layer can include fibroblasts. In some embodiments, the three-dimensional culture can include a hydrogel and / or extracellular matrix components. At least a portion of the three-dimensional culture can be degradable by metalloproteinases. In some embodiments, the three-dimensional culture can include dextran, gelatin, collagen, hyaluronic acid, alginate, polyethylene glycol, and / or a porous membrane.

[0034] In such systems, the three-dimensional cultures can be configured to provide stimulatory effects to immune cells, such as antigen-specific stimulation, CD3 / CD28 stimulation, CD3 / CD28 stimulation with CD137, and costimulation with one or more cytokines (e.g., IL2, IL7, and / or IL5). In some embodiments, the three-dimensional cultures comprise cytokine-producing cells. Suitable cytokine-producing cells include fibroblasts, endothelial cells, cytokine-producing immune cells, and dendritic cells.

[0035] In such systems, the immune cells having a selected phenotype can be memory T cells. Such memory T cells can be stem cell memory T cells, central memory T cells, effector memory T cells, and transitional memory T cells. In some embodiments, the immune cells having a selected phenotype are selected from the group consisting of one or more of cytolytic effector T cells, NK cells, and macrophages.

[0036]

[0010] Embodiments of the inventive concept include the use of immune cells having a specific phenotype to manufacture an immunotherapeutic composition, which involves isolating immune cells having a specific phenotype produced by the methods described above and providing the immune cells having a specific phenotype in a pharmaceutically acceptable carrier. Such a pharmaceutically acceptable carrier may be a liquid medium suitable for injection. Alternatively, the pharmaceutically acceptable carrier may be a biocompatible hydrogel or tissue scaffold.

[0037] Embodiments of the inventive concept include three-dimensional cell-based assay systems having a first planar or essentially planar layer of extracellular matrix or hydrogel (which can be a product of standard or customized molding, photolithography, or bioprinting) within a culture vessel, first cells (e.g., immune cells, target cells, or a combination of immune and target cells) beneath or mixed within the first layer, and a second planar layer containing second cells (e.g., immune cells, target cells, a combination of immune and target cells, tumor cells, non-tumor diseased mammalian cells, pathogens, bacteria, viruses, a source of chemokines, a source of chemoattractants, and / or a source of stimuli that enable an immune response). Such second cells can be of human, non-human animal, or non-animal origin. Such systems may include one or more other cell types, such as stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, viruses, T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and their derivatives. In some embodiments, the second layer may include an extracellular matrix or hydrogel. Such systems may include biomaterials or biocompatible materials, such as polymers of natural or synthetic origin; decellularized tissues, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. At least one layer of such systems may include a polymer polymerized using electromagnetic radiation, temperature, or time. At least one layer of such systems may be selected for biocompatibility, modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. In such a system, the first layer may have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter.

[0038] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like numerals represent like elements. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows a schematic representation of an exemplary planar three-dimensional construct of the inventive concept. [Figure 2] FIG. 2 shows a schematic representation of cell movement within a planar three-dimensional construct in accordance with the present concept. [Figure 3A] Figures 3A-3D provide photomicrographs of infiltrating immune cells and the effect of immune checkpoint inhibitors on the infiltration of such cells and the subsequent effect on tumor cells. Figure 3A shows a photomicrograph of immune cell infiltration through an extracellular matrix (ECM) barrier disposed between a layer containing tumor cells and a layer containing immune cells in a planar three-dimensional construct of the present concepts. [Figure 3B] Figure 3B provides a histogram of representative data showing the effect of applying immune checkpoint inhibitors on immune cell infiltration in such constructs. [Figure 3C] Figure 3C provides a histogram of representative data showing the effect of immune checkpoint inhibitors on tumor cell death in such constructs. [Figure 3D] Figure 3D shows exemplary data demonstrating the effect of checkpoint inhibitors on tumor area in such constructs. [Figure 4] Figure 4 provides a schematic and photomicrograph of clonal expansion of immune cells using a planar three-dimensional construct of the inventive concept. Clonal expansion is evident when the underlying layer contains tumor cells. [Figure 5] Figure 5 shows typical results from a study of the effect of fibroblasts on tumor sensitivity to drug therapy. As shown, the presence of fibroblasts in the layer overlying the tumor cells provides a protective effect. [Figure 6]Figure 6 shows typical results of an immune cell distribution study in tumor cell-containing planar three-dimensional constructs without or with an intervening fibroblast-containing layer. The left panel shows a photomicrograph of such a construct without an intervening fibroblast layer, in which immune cells are randomly distributed. The right panel shows a photomicrograph of such a construct with an intervening fibroblast-containing layer, in which tumor cells are non-randomly distributed along the fibroblast spindles. [Figure 7] FIG. 7 provides a photomicrograph of a planar three-dimensional construct of the present concepts in which tumor cells are cultured with a layer of fibroblasts in the presence of immune cells (eg, PBMCs). [Figure 8] FIG. 8 provides a photomicrograph of a planar three-dimensional construct of the present concepts in which tumor cells are cultured with a layer of fibroblasts in the absence of immune cells. [Figure 9] FIG. 9 provides a photomicrograph showing significant aggregation of immune cells (PBMCs) along with a fibroblast network covering tumor cells within a planar three-dimensional construct of the present concepts. [Figure 10A] FIG. 10 shows typical results of a flow cytometry study performed to characterize immune cells cultured in a planar three-dimensional construct of the present concept incorporating cultured tumor cells. [Figure 10B] Same as above. [Figure 11A] FIG. 11 shows typical results of a flow cytometry study performed to characterize immune cells cultured in a planar three-dimensional construct of the present concept incorporating tumor cells taken from a patient. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 12] FIG. 12 shows typical results of a flow cytometry study demonstrating selectively enhanced clonal expansion of immune cells cultured in planar three-dimensional constructs of the present concept incorporating tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description The following description contains information that is helpful in understanding the present invention, but is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0041] The present subject matter provides devices, systems, and methods for providing three-dimensional tumor models that can incorporate analogs of tissue barriers (e.g., stromal barriers) around tumor cells in culture and replicate an individual's immune cell response to the tumor cells. Such tumor models can be used to accurately evaluate appropriate treatment modes and protocols that may be effective against an individual's tumor. In some embodiments, such tumor models can be used to selectively expand immune cells that respond to the tumor cells utilized in the model. Such expanded immune cell populations can then be utilized therapeutically (e.g., by returning at least a portion of the expanded immune cell population to the tumor-bearing individual).

[0042] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like numerals represent like elements.

[0043] In some embodiments, numerical values ​​expressing properties such as amounts and concentrations of ingredients, reaction conditions, and the like, used to describe and claim particular embodiments of the present invention should be understood to be modified in some cases by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. The numerical values ​​set forth in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0044] As used throughout this description and the claims that follow, the meanings of "a," "the," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0045] Unless the context indicates to the contrary, all ranges set forth herein should be construed as inclusive, and open-ended ranges should be construed as including only commercially practical values. Similarly, unless the context indicates to the contrary, all lists of values ​​should be considered as inclusive of intermediate values.

[0046] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value within the range. Unless otherwise indicated herein, each individual value with a range is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein with respect to specific embodiments, or the use of exemplary language (e.g., "etc.") are intended solely to better clarify the invention and do not impose limitations on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0047] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements described herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion is made, the specification is deemed to include the group so modified, and accordingly satisfies the recitation requirement of all Markush groups used in the appended claims.

[0048] It should be appreciated that the disclosed technology provides many advantageous technical effects, including the accurate and effective treatment of cancer. As used herein, the term "essentially planar" refers to a feature surface that deviates from planarity by 10% or less.

[0049] The following description provides many exemplary embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C and a second embodiment includes elements B and D, the inventive subject matter is considered to include any other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0050] As used herein, unless the context indicates otherwise, the term "coupled to" is intended to include both direct coupling (where the two elements coupled to each other touch each other) and indirect coupling (where at least one additional element is located between the two elements). Thus, the terms "coupled to" and "coupled with" are used interchangeably.

[0051] As used throughout this description and the claims that follow, the meanings of "a," "the," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0052] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements described herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion is made, the specification is deemed to include the group so modified, and accordingly satisfies the recitation requirement of all Markush groups used in the appended claims.

[0053] Central to the conceptual embodiment of the present invention is the generation of three-dimensional tissue models. Any suitable method for doing so can be used. In a preferred embodiment, the apparatus and method are described in U.S. Pat. Nos. 10,073,346, 10,423,071, and U.S. Patent Application No. 16 / 156,663, which are works by the present inventors and are incorporated herein by reference. These describe methods for introducing cells into a suspension containing one or more photoactivatable polymer precursors. Exposure to light of an appropriate wavelength through a photomask with a specific configuration results in polymerization and the formation of a three-dimensional solid of the desired profile (provided by the photomask) in which the cells are suspended. The dimensions of the three-dimensional solid vary depending on many factors, including the area irradiated, the amount of cell suspension provided, the amount of light provided (by intensity, exposure, etc.), and the presence and configuration of pillars inserted into the culture plate wells.

[0054] Such methods allow for the production of cell-laden solids of a wide variety of shapes and sizes. Repeated cycles of solid production generate a wide variety of complex shapes resulting from the successive use of photomasks of different configurations and the subsequent production of overlying three-dimensional solids (which can incorporate suspension cells, growth factors, chemotactic factors, etc.). These methods also allow for the production of a wide variety of complex tissue models by incorporating different cell types into different portions of the resulting three-dimensional solid.

[0055] Embodiments of the inventive concepts can be directed to a wide variety of disease states. While application to cancer (e.g., through the use of tumor models) is described below, the inventors also contemplate application of such cell-based models to non-tumor disease states. Other non-tumor disease models may include fibrosis, inflammation, CNS diseases, and other diseases that preclude the utility of mutant normal cells that behave with dysfunctional phenotypes or genotypes. The models can be supplemented with microenvironment cells, i.e., stromal cells (e.g., fibroblasts) and immune cells (T cells, B cells, NK cells, macrophages, monocytes, etc.), in a manner similar to tumor models.

[0056] In some embodiments, non-diseased cell models can be developed in a similar manner to serve as toxicity assays for compounds tested in disease (tumor) models. The models can include patient-derived or allogeneic donor cells, or cell lines of specific healthy tissue lineages (e.g., cardiac, neural (brain, CNS), skin, bone, etc.). Additionally, healthy cells can be derived from stem cells, such as induced pluripotent stem cells (iPSCs). Healthy 3D models can be in static or dynamic fluid flow cultures, separate from or coupled to disease (tumor) models, simulating a human-like closed system for drug compound testing.

[0057] It should be understood that systems of the present inventive concept, in addition to including the planar three-dimensional constructs described below, can also include mechanisms supporting methods performed utilizing such constructs. Such components include liquid handling and dispensing instruments (e.g., pipettors) positioned to dispense liquids (e.g., cells in suspension, drugs in solution, etc.) into containers containing such constructs and / or to collect liquids from such containers (e.g., to recover propagated clonal cells, etc.). Such components can also include sensor equipment, e.g., appropriate optical devices (cameras, microscopes, etc.), for observing or monitoring the planar three-dimensional constructs. Similarly, such systems can include control systems communicatively coupled to such liquid handling and sensor equipment devices. Such control systems can enable manual control via an appropriate interface and / or can include computing devices for performing user-specified tasks. Such user-specified tasks can include maintenance and / or monitoring of the planar three-dimensional constructs, as well as functional tasks utilizing the planar three-dimensional constructs. Such functional tasks may include, as described below, adding specific drugs (e.g., for screening studies), dispensing immune cells for clonal expansion, collecting clonally expanded cells, etc. Such a controller may include a memory device that encodes instructions for performing such tasks.

[0058] Such a controller may be co-located with other system components or may be located remotely. Such remote controllers may be accessed via a server. Throughout the following description, numerous references are made to servers, services, interfaces, portals, platforms, or other systems formed from computing devices. It should be understood that the use of such terms is to be considered to represent one or more computing devices having at least one processor configured to execute software instructions stored on a computer-readable, tangible, non-transitory medium. For example, a server may include one or more computers operating as a web server, database server, or other type of computer server in a manner that performs the described roles, responsibilities, or functions.

[0059] One embodiment of the inventive concept is a method for predicting effective treatment for an individual patient. In a first step, the characteristics and properties of an individual patient's tumor are characterized. This can be accomplished by any suitable method, including genomic, expressomic, and / or proteomic analysis (e.g., identification of specific surface markers by immunohistochemistry, flow cytometry, immunoassays, etc.) and / or imaging of samples taken from the tumor and / or circulating cancer cells. Such characterization can be used to determine the presence or absence of cells of the immune system, allowing the tumor to be classified as immunoinflamed (showing evidence of inflammation and / or the presence of immune cells within the tumor), immune excluded (e.g., immune cells are present but arrested at the stromal cell barrier surrounding the tumor), or immune "desert" (i.e., no immune cells are visible).

[0060] Following characterization of the tumor and its immune status, a three-dimensional model of the tumor in an individual patient within that patient's normal tissue can be created. As noted above, this model can be created by any suitable method. The three-dimensional model is deposited on a suitable solid surface, such as the floor of a test well or the surface of a membrane. In such a model, naturally occurring tissue layers can be represented by deposited layers of cells and / or one or more polymer matrices containing such cells. Such polymer matrices can include supporting components such as components of the extracellular matrix, biomaterials, biopolymer scaffolds, photopolymer precursors, etc.

[0061] Tumor cells can be obtained from a patient or in the form of a tumor cell line. Such a tumor cell line can represent the cells of a patient's tumor, for example, in terms of tissue of origin, tumor type, and / or tumor marker expression. In preferred embodiments, tumor cells are obtained from an individual patient, for example, through surgery, biopsy, and / or collection of circulating tumor cells. In some embodiments, tumor cells can be provided as cells or clumps of cells held in suspension. In other embodiments, tumor cells can be provided as one or more intact parts of a tumor mass. In other embodiments, the tumor cells or clumps can be passaged in an animal model (e.g., mouse) to generate patient-derived xenograft (PDX) models, from which cells can then be excised for in vitro cell culture studies. In yet another embodiment, cells can be propagated and established in cell culture to allow proliferation (either permanently as an "immortalized" cell line or as a primary cell line that senesces after a certain number of passages). The initial layer deposited on the solid surface (or on a layer of cell-free matrix material deposited on the solid surface) comprises viable tumor cells obtained from an individual patient. If tumor characterization indicates that the tumor is inflamed / infiltrated with immune cells, this initial layer may include such immune cells (e.g., stimulatory and suppressor cells of the innate and adaptive immune systems, such as T cells, NK cells, B cells, dendritic cells, mast cells, neutrophils, macrophages, etc.). These immune cells may be obtained from the patient. Alternatively, such immune cells may be modified after collection from the patient to model the outcomes of cell-directed immunotherapy utilizing such modified immune cells. Alternatively, in some embodiments, such an initial layer may include tumor cells and stromal cells. In some embodiments, the initial layer may include tumor cells, stromal cells, and immune cells.

[0062] In some embodiments, one or more additional layers can be deposited over all or at least a portion of the initial layer. Such additional layers can replicate aspects of the environment near a tumor in an individual (e.g., encapsulation by a layer of stromal cells). Such additional layers can be deposited over the portion of the model containing the initial tumor cells. For example, if characterization indicates that the tumor is encapsulated by a layer containing stromal cells, such additional layers can include stromal cells. Suitable stromal cells include fibroblasts, endothelial cells, mesenchymal stem cells (MSCs), adipocytes, and / or pericytes. In some embodiments, this additional layer can include components of an extracellular matrix, a biopolymer scaffold, and / or a biomaterial. In preferred embodiments of the inventive concept, cells for such additional layers are obtained from the patient, although in other embodiments, cell culture or corresponding cells obtained from a different individual can be used. In some embodiments, this additional layer can include both stromal cells and immune cells.

[0063] As noted above, characterization of an individual's tumor may indicate that immune cells are present near the tumor but not within the tumor itself. In such cases, these immune cells are often located at or near a barrier of stromal or similar cells that surrounds at least a portion of the tumor. Such a barrier can be represented in the three-dimensional tumor model by the additional layer described above. If characterization indicates that immune cells are present and in this region, a second additional layer containing such immune cells can be deposited, positioned such that the layer containing stromal cells is located between the layer containing tumor cells and the layer containing immune cells. Such immune cells are preferably obtained from the individual. Alternatively, to replicate the results of cell-directed immunotherapy in the individual, such immune cells can be obtained from the individual and modified before being incorporated into the three-dimensional model. Alternatively, such immune cells can be obtained from another individual and / or from tissue culture.

[0064] Once the three-dimensional tumor model is generated, a liquid medium (e.g., tissue culture medium) is introduced to cover the model and provide oxygen and nutrients. Such medium can be used as a vehicle for therapeutic compounds and therapies (e.g., chemotherapeutic drugs, repurposed drugs from other uses, immunomodulators, gene-editing enzymes, cell therapies, etc.) and can be modified as needed. In some embodiments, this liquid medium can contain cells, such as immune cells and / or stromal cells, in suspension. Such immune cells in suspension can (at least partially) mimic the presence of circulating immune cells.

[0065] Once the three-dimensional tumor model is generated, the proposed treatment can be applied to the individual's cancer. Suitable treatment modes include, but are not limited to, chemotherapy, immunotherapy, radiation therapy, hyperthermia, and / or combinations thereof. In some embodiments, two or more treatment modes can be combined. For example, two or more chemotherapeutic agents can be utilized. Alternatively, two or more of chemotherapy, immunotherapy, radiation therapy, and / or hyperthermia can be combined and applied to the three-dimensional tumor model. In preferred embodiments, multiple tumor models are generated, and two or more treatment approaches are tested in parallel. For example, a multi-well dish or plate can be utilized, with individual three-dimensional tumor models deposited in the wells of the dish or plate for parallel treatment.

[0066] During and / or after administration of the proposed treatment, the tumor model can be observed or characterized to determine the effectiveness of the proposed treatment. Any suitable method of observation or characterization can be utilized. For example, when deposited on a suitable transparent surface, tumor cells can be directly observed under a microscope to observe tumor cell death, infiltration of previously isolated immune cells into the tumor cell-containing portion of the model, etc. Alternatively, samples can be obtained from the tumor model and characterized (e.g., by immunohistochemical staining, gene expression characterization, etc.). In yet other embodiments, samples of fluid in contact with the three-dimensional tumor model can be obtained and characterized for the content of markers of inflammation, oxidative stress, apoptosis, etc. Suitable methods of characterization include high-content imaging, single-cell sorting (e.g., flow cytometry), proteomic and / or genomic analysis to quantify immune cell infiltration, observation of tumor cell death, and changes in the tumor microenvironment. Such measurements can be obtained before (e.g., baseline), during, and after administration of the treatment.

[0067] Analysis of the results of such proposed treatments allows for the selection of one or more treatment modes likely to be effective in treating the tumor in a particular patient. It should be appreciated that such an approach provides a highly predictive model by reproducing not only the tumor type, but also the tumor's architecture and / or the ongoing response of the patient's immune system to the tumor cells. In preferred embodiments of the inventive concepts, one or more of the steps of generating multiple three-dimensional tumor models, applying various treatments to such tumor models, characterizing the tumor model's response to such treatments, analyzing the data obtained from such characterization, and recommending one or more treatment protocols are provided by an automated system.

[0068] In some embodiments, historical or previously collected data, data from characterization of tumor models of the present invention upon application of treatment protocols, recommended and / or selected protocols, and outcomes from a large number of individual patients can be aggregated as a single data collection in one or more databases. Such a data collection can be used as the basis for training an artificial intelligence system, which can then use this data collection to determine or recommend one or more treatment protocols for individuals not represented in the data collection (based at least in part on data collected from the personalized three-dimensional tumor models). Such an artificial intelligence system can utilize evolutionary algorithms and can have a neural network architecture. Such an artificial intelligence system can be provided locally or accessed remotely via a suitable user interface (e.g., a web page or application for a portable device). In some embodiments, a remotely accessible artificial intelligence system can be offered as a subscription service. In some embodiments, such an artificial intelligence system can be used to evaluate and recommend a treatment protocol for an individual based solely on data from characterization of the individual's tumor, without generating and utilizing a personalized three-dimensional tumor model.

[0069] Another embodiment of the inventive concept utilizes multi-layered, three-dimensional constructs of immune cells (e.g., peripheral blood mononuclear cells (PBMCs), T cells, NK cells, etc.) in combination with tumor cells and / or tumor-associated chemoattractants, and in some embodiments, additional cell types associated with solid tumors in vitro (e.g., epithelial cells, etc.). The layers here can be planar or essentially planar. These are used in a variety of applications, such as when simulations of in vivo interactions between tumor cells and / or tumor cell products and cellular components of the human immune system are studied and / or utilized, and when accurate and convenient imaging of layers within the construct is required for accuracy and efficacy.

[0070] Prior art methods and compositions for generating three-dimensional layered cell-containing tumor or tissue models inevitably generate concave or convex layers of varying thickness due to meniscus formation at the top surface of the fluid component prior to polymerization. In such configurations or models, the distribution of cells within a given layer is, at least in part, an artifact of the geometry of the concave or convex surface. Because microscopy is necessarily limited to a designated focal plane, accurate assessment of changes in cell population and / or distribution within such concave or convex layers requires disassembling the layered model. For example, adding cells in suspension to such models may result in pooling toward the center of the concave surface or the periphery of the convex surface, or the cells may aggregate due to simple sedimentation. Furthermore, if the concave or convex surface is very steep, different layers of the model may lie within the same focal plane, making microscopy impractical for accurate quantification.

[0071] Thus, in some embodiments of the inventive concept, a three-dimensional arrangement of distinct planar layers is provided, with all or a portion of adjacent layers in direct physical contact with each other and arranged so that the layers are parallel to the focal plane of a microscope placed within or above the layers for observation. Individual layers comprise polymeric components (which may be crosslinked) that provide a mechanically stable gel and may contain tumor cells (e.g., cells from an individual, cells from an established tumor cell line, etc.), cells associated with tumors in vivo (e.g., epithelial cells, stromal cells, etc.), growth factors, chemoattractants, chemokines, cytokines, antibodies, and / or cells of the immune system (PBMCs, T cells, etc.). In some embodiments, one or more layers may comprise crosslinked polymeric components and essentially function as a physical barrier to exclude tumor cells, growth factors, chemoattractants, and / or cells of the immune system.

[0072] In some embodiments, the composition and / or degree of crosslinking provides a desired density, stiffness, and / or permeability (e.g., to cells, growth factors, biomolecules, chemotherapeutic agents, immunotherapeutic agents, etc.). In some embodiments, cells may be provided as embedded cells within and through designated layers of the construct. In other embodiments, cells may be deposited on exposed surfaces of layers of the construct and, in some cases, may migrate into such layers.

[0073] In some embodiments, layers of cells can be deposited between layers of the construct such that both adjacent layers are in contact with the layer of cells. In other embodiments, regions of cells can be provided within a layer of the construct and positioned such that the region of cells is in close proximity to an adjacent layer of the construct. For example, cells representative of cells found in the capsule or region immediately surrounding a tumor (e.g., epithelial cells, stromal cells, fibroblasts, etc.) can be deposited in a manner that mimics the barrier to therapy found in an in vivo tumor.

[0074] In some embodiments of the inventive concepts, one or more planar three-dimensional constructs can be used to evaluate the effectiveness of cell-based therapies against cancer cells (e.g., patient-specific tumor cells, tumor cell lines, etc.). For example, immune cells utilized in such therapies (e.g., PBMCs, T cells, NK cells, macrophages, etc.) can be introduced into a surface or upper layer of the planar three-dimensional construct (e.g., by incorporation into a layer and / or application as a suspension to an exposed surface of a layer). Other layers of the planar three-dimensional construct can contain tumor / cancer cells and / or chemoattractant compounds associated with such tumor / cancer cells. The migration of immune cells and / or intervening layers can be monitored over time to determine the aggregation, migration, proliferation, and / or cytotoxic effects of such immune cells.

[0075] In some such embodiments, layers or interfaces between layers interposed between tumor / cancer cells and immune cells can contain cells associated with the tumor in vitro (e.g., epithelial cells, stromal cells, etc.), and migration through and / or aggregation at such layers or interfaces can be used to determine the effectiveness of immune cells in overcoming such barriers during in vivo cancer treatment. In some embodiments, such constructs can contain or be exposed to biomolecules and / or drugs directed against tumor or cancer cells. Examples include specific antibodies, immune checkpoint protein analogs (such as analogs of PD-1 and / or PDL-1), in situ gene editing compositions, and / or chemotherapeutic compounds.

[0076] Another embodiment of the inventive concept is the use of planar three-dimensional constructs in the selective clonal expansion of immune cells (e.g., PBMCs, T cells, TCM cells, NK cells). Such clonally expanded cells can be used, for example, in cell-based immunotherapy. In such embodiments, immune cells can be provided in a layer that is in direct or indirect contact with a layer containing tumor or cancer cells and / or one or more tumor-associated growth factors. Such immune cells can be integrated into a layer of the planar three-dimensional construct and / or applied as a suspension of immune cells that is applied to the exposed surface of a layer of such a construct. In some embodiments, a layer of cells characteristic of the tissue surrounding the tumor can be interposed between the tumor cells and the immune cells presented for clonal expansion. Examples of such cells include stromal cells, such as fibroblasts.

[0077] Responsive or active immune cells can be observed to divide and form an expanded population of reactive or active immune cells over time. Surprisingly, the inventors have discovered that this time course can be significantly shortened compared to conventional techniques for immune cell expansion. For example, immune cell populations can be expanded at least 10-fold over a period of about 7 to about 14 days (versus 6 to 8 weeks with conventional techniques). Such methods also advantageously allow for the effective coculture of such immune cells with tumor cells, while avoiding undesirable immunogenic responses to non-tumor antigens present in conventional basement membrane-derived products (e.g., derived from Engelbreth-Holm-Swarm mouse tumors) used to support tumor cells with undesirable levels of growth factors.

[0078] An example of a planar three-dimensional construct in accordance with the present invention is shown in Figure 1 below. As shown, the planar three-dimensional construct can include a bottom planar primary layer comprising a gel or polymer matrix. Such a matrix may not be loaded with cells or active compounds. In some embodiments, such a polymer matrix can incorporate cells or active compounds. Suitable cells for incorporation include tumor or other target cells, tumor-associated non-tumor cells (e.g., stromal cells, etc.), immune cells (e.g., T cells, NK cells, peripheral blood mononuclear cells, or PBMCs, etc.), active compounds that attract immune cells (e.g., chemokines, cytokines, proteins, low molecular weight compounds), and / or active compounds that repel immune cells. Such a primary layer can be in direct contact with the liquid medium. In other embodiments, a planar secondary layer can be provided in contact with the primary layer and can also include cells and / or active compounds. In such embodiments, the exposed surface of the secondary layer is in contact with the liquid medium. In the left panel, a layer of gel or polymer incorporating target (e.g., tumor) cells is overlaid with a layer of immune cells applied as a suspension.

[0079] An example of a planar three-dimensional construct in accordance with the inventive concept is shown in Figure 1. In the left panel, a single gel or polymer layer is provided. As described above, such a polymer or gel layer may contain target cells, immune cells, disease-associated non-target cells, and / or active compounds that affect the function or activity of immune cells. As shown, a layer of immune cells is provided on the exposed surface of this gel layer by applying a suspension of such cells in a liquid medium. The middle panel shows an alternative embodiment in which the above-described primary gel or polymer sublayer is covered with a secondary gel or polymer layer that may also contain target cells, immune cells, non-target cells, and / or active compounds that affect the function or activity of immune cells. In this example, the secondary layer includes immune cells embedded within the secondary layer. The right side of Figure 1 shows another embodiment similar to that shown in the middle panel, except that the immune cells are applied to the exposed surface of the secondary layer by application of a cell suspension in a liquid medium. It should be understood that in some embodiments, a layer of cells (e.g., tumor-associated non-tumor cells, stromal cells, epithelial cells) can be interposed between the primary and secondary layers or distributed on the exposed surface of the layers. Alternatively, the immune cells may be embedded in a first layer and the tumor cells embedded on top of that layer or in a second layer.

[0080] During use, cells located at one location in a planar three-dimensional construct can migrate within the construct, for example, between layers of the construct or from the exposed surface of a layer to the interior of the layer. For example, immune cells embedded in the second layer of the embodiment shown in the middle panel of FIG. 1 can migrate into the primary layer containing tumor cells and / or chemoattractant compounds. Similarly, immune cells distributed on the exposed surface of the primary or secondary layer can migrate into the interior of the layer if the layer contains tumor cells and / or chemoattractant compounds. It should be understood that cells provided as a layer above a secondary gel or polymer layer of the construct can migrate into the secondary layer and subsequently into the primary layer. For example, immune cells applied as a suspension to the exposed surface of the secondary layer can migrate into the secondary layer and subsequently into the primary layer containing tumor cells and / or chemoattractant compounds. An example of such migration and infiltration is shown in FIG. 2. Similarly, it should be understood that immune cells can be embedded with tumor cells within a layer and migrate within the layer.

[0081] Examples of immune cell migration and infiltration into the primary layer of a planar three-dimensional construct containing tumor cells are shown in Figures 3A-3D. As shown in Figure 3A, on day 12 after introduction, immune cells migrated into the primary layer and formed a coculture with the tumor cells provided to the primary layer. This migration and infiltration can be enhanced by including active compounds such as immune checkpoint inhibitors (see Figure 3B). Suitable checkpoint inhibitors may include drugs that block PD-1 / PD-L1 and / or CTLA-4 / B7-1 / B7-2. Such improved immune cell infiltration can effectively increase the effectiveness of immune cells in destroying tumor cells in the primary layer (see Figure 3C). This is illustrated in Figure 3D, which provides a histogram of typical results from such a study. As shown, increasing drug (checkpoint inhibitor) concentrations result in decreased tumor size. It should be understood that such findings can be applied to evaluate efficacy and identify appropriate checkpoint inhibitors for use in individual patients. Similarly, such findings can be applied to screening for new checkpoint inhibitor compounds.

[0082] Another embodiment of the inventive concepts is the use of target cells (e.g., tumor cells or other disease cells) embedded in a layer of a planar three-dimensional construct of the inventive concepts to stimulate division and / or clonal expansion in effector cells (e.g., immune cells) at the exposed surface of the layer. Such clonally expanded cell populations are easily harvested, for example, by pipetting, manual harvesting, and / or robotic harvesting (e.g., using enzymatic or mechanical means). In some embodiments, such clonal expansion occurs at the exposed surface of a gel or polymer layer containing tumor or other disease cells. In other embodiments, such clonal expansion occurs within and / or on the exposed surface of a secondary layer that is in contact or at least partially in contact with such a primary layer of a planar three-dimensional construct of the inventive concepts. An example of such clonal expansion is shown in Figure 4. As shown, in the absence of target cells in the gel / polymer layer, the surface exhibits individual, isolated immune cells. Surprisingly, the presence of target cells in the gel / polymer layer results in the development of large colonies of clonally expanded immune cells.

[0083] As described above, in some embodiments, non-target cells may be incorporated into one or more layers of a planar three-dimensional construct of the present invention, at the exposed surface of such a layer, and / or at the interface between two layers of the construct. Such non-target cells may be associated with a disease state but are not representative of diseased cells. For example, such non-target cells may be cells found in an otherwise normal layer of cells (e.g., stromal or epithelial cells) surrounding a tumor. In some embodiments, such non-target cells may interfere with the migration and / or activity of effector cells (e.g., immune cells). Therefore, including such non-target cells in a planar three-dimensional construct of the present invention can be useful as a tool for investigating ways to overcome such interference and / or for identifying patient-specific therapeutic approaches. An example of such interference is shown in Figure 5. As shown, the presence of immune cells and checkpoint inhibitors is effective in inducing tumor cell death when tumor cells are embedded in the gel / polymer layer of a planar three-dimensional construct of the present invention. However, this effect is blocked by the inclusion of fibroblasts within the primary gel / polymer layer, on the exposed surface of the primary layer, or at the interface between the primary and secondary layers.

[0084] Another example of this phenomenon is shown in Figure 6. This figure provides micrographs of the distribution of immune cells within or on a gel / polymer layer containing tumor cells. In the left panel, the gel / polymer layer does not contain fibroblasts, while in the right panel, the gel / polymer layer contains fibroblasts. As shown, in the absence of fibroblasts, immune cells are uniformly distributed throughout the field of view. Surprisingly, when fibroblasts are present in the gel / polymer layer, immune cells are found to aggregate along the fibroblast spindles. Without wishing to be bound by theory, the inventors believe that such aggregation adversely affects immune cell function and / or activity.

[0085] As described above, in some embodiments, planar three-dimensional constructs of the present invention may contain non-tumor cells (e.g., fibroblasts) associated with tumors in vitro. The inventors have discovered that planar three-dimensional constructs of the present invention demonstrate that, in the absence of active immune cells, the presence of tumor-associated non-tumor cells in the construct aids in reducing tumor cell mortality and aids in the formation of larger tumor cell aggregates. This effect is illustrated in Figure 7. As shown in Figure 8, when immune cells are introduced into a planar three-dimensional construct of the present invention as shown in Figure 7, the immune cells aggregate at the location of fibroblasts (HDFs) present near the interface with the immune cells. This phenomenon is illustrated in more detail in Figure 9. This figure shows significant aggregation of immune cells at the interface with fibroblasts interposed between the immune cells and tumor cells, although some infiltration is observed. This indicates that planar three-dimensional constructs of the present invention can be used as a tool to investigate and evade cell-based immunotherapy by isolating tumors from cellular components of the immune system and / or by non-tumor cells associated with the tumor.

[0086] Figure 14 The inventors also surprisingly discovered that stimulation of peripheral blood mononuclear cells (PBMCs) with tumor cells presented as three-dimensional constructs described herein can result in the clonal expansion of specific immune cell phenotypes, such as memory T cells (TCMs). Such memory T cells are highly desirable for use in immunotherapy. The inventors also unexpectedly discovered that this effect can be enhanced by including an intervening layer of cells found in peritumoral tissue (e.g., fibroblasts), which are positioned between the tumor cells and the PBMCs and provide both a physical and chemical barrier. Figure 10 shows the expansion of specific T cell phenotypes from stimulated PBMCs using three-dimensional tissue constructs incorporating patient-derived tumor cells, with and without an intervening fibroblast-containing layer. As shown, tumor cells and CD3+ tumor cells are evident in the coculture. Specific T cell phenotypes can be identified by the presence or absence of characteristic cell surface markers. For example, TCM cells can be identified by the presence of specific surface markers, such as CD4, CD8, CD45RA, CD45RO, CD197, CD62L, CD27, and CD95. An increase in CD4+ and CD8+ T cells is evident when cocultured with tumor cells. This effect is enhanced when activated PBMCs are cocultured with tumor cells in a three-dimensional construct that provides an intervening layer of fibroblasts. Corresponding results are seen in similar studies performed using three-dimensional tissue constructs containing patient-derived tumor cells, as shown in Figure 11. In both studies, we found that the use of an intervening layer incorporating fibroblasts increased infiltrating T cells and promoted their differentiation toward specific T cell phenotypes.

[0087] In some embodiments of the present invention, planar three-dimensional constructs of the present invention can be used to induce selective clonal expansion of immune cells. As shown in Figure 12, co-culture with tumor cells provided in three-dimensional cultures of the present invention provides selective enhancement of clonal expansion of TCM T cell phenotypes from PBMCs, particularly when fibroblasts are provided in the three-dimensional culture. Specifically, co-culture of PBMCs with tumor cells provided in three-dimensional cultures of the present invention resulted in an approximately four-fold increase in clonal expansion of TCM cells compared to PBMC cultures in the absence of such three-dimensional cultures. Co-culture of PBMCs with three-dimensional cultures of the present invention incorporating tumor cells and fibroblasts (as described above) resulted in an approximately 15-fold increase in clonal expansion of TCM cells compared to PBMC cultures in the absence of such three-dimensional cultures.

[0088] One embodiment of the inventive concept is a method for generating therapeutic cell products by stimulating immune cells ex vivo, co-culturing the stimulated immune cells with antigen-producing cells or pathogens (e.g., tumor cells or pathogen cells, or cells derived therefrom) in a three-dimensional culture (e.g., incorporating one or more tumor cell types and / or cells such as fibroblasts present in the capsular tissue surrounding such tumors), and then expanding the cells. The three-dimensional culture can incorporate an extracellular matrix and / or hydrogel. Specific phenotypes with therapeutic value can be selected from the resulting expanded population, for example, by flow cytometry to identify one or more specific cell surface markers.

[0089] In such methods, ex vivo stimulation of immune cells can be performed by any suitable method. Examples of suitable methods for stimulating immune cells include (but are not limited to) antigen-specific stimulation, CD3 / CD28 stimulation (with or without CD137), and / or exposure to stimulatory cytokines (e.g., IL2, IL7, IL15). Immune cells suitable for stimulation can be derived from PBMCs and / or tumor-infiltrating leukocytes. Similarly, gene editing methods (e.g., CRISPR) can be applied in situ to immune cells in the planar three-dimensional constructs of the present invention to modulate their function. For example, one or more CAR-T cells can be generated in situ in such constructs to determine their relative efficacy (which can then be used to determine treatment strategies).

[0090] The three-dimensional cultures utilized in such methods can include additional components, such as cytokine-producing cells (e.g., stromal cells, fibroblasts, endothelial cells, etc.) and / or other immune cells (e.g., dendritic cells). The extracellular matrix or hydrogel components of such three-dimensional cultures (e.g., dextran, gelatin, collagen, hyaluronic acid, polyethylene glycol, alginate, and / or chemically modified derivatives thereof) can provide mechanical stress components that can affect the differentiation and behavior of cells in culture. Thus, such structural components can provide stiffnesses ranging from about 200 Pa to about 20 kPa, or any range within this range. In some embodiments, the structural components of the three-dimensional cultures can include one or more components degradable by matrix metalloproteinases, which allow for the proliferation of antigen-producing cells (e.g., tumor cells).

[0091] The three-dimensional cultures utilized in such methods may include one or more layers comprising extracellular matrix and / or hydrogel components and having a thickness or depth of about 50 μm to about 2 mm. In some embodiments, the thickness of a layer may vary across its width and / or length. In three-dimensional cultures having multiple layers, the layers may have the same or different thicknesses. The extracellular matrix and / or hydrogel components of such three-dimensional cultures may be provided as continuous or discontinuous structures (e.g., patterned into distinct regions and / or orientations). Such discontinuous or patterned cultures may provide spatial separation of different cell populations and may be generated using any suitable technique (e.g., photopatterning, micropipetting, positioning of preformed segments, bioprinting, etc.). In some embodiments, the three-dimensional tissue cultures may incorporate one or more porous membranes, which may be positioned within the three-dimensional tissue culture (e.g., between two cell populations incorporated into the three-dimensional culture) or on the surface of the three-dimensional tissue culture. Such porous membranes can have pore sizes ranging from about 1 μm to about 10 μm, which can enhance cell selectivity.

[0092] Immune cells provided by and / or during clonal expansion using such devices and methods can include effector T cells and memory T cells. Such memory T cells can be memory T cell stem cells, central memory T cells, effector memory T cells, and / or transitional memory T cells. Other cell populations provided by and / or during clonal expansion using such devices and methods include cytolytic effector T cells, dendritic cells, NK cells, and / or macrophages. In some embodiments, immune cell populations provided by and / or during clonal expansion using such devices and methods can include a combination of two or more of these cell types.

[0093] Immune cells provided by selective clonal expansion as described above can be used for immunotherapy. For example, TCM cells derived from selective clonal expansion of PBMCs co-cultured with three-dimensional cultures containing tumor cells obtained from an individual can be used to provide patient-specific immunotherapy for that individual. In such embodiments, such immune cells can be isolated (e.g., using flow cytometry) and suspended in a pharmaceutically acceptable carrier. Such suspended cells can then be administered to an individual in need of treatment. For example, such immune cells can be suspended in a liquid medium and administered by intravenous infusion. Alternatively, such immune cells can be provided in a biocompatible, pharmaceutically acceptable hydrogel and / or tissue scaffold and applied locally to the tissue to be treated.

[0094] As discussed above, the planar three-dimensional constructs of the present invention provide novel and useful juxtapositions between pathogenic cells (e.g., tumor cells) and components of the cellular immune system that mimic those of an infection or disease process. Such constructs can also provide tissue- or disease-specific features that may reduce the effectiveness of a therapeutic approach, such as the presence of a capsule or other tissue barrier surrounding the affected tissue. These features provide a method for identifying or screening effective therapeutic modes, including personalized medicine. Finally, such planar three-dimensional constructs provide a mechanism for selective clonal expansion of immune cells that react with pathogenic cells. Such expanded clones can be recovered for use in therapeutic applications or therapeutic formulations. Such discoveries support a wide range of systems, methods, and compositions.

[0095] An embodiment of the inventive concept includes a method for predicting the diagnosis and treatment of a cancer patient by obtaining data regarding the pathology of the tumor of the cancer patient, determining the distribution of cancer cells, stromal cells, or immune cells within or near the tumor, and generating a plurality of three-dimensional models of the tumor containing two or more of: (i) tumor cells obtained from the cancer patient, (ii) stromal cells, and / or (iii) immune cells. The cancer cells, immune cells, and / or stromal cells can be obtained from the cancer patient or from a non-patient source (i.e., an allogeneic source, tissue or cell culture, explanted tissue, etc.). In such a method, each of the three-dimensional models reflects the distribution of cancer cells, stromal cells, or immune cells indicated by a pathology, and each of the plurality of three-dimensional models is deposited on a test surface, and tumor cells or an acellular layer of each of the plurality of three-dimensional models is attached to the test surface. The plurality of three-dimensional models is exposed to a plurality of anti-cancer treatments, and the effects of the anti-cancer treatments on the three-dimensional tumor models are characterized. The therapeutic effects of one or more anti-cancer treatments in the cancer patient can be predicted based on the effects on the three-dimensional tumor models.

[0096] Each of the multiple three-dimensional tumor models can include a first compartment and a second compartment, the first compartment having a first side and a second side, the first side being in contact with the test surface and the second side being in contact with the second compartment, and one or both of the first and second compartments (optionally) including one or more of an extracellular matrix, a biomaterial, and a biopolymer scaffold. Coexistence of tumor cells and immune cells in the first compartment can provide a model of an immunoinflammatory tumor. Alternatively, to provide a model of an immune-exclusion tumor, tumor cells can be present in the first compartment and immune cells can be absent from the first compartment, with immune cells present in or on the surface of the second compartment. In some embodiments, stromal cells (e.g., fibroblasts, endothelial cells, mesenchymal stem cells (MSCs), adipocytes, and / or pericytes) are positioned near the first compartment such that the stromal cells are sandwiched between the tumor cells and the immune cells (e.g., stimulatory immune cells of the innate or acquired immune system, inhibitory immune cells of the innate or acquired immune system, peripheral blood mononuclear cells (PBMCs), T cells, NK cells, B cells, dendritic cells, mast cells, neutrophils, and / or macrophages). Alternatively, in some embodiments, the tumor cells and stromal cells can coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, the tumor cells and immune cells coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, the immune cells and stromal cells coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, the tumor cells, stromal cells, and immune cells coexist in at least a portion of the plurality of three-dimensional tumor models. In some embodiments, immune cells are absent from the tumor model to provide a model of an immune desert tumor.

[0097] Such methods can include providing a liquid medium to a portion of a three-dimensional model. The liquid medium can include immune cells and, optionally, stromal cells. The tumor cells, stromal cells, and / or immune cells can be obtained from a surgical tumor sample, a tumor biopsy, normal tissue from a cancer patient, from the circulation of a cancer patient, or from an established cell line. In some embodiments, the immune cells are activated T cells that are prevented from infiltrating three-dimensional tumors under controlled, untreated conditions. Alternatively, the immune cells can be activated T cells that can infiltrate three-dimensional tumors under controlled, untreated conditions.

[0098] The data utilized in such methods can be obtained by one or more of immunohistochemistry (IHC), flow cytometry, gene expression, and other methods that provide information about the tumor microenvironment or tumor composition of an individual patient. The IHC data so provided can relate to the relative orientation and location of tumor, immune, and stromal compartments in cancer patients. Such data can be collected by automated analysis, such as high-content imaging, cell sorting, flow cytometry, proteomic analysis, expression analysis, and / or genomic sequencing. In some embodiments, data collected by automated analysis can be evaluated to quantify one or more of immune cell infiltration, tumor cell death, and other tumor microenvironment changes within multiple three-dimensional tumor models.

[0099] The anti-cancer treatments evaluated in such methods can be targeted cancer therapies, immunomodulators, chemotherapy, repurposed drugs traditionally used to treat conditions other than cancer, radiation, or a combination of two or more of these. The three-dimensional tumor models utilized in such methods can be generated and automated using a liquid handling system, such as a bioprinter. Such liquid handling systems can include a photomask and a light source. Such liquid handling systems can deposit one or more of an extracellular matrix, a biomaterial, or a biopolymer scaffold onto the test surface.

[0100] Some embodiments of the inventive concepts are methods for optimizing cancer treatment for a cancer patient by generating a first dataset including predicted past treatment outcomes developed using the methods described above for a plurality of cancer patients with a history of the disease, generating a second dataset including treatment outcomes for the plurality of cancer patients with a history of the disease, and providing an artificial intelligence system with a learning algorithm configured to access the first and second datasets and generate a proposed treatment planning algorithm that correlates or otherwise relates the predicted treatment outcomes to the treatment outcomes, and applying the treatment planning algorithm to the predicted treatment outcomes to provide or suggest one or more treatment protocols believed to be effective for treating the cancer patient. Such an artificial intelligence system can be configured as a neural network. In some embodiments, the artificial intelligence system is accessed via an information network and may also be accessed via a subscription service.

[0101] Some embodiments of the inventive concepts are methods for optimizing cancer treatment for a cancer patient by generating a first dataset including predicted past treatment responses using the methods described above for a plurality of cancer patients with a history of the disease, generating a second dataset including recorded treatment outcomes for the plurality of cancer patients with a history of the disease, and providing the first and second datasets to an artificial intelligence system including a learning algorithm to generate a treatment planning algorithm that correlates the predicted treatment responses with the treatment outcomes, and applying the treatment planning algorithm to data related to tumor pathology of the cancer patient to report or propose a treatment plan for the cancer patient. Such an artificial intelligence system can be configured as a neural network. In some embodiments, the artificial intelligence system can be accessed via an information network and can also be accessed via a subscription service.

[0102] Embodiments of the inventive concept include methods for conducting three-dimensional cell-based assays by obtaining a planar three-dimensional construct comprising a planar or essentially planar first layer of extracellular matrix or hydrogel within a culture vessel, where first cells (e.g., immune cells, target cells, or a combination of immune and target cells) are provided below or mixed within the first layer, and second cells (e.g., immune cells, target cells, and a combination of immune and target cells) are provided on top of the first layer, and measuring or otherwise characterizing interactions between the first and second cells over a period of time. The second cells can be tumor cells, non-tumor diseased mammalian cells, pathogens, bacteria, viruses; a source of chemokines, chemoattractants, stimuli that enable an immune response, and / or immune cells (e.g., T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and / or their derivatives). The second cells can be of human, non-human, or non-animal origin. Such constructs may further comprise stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and / or viruses.

[0103] Such constructs may include biomaterials such as polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and / or chemically modified derivatives thereof formulated for chemical or physical crosslinking. Such constructs may include at least one layer comprising a polymer polymerized using electromagnetic radiation, temperature, and / or time. Such constructs may include a planar or essentially planar second layer. The at least one layer may be selected for biocompatibility, modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers of the construct.

[0104] Constructs suitable for these methods can be fabricated using standard or customized molding methods, photolithography, bioprinting, and / or techniques to control the size, shape, and planarity of one or more layers of the construct. The first layer of such a construct can have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter, and a stiffness of between 50 Pa and 20 kPa.

[0105] An embodiment of the inventive concept includes a three-dimensional cell-based assay system comprising a first planar or essentially planar layer of extracellular matrix or hydrogel within a culture vessel; first cells (e.g., immune cells, target cells, or a combination of immune and target cells) below, mixed within, or above the first layer; a second layer of extracellular matrix or hydrogel above the first layer; and second cells (e.g., immune cells, target cells, a combination of immune and target cells, tumor cells, non-tumor diseased mammalian cells, pathogens, bacteria, viruses; a source of chemokines, a source of chemoattractants, a source of stimuli that enable an immune response) mixed in or disposed on the second layer. Such second cells can be of human, non-human animal, or non-animal origin. Suitable immune cells include stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and / or viruses. Such assay systems may further comprise stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and / or viruses. Such assay systems may include a planar or essentially planar second layer. Such assay systems may include immune cells such as T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and / or derivatives thereof.

[0106] Such assay systems may include biomaterials such as polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. Such assay systems may include at least one layer comprising a polymer produced by polymerization using electromagnetic radiation, temperature, or time.

[0107] At least one layer of such an assay system is selected for biocompatibility, modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. Such assay systems can be constructed using standard or customized molding methods, photolithography, bioprinting, or other techniques to control the size, shape, and planarity of the layers of the construct. The first layer of such an assay system can have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter, and a stiffness of between 50 Pa and 20 kPa.

[0108]

[0010] Embodiments of the present concepts include methods for assessing the effectiveness of a therapy by measuring at least one of cell viability, cell structure, and the presence of a marker of cell apoptosis in either a first or second cell in the manner described above. Such measurements may be obtained by at least one of live cell imaging, immunofluorescence, flow cytometry, proteomic analysis, and genomic analysis. Such methods may include obtaining measurements of immune cell infiltration into a layer of a construct or into a collection of target cells, analyzing protein secretion, characterizing cytokines, observable changes to the target or immune cells, characterizing apoptosis in the target or immune cells, characterizing proliferation in the target or immune cells, characterizing cell aggregate size in the target or immune cells, characterizing phenotypic changes in the target or immune cells, and / or characterizing genotypic changes in the target and immune cells. Such methods may include subjecting at least a portion of a layer of a three-dimensional construct or assay system to at least one of chemical digestion, enzymatic digestion, mechanical digestion, or disruption by addition of a chelating agent. Such steps may include robotic selection or aspiration to collect cells released by the digestion or disruption of the layer.

[0109] Embodiments of the inventive concepts include methods of providing personalized medicine by carrying out the above-described methods incorporating patient- or pathogen-derived target cells and autologous or allogeneic immune cells, subjecting the construct or assay system used to one or more therapies (e.g., radiation therapy, chemotherapy, targeted therapy, and / or immunotherapy), determining the efficacy of the one or more therapies with the construct or assay system, and providing efficacy information based on such determinations to medical professionals to aid in clinical decision-making for a particular patient or group of patients.

[0110] Embodiments of the inventive concept include expanding an immune cell population by performing the above-described method, wherein the three-dimensional construct or assay system comprises immune cells cultured near target cells in a first or second layer, providing an incubation period sufficient for the immune cells to proliferate and generate an expanded immune cell population, and recovering the expanded immune cell population from the three-dimensional construct or assay system. The expanded immune cell population is modified after recovery, for example, by genetic modification, further expansion, or a combination thereof. Such immune cells may be autologous or allogeneic.

[0111] Embodiments of the inventive concept include methods of producing antibodies by performing the above-described clonal expansion method, where the three-dimensional construct or assay system includes immune cells, which are cultured B cells or hybridoma cells, to generate an expanded immune cell population, selecting a cell subpopulation from the expanded immune cell population, and harvesting antibodies from the selected cell subpopulation. Such selection can be performed using live imaging, manual picking, and / or robotic picking.

[0112] Embodiments of the inventive concept include methods of endogenous cell therapy by performing the clonal expansion method described above, where the cells to be expanded are immune cells derived from peripheral blood, spleen, or a disease microenvironment, expanding the immune cells to generate an expanded immune cell population, and infusing at least a portion of the expanded immune cell population into an individual in need of treatment. In some embodiments, a subpopulation of immune cells (which can be evaluated for efficacy in a patient) is separated from the expanded immune cell population and infused into a patient in need of treatment. Evaluation of patient efficacy can be performed after or before infusion.

[0113] Embodiments of the inventive concept include methods of providing immune cells having a particular phenotype (e.g., peripheral blood mononuclear cells or tumor-infiltrating lymphocytes) by generating a three-dimensional culture including a layer of antigen-producing cells (e.g., tumor cells or pathogen cells), contacting the three-dimensional culture with immune cells to generate a co-culture, and incubating the co-culture for a period of time sufficient to generate immune cells having the particular phenotype. Suitable phenotypes include cytolytic effector T cells, NK cells, macrophages, memory T cells, e.g., stem cell memory T cells, central memory T cells, effector memory T cells, and transitional memory T cells. The period of time can be selected to produce a stimulatory effect on the immune cells, such as antigen-specific stimulation, CD3 / CD28 stimulation, CD3 / CD28 stimulation with CD137, and costimulation with one or more cytokines (e.g., IL2, IL7, and IL5). Such three-dimensional cultures may include a stromal cell layer (which may include fibroblasts) interposed between the antigen-producing cell layer and the immune cells. In some embodiments, the three-dimensional cultures may include cytokine-producing cells (e.g., stromal cells, fibroblasts, endothelial cells, cytokine-producing immune cells, and / or dendritic cells).

[0114] Such three-dimensional cultures may include hydrogel or extracellular matrix components. The three-dimensional cultures may include dextran, gelatin, collagen, hyaluronic acid, and polyethylene glycol, and at least a portion of the three-dimensional culture may be degradable by metalloproteinases. The three-dimensional cultures may have a thickness of 20 μm to 2 mm and a stiffness of 50 Pa to 20 kPa. The three-dimensional cultures may be patterned into distinct regions and / or directions, for example, within a container or well or on a surface. In some embodiments, the three-dimensional cultures may include a porous membrane.

[0115] Embodiments of the inventive concept include methods of providing immunotherapy by isolating immune cells having a particular phenotype produced by the methods described above to provide isolated immune cells, contacting the isolated immune cells with a pharmaceutically acceptable carrier to form an immunotherapeutic composition, and administering the immunotherapeutic composition (e.g., by infusion) to an individual in need of treatment. Suitable pharmaceutically acceptable carriers include liquid media suitable for injection or infusion. In some embodiments, the pharmaceutically acceptable carrier is a biocompatible hydrogel or tissue scaffold, which can be administered by topical application to a portion of the individual to be treated.

[0116] Embodiments of the present concepts include systems for providing immune cells with specific phenotypes, including three-dimensional cultures comprising a layer of antigen-producing cells (e.g., a pathogenic cell layer, a tumor cell layer, etc.) and immune cells (e.g., peripheral blood mononuclear cells or tumor-infiltrating lymphocytes) co-cultured in the three-dimensional culture, the three-dimensional culture being configured to enhance clonal expansion of the immune cells with the specific phenotype. The three-dimensional cultures in such systems may include a stromal cell layer interposed between the antigen-producing or tumor cell layer and the immune cells. The three-dimensional cultures may have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter, and may have a stiffness of between 50 Pa and 20 kPa. In such systems, the three-dimensional cultures may be patterned into distinct regions and / or directions.

[0117] In such systems, the three-dimensional culture can include a stromal cell layer interposed between the antigen-producing or tumor cell layer and the immune cells. Such a stromal cell layer can include fibroblasts. In some embodiments, the three-dimensional culture can include a hydrogel and / or extracellular matrix components. At least a portion of the three-dimensional culture can be degradable by metalloproteinases. In some embodiments, the three-dimensional culture can include dextran, gelatin, collagen, hyaluronic acid, alginate, polyethylene glycol, and / or a porous membrane.

[0118] In such systems, the three-dimensional cultures can be configured to provide stimulatory effects to immune cells, such as antigen-specific stimulation, CD3 / CD28 stimulation, CD3 / CD28 stimulation with CD137, and costimulation with one or more cytokines (e.g., IL2, IL7, and / or IL5). In some embodiments, the three-dimensional cultures comprise cytokine-producing cells. Suitable cytokine-producing cells include fibroblasts, endothelial cells, cytokine-producing immune cells, and dendritic cells.

[0119] In such systems, the immune cells having a selected phenotype can be memory T cells. Such memory T cells can be stem cell memory T cells, central memory T cells, effector memory T cells, and transitional memory T cells. In some embodiments, the immune cells having a selected phenotype are selected from the group consisting of one or more of cytolytic effector T cells, NK cells, and macrophages.

[0120]

[0010] Embodiments of the inventive concept include the use of immune cells having a specific phenotype to manufacture an immunotherapeutic composition, which involves isolating immune cells having a specific phenotype produced by the methods described above and providing the immune cells having a specific phenotype in a pharmaceutically acceptable carrier. Such a pharmaceutically acceptable carrier may be a liquid medium suitable for injection. Alternatively, the pharmaceutically acceptable carrier may be a biocompatible hydrogel or tissue scaffold.

[0121] Embodiments of the inventive concept include three-dimensional cell-based assay systems having a first planar or essentially planar layer of extracellular matrix or hydrogel (which can be a product of standard or customized molding, photolithography, or bioprinting) within a culture vessel, first cells (e.g., immune cells, target cells, or a combination of immune and target cells) beneath or mixed within the first layer, and a second planar layer containing second cells (e.g., immune cells, target cells, a combination of immune and target cells, tumor cells, non-tumor diseased mammalian cells, pathogens, bacteria, viruses, a source of chemokines, a source of chemoattractants, and / or a source of stimuli that enable an immune response). Such second cells can be of human, non-human animal, or non-animal origin. Such systems may include one or more other cell types, such as stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, viruses, T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and their derivatives. In some embodiments, the second layer may include an extracellular matrix or hydrogel. Such systems may include biomaterials or biocompatible materials, such as polymers of natural or synthetic origin; decellularized tissues, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. At least one layer of such systems may include a polymer polymerized using electromagnetic radiation, temperature, or time. At least one layer of such systems may be selected for biocompatibility, modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. In such a system, the first layer may have a defined thickness of between 10 micrometers and 2 millimeters, preferably between 100 micrometers and 1 millimeter.

[0122] It will be apparent to those skilled in the art that many modifications beyond those already described are possible without departing from the inventive concepts herein. Accordingly, the present subject matter should not be limited except by the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the term "comprising" should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly referenced. When the specification and claims refer to at least one selected from the group consisting of A, B, C..., and N, the text should be interpreted as requiring only one element from that group, rather than A plus N, B plus N, etc. Exemplary embodiments: 1. A method for predicting the diagnosis and treatment of a cancer patient, comprising: obtaining data regarding tumor pathology of said cancer patient; determining the distribution of cancer cells, stromal cells, or immune cells within or near the tumor; generating a plurality of three-dimensional models of the tumor comprising two or more of tumor cells, stromal cells, and immune cells obtained from the cancer patient; generating a plurality of three-dimensional models, each of which reflects the distribution of cancer cells, stromal cells, or immune cells indicated by the pathology, and depositing each of the plurality of three-dimensional models on a test surface, and binding tumor cells or an acellular layer of each of the three-dimensional models to the test surface; exposing the plurality of three-dimensional models to a plurality of anti-cancer treatments; Measuring the effect of the anti-cancer treatment on the three-dimensional tumor model; and Predicting the therapeutic effect of one or more of the anti-cancer treatments in the cancer patient based on their effect on the three-dimensional tumor model. A method comprising: 2. The method of embodiment 1, wherein each of the plurality of three-dimensional tumor models comprises a first compartment and a second compartment, the first compartment having a first side and a second side, the first side being in contact with the test surface and the second side being in contact with the second compartment, and one or both of the first compartment and the second compartment optionally comprising one or more of an extracellular matrix, a biomaterial, and a biopolymer scaffold. 3. The method of embodiment 2, wherein tumor cells and immune cells are co-present in said first compartment, thereby providing a model of immune inflamed tumors. 4. The method of embodiment 2, wherein tumor cells and no immune cells are present in the first compartment, and immune cells are present in or on the surface of the second compartment, so as to provide a model of immune excluded tumor. 5. The method of embodiment 4, wherein stromal cells are positioned near said first compartment such that the stromal cells are sandwiched between the tumor cells and the immune cells. 6. The method of embodiment 1, wherein tumor cells and stromal cells coexist in at least some of said multiple three-dimensional tumor models. 7. The method of embodiment 1, wherein tumor cells and immune cells coexist in at least some of said multiple three-dimensional tumor models. 8. The method of embodiment 1, wherein immune cells and stromal cells coexist in at least some of the multiple three-dimensional tumor models. 9. The method of embodiment 1, wherein tumor cells, stromal cells, and immune cells coexist in at least some of the multiple three-dimensional tumor models. 10. The method of embodiment 1, wherein immune cells are absent from said tumor model so as to provide a model of an immune desert tumor. 11. The method of any one of the preceding claims, further comprising providing a liquid medium to a portion of the three-dimensional model. 12. The method of embodiment 11, wherein the liquid medium comprises immune cells, and optionally stromal cells. 13. The method of any one of embodiments 1-12, wherein the tumor cells, stromal cells, or immune cells are obtained from a surgical sample of the tumor, a biopsy of the tumor, normal tissue of the cancer patient, or are obtained from the circulation of the cancer patient. 14. The method of any of embodiments 1 to 13, wherein the immune cells are activated T cells that are prevented from infiltrating three-dimensional tumors under control conditions in the absence of treatment. 15. The method of any of embodiments 1 to 13, wherein the immune cells are activated T cells capable of infiltrating three-dimensional tumors under controlled conditions without treatment. 16. The method of any of embodiments 1 to 15, wherein the data is obtained by at least one of immunohistochemistry (IHC), flow cytometry, gene expression, and a method that provides information about the tumor microenvironment or the makeup of an individual patient's tumor. 17. The method of embodiment 16, wherein the IHC provides data related to the relative orientation and location of tumor, immune, and stromal compartments in said cancer patient. 18. The method of any one of embodiments 1-17, wherein at least one of the plurality of anti-cancer treatments is selected from the group consisting of targeted cancer therapy, immunomodulatory agents, chemotherapy, repurposed drugs traditionally utilized to treat conditions other than cancer, radiation, and combinations of two or more thereof. 19. The method of any one of embodiments 1 to 18, wherein the three-dimensional tumor model is generated using a liquid handling system. 20. The method of embodiment 19, wherein the liquid handling system is a bioprinter. 21. The method of embodiment 19, wherein the liquid handling system comprises a photomask and a light source. 22. The method of any one of embodiments 19-21, wherein the liquid handling system is automated. 23. The method of any of embodiments 1-22, comprising depositing at least one of an extracellular matrix, a biomaterial, or a biopolymer scaffold onto the test surface. 24. The method of any of embodiments 1-23, wherein the stromal cells are selected from the group consisting of fibroblasts, endothelial cells, mesenchymal stem cells (MSCs), adipocytes, and pericytes. 25. The method of any one of embodiments 1 to 24, wherein the immune cells are selected from the group consisting of stimulatory immune cells of the innate or acquired immune system, inhibitory immune cells of the innate or acquired immune system, peripheral blood mononuclear cells (PBMCs), T cells, NK cells, B cells, dendritic cells, mast cells, neutrophils, and macrophages. 26. The method of any one of embodiments 1 to 25, wherein the data is collected by automated analysis, and the automated analysis is selected from the group consisting of high-content imaging, cell sorting, flow cytometry, proteomic analysis, expression analysis, and genomic sequencing. 27. The method of embodiment 26, comprising evaluating data collected by automated analysis to quantify one or more of immune cell infiltration, tumor cell death, and other tumor microenvironment changes within the plurality of three-dimensional tumor models. 28. The method of any of embodiments 1-27, wherein the stromal cells are obtained from the cancer patient. 29. The method of any of embodiments 1-27, wherein the stromal cells are obtained from a first alternative source that is not a cancer patient. 30. The method of any one of embodiments 1 to 29, wherein the immune cells are obtained from the cancer patient. 31. The method of any of embodiments 1-29, wherein the immune cells are obtained from a second, alternative source that is not a cancer patient. 32. A method for optimizing cancer treatment in a cancer patient, comprising: generating a first dataset comprising predicted past treatment effects for a plurality of cancer patients with a history of the disease using the method of any one of embodiments 1 to 31; generating a second dataset comprising treatment outcomes for the plurality of cancer patients with said history; and providing the first and second data sets to an artificial intelligence system including a learning algorithm to generate a treatment planning algorithm that correlates predicted treatment effect with treatment outcome; and Applying said treatment planning algorithm to the predicted treatment effect from application of the method according to any one of embodiments 1 to 31 to provide an optimized treatment plan recommendation. A method comprising: 33. A method for optimizing cancer treatment in a cancer patient, comprising: generating a first dataset comprising predicted past treatment effects for a plurality of cancer patients with a history of the disease using the method of any one of embodiments 1 to 31; generating a second dataset comprising treatment outcomes for the plurality of cancer patients with said history; and providing the first and second data sets to an artificial intelligence system including a learning algorithm to generate a treatment planning algorithm that correlates predicted treatment effect with treatment outcome; and and applying a treatment planning algorithm to data related to the pathology of the tumor of the cancer patient to provide an optimized treatment plan recommendation. A method comprising: 34. The method of embodiment 32 or 33, wherein the artificial intelligence system is configured as a neural network. 35. A method according to any one of embodiments 32 to 34, wherein the artificial intelligence system is accessed via an information network. 36. The method of embodiment 35, wherein the information network includes a user interface, and the user interface includes a subscription service. 37. A method for performing a three-dimensional cell-based assay, comprising: a planar three-dimensional construct comprising a planar or essentially planar first layer of extracellular matrix or hydrogel within a culture vessel, wherein first cells are provided below or mixed within the first layer, and second cells are provided above the first layer; evaluating the interaction between said first cell and said second cell over a period of time in liquid culture medium. 38. The method of embodiment 37, wherein the first cell is selected from the group consisting of an immune cell, a target cell, and a combination of an immune cell and a target cell. 39. The method of embodiment 37 or 38, wherein the second cell is selected from the group consisting of an immune cell, a target cell, and a combination of an immune cell and a target cell. 40. The method of any of embodiments 37-39, wherein the construct further comprises a cell type selected from the group consisting of stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and viruses. 41. The method of any of embodiments 37-40, wherein the construct comprises a planar or essentially planar second layer. 42. The method of any of embodiments 39 to 41, wherein the second cell is selected from the group consisting of a tumor cell, a non-tumor diseased mammalian cell, a pathogen, a bacterium, a virus, a source of a chemokine, a source of a chemoattractant, and a source of a stimulus that enables an immune response. 43. The method of any of embodiments 39 to 42, wherein the construct comprises an immune cell selected from the group consisting of a T cell, a B cell, a dendritic cell, a macrophage, a NK cell, a peripheral blood mononuclear cell (PBMC), and derivatives thereof. 44. The method of any of embodiments 39-43, wherein the second cell is of human, animal, or non-animal origin. 45. The method of any of embodiments 37 to 44, wherein the construct comprises a biomaterial selected from the group consisting of polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. 46. ​​The method of any of embodiments 37-45, wherein the construct comprises at least one layer comprising a polymer polymerized using electromagnetic radiation, temperature, or time. 47. The method of embodiment 46, wherein the at least one layer is selected for biocompatibility, elastic modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. 48. The method of any of embodiments 37-47, comprising manufacturing the construct using standard or customized molding methods, photolithography, bioprinting, or techniques that control the size, shape, and planarity of the layers of the construct. 49. The method of any one of embodiments 37-48, wherein the first layer has a specified thickness of between 10 micrometers and 2 millimeters. 50. The method of any one of embodiments 37-48, wherein the first layer has a specified thickness between 100 micrometers and 1 millimeter. 51. A three-dimensional cell-based assay system comprising: a first planar or essentially planar layer of extracellular matrix or hydrogel within a culture vessel; first cells below, mixed in, or above the first layer; and a second layer comprising second cells mixed into or disposed on the second layer; Including, the system. 52. The system of embodiment 51, wherein the first cell is selected from the group consisting of an immune cell, a target cell, and a combination of an immune cell and a target cell. 53. The system of embodiment 51 or 52, wherein the second cells are selected from the group consisting of immune cells, target cells, and a combination of immune cells and target cells. 54. A system described in any of embodiments 51 to 53, comprising a cell type selected from the group consisting of stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and viruses. 55. The system of any of embodiments 51-54, comprising a planar or essentially planar second layer. 56. The system described in any of embodiments 53 to 55, wherein the second cell is selected from the group consisting of a tumor cell, a non-tumor diseased mammalian cell, a pathogen, a bacterium, a virus, a source of a chemokine, a source of a chemoattractant, and a source of a stimulus that enables an immune response. 57. The system according to any of embodiments 53 to 56, comprising immune cells selected from the group consisting of T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and derivatives thereof. 58. The system of any of embodiments 53-57, wherein the second cell is of human, animal, or non-animal origin. 59. The system according to any of embodiments 51 to 58, comprising a biomaterial selected from the group consisting of polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. 60. The system of any of embodiments 51-59, wherein the system comprises at least one layer comprising a polymer polymerized using electromagnetic radiation, temperature, or time. 61. The system of embodiment 60, wherein the at least one layer is selected for biocompatibility, elastic modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. 62. The system of any of embodiments 51-61, wherein the first or second layer is the product of standard or customized molding, photolithography, or bioprinting. 63. The system of any of embodiments 51-62, wherein the first layer has a specified thickness between 10 micrometers and 2 millimeters. 64. A system described in any of embodiments 51-62, wherein the first layer has a specified thickness between 100 micrometers and 1 millimeter. 65. A method for evaluating the effectiveness of a therapy according to any one of embodiments 37 to 50, comprising determining at least one of cell viability, cell structure, and the presence of a cell apoptosis marker in either the first or second cell. 66. The method of embodiment 64, wherein the evaluation is performed by at least one of live cell image analysis, immunofluorescence, flow cytometry, proteomic analysis, and genomic analysis. 67. The method of embodiment 65 or 66, comprising at least one of characterizing immune cell infiltration into the layer of the construct, immune cell infiltration into the target cell aggregates, analysis of protein secretion, characterization of cytokines, observable changes to the target cells or immune cells, characterization of apoptosis in the target cells or immune cells, characterization of proliferation in the target cells or immune cells, characterization of cell aggregate size in the target cells or immune cells, characterization of phenotypic changes in the target cells or immune cells, and characterization of genotypic changes in the target and immune cells. 68. A method for recovering cells from a system described in any of embodiments 51 to 64, comprising subjecting at least a portion of a layer of the construct to at least one of chemical digestion, enzymatic digestion, mechanical digestion, or disruption by addition of a chelating agent. 69. The method of embodiment 68, comprising at least one of robotic selection or aspiration to collect cells released by digestion or disruption of the layer. 70. A method of providing personalized medicine, comprising: Carrying out the method of any of embodiments 37-50, comprising target cells derived from a patient or a pathogen and autologous or allogeneic immune cells; subjecting the construct to a therapy selected from the group consisting of radiation therapy, chemotherapy, targeted therapy, and immunotherapy; and determining the effectiveness of said treatment; and providing efficacy information to healthcare professionals to support clinical decision-making for specific patients or patient groups. A method comprising: 71. A method for expanding an immune cell population, comprising: 51. Carrying out the method according to any of embodiments 37-50, wherein immune cells are cultured in the vicinity of target cells in said first or second layer. providing an incubation period sufficient for the immune cells to proliferate and generate an expanded immune cell population; and recovering the expanded immune cell population from the construct. A method comprising: 72. The method of embodiment 71, wherein the expanded immune cell population is modified after harvesting. 73. The method of embodiment 72, wherein the expanded immune cell population is modified by genetic modification, further expansion, or a combination thereof. 74. The method of any of embodiments 70-72, wherein the immune cells are of autologous or allogeneic origin. 75. A method for producing an antibody, comprising: 74. Performing the method according to any one of embodiments 70-73, wherein the immune cells are cultured B cells or hybridoma cells to generate an expanded immune cell population. selecting a cell subpopulation from the expanded immune cell population; and harvesting said antibody from said cell subpopulation. A method comprising: 76. The method of embodiment 74, wherein the selecting is performed by live imaging, manual picking, and robotic picking. 77. A method of endogenous cell therapy comprising: Performing the method according to any one of embodiments 70-73, wherein the immune cells are derived from peripheral blood, spleen, or a disease microenvironment. Proliferating the immune cells to generate an expanded immune cell population; and infusing at least a portion of said expanded immune cell population into an individual in need of treatment. A method comprising: 78. The method of embodiment 77, comprising separating a subpopulation of immune cells relative to the expanded immune cell population, and infusing the subpopulation of immune cells into a patient in need of treatment. 79. The method described in embodiment 78, comprising assessing a subpopulation of immune cells by a method described in any one of embodiments 37 to 50 to assess patient efficacy. 80. The method of embodiment 79, wherein the assessment of patient efficacy is performed after infusion. 81. The method of embodiment 79, wherein the assessment of patient efficacy is performed before infusion. 82. A method for providing immune cells having a specific phenotype, comprising: generating a three-dimensional culture comprising an antigen-producing cell or tumor cell layer; contacting the three-dimensional culture with immune cells to generate a co-culture; incubating said co-culture for a period of time sufficient to generate said immune cells having said particular phenotype. A method comprising: 83. The method of embodiment 82, wherein the three-dimensional culture comprises a stromal cell layer, the stromal cell layer being interposed between the antigen-producing cells or tumor cell layer and the immune cells. 84. The method of embodiment 83, wherein the stromal cell layer comprises fibroblasts. 85. The method of any of embodiments 82-84, wherein the three-dimensional culture comprises a hydrogel or extracellular matrix component. 86. The method of any of embodiments 82 to 85, wherein incubating the co-culture for a period of time sufficient to generate the immune cells having the particular phenotype results in a stimulatory effect on the immune cells, the stimulatory effect being selected from the group consisting of antigen-specific stimulation, CD3 / CD28 stimulation, CD3 / CD28 stimulation with CD137, and co-stimulation with one or more cytokines. 87. The method of embodiment 86, wherein the one or more cytokines are selected from the group consisting of IL2, IL7, and IL5. 88. The method of any of embodiments 82 to 87, wherein the immune cells are peripheral blood mononuclear cells or tumor-infiltrating lymphocytes. 89. The method of any of embodiments 82-88, wherein the antigen-producing cells are pathogen cells. 90. The method of any of embodiments 82-89, wherein the three-dimensional culture comprises cytokine-producing cells. 91. The method of embodiment 90, wherein the cytokine-producing cells are selected from the group consisting of stromal cells, fibroblasts, endothelial cells, cytokine-producing immune cells, and dendritic cells. 92. The method of embodiment 92, wherein the three-dimensional culture has a stiffness of 50 Pa to 20 kPa. 93. The method of any of embodiments 82-92, wherein at least a portion of the three-dimensional culture is degradable by matrix metalloproteinases. 94. The method of any of embodiments 82-93, wherein the three-dimensional culture comprises one or more components selected from the group consisting of dextran, gelatin, collagen, hyaluronic acid, and polyethylene glycol. 95. The method of any of embodiments 82 to 94, wherein the three-dimensional culture has a thickness of 20 μm to 2 mm. 96. The method of any of embodiments 82-95, wherein the three-dimensional culture is patterned into distinct regions and / or directions. 97. The method of any of embodiments 82-96, wherein the three-dimensional culture comprises a porous membrane. 98. The method of any of embodiments 82-97, wherein said immune cells having a selected phenotype are memory T cells. 99. The method of embodiment 98, wherein the memory T cells are selected from the group consisting of stem cell memory T cells, central memory T cells, effector memory T cells, and transitional memory T cells. 100. The method of any of embodiments 82-98, wherein the immune cells having a selected phenotype are selected from the group consisting of one or more of cytolytic effector T cells, NK cells, and macrophages. 101. A method of providing immunotherapy, comprising: providing isolated immune cells by isolating immune cells having a specific phenotype produced by the method according to any one of embodiments 82 to 101; contacting the isolated immune cells with a pharmaceutically acceptable carrier to produce an immunotherapeutic composition; and administering said immunotherapeutic composition to an individual in need of treatment. A method comprising: 102. The method of embodiment 101, wherein the pharmaceutically acceptable carrier is a liquid medium and administration is by injection into the individual being treated. 103. The method of embodiment 101, wherein the pharmaceutically acceptable carrier is a biocompatible hydrogel or tissue scaffold and administration is by topical application to a portion of the individual to be treated. 104. A system for providing immune cells with a specific phenotype, comprising: a three-dimensional culture comprising an antigen-producing cell or tumor cell layer, and Immune cells co-cultured with the three-dimensional culture wherein the three-dimensional culture is configured to enhance clonal expansion of the immune cells having the particular phenotype. 105. The system of embodiment 104, wherein the three-dimensional culture comprises a stromal cell layer, the stromal cell layer being interposed between the antigen-producing cells or tumor cell layer and the immune cells. 106. The system of embodiment 105, wherein the stromal cell layer comprises fibroblasts. 107. A system described in any of embodiments 104 to 106, wherein the three-dimensional culture comprises a hydrogel or extracellular matrix component. 108. A system described in any of embodiments 104 to 107, wherein the three-dimensional culture is configured to provide a stimulatory effect on the immune cells, the stimulatory effect being selected from the group consisting of antigen-specific stimulation, CD3 / CD28 stimulation, CD3 / CD28 stimulation with CD137, and co-stimulation with one or more cytokines. 109. The system of embodiment 108, wherein the one or more cytokines are selected from the group consisting of IL2, IL7, and IL5. 110. A system described in any of embodiments 104 to 109, wherein the immune cells are peripheral blood mononuclear cells or tumor-infiltrating lymphocytes. 111. A system described in any of embodiments 104 to 110, wherein the antigen-producing cells are pathogen cells. 112. The system of any of embodiments 104 to 111, wherein the three-dimensional culture comprises cytokine-producing cells. 113. The system described in embodiment 112, wherein the cytokine-producing cells are selected from the group consisting of stromal cells, fibroblasts, endothelial cells, cytokine-producing immune cells, and dendritic cells. 114. The system of any of embodiments 104-113, wherein the three-dimensional culture has a stiffness of 50 Pa to 20 kPa. 115. A system according to any of embodiments 104 to 114, wherein at least a portion of the three-dimensional culture is degradable by a metal metalloproteinase. 116. A system described in any of embodiments 104 to 115, wherein the three-dimensional culture comprises one or more components selected from the group consisting of dextran, gelatin, collagen, hyaluronic acid, alginate, and polyethylene glycol. 117. A system described in any of embodiments 104 to 116, wherein the three-dimensional culture has a thickness of 20 μm to 2 mm. 118. A system described in any of embodiments 104 to 117, wherein the three-dimensional culture is patterned into distinct regions and / or directions. 119. The system of any of embodiments 104-118, wherein the three-dimensional culture comprises a porous membrane. 120. A system described in any of embodiments 104 to 119, wherein the immune cells having a selected phenotype are memory T cells. 121. The system of embodiment 120, wherein the memory T cells are selected from the group consisting of stem cell memory T cells, central memory T cells, effector memory T cells, and transitional memory T cells. 122. A system described in any of embodiments 104 to 121, wherein the immune cells having a selected phenotype are selected from the group consisting of one or more of cytolytic effector T cells, NK cells, and macrophages. 123. Use of immune cells having a specific phenotype for producing an immunotherapeutic composition, comprising isolating immune cells having a specific phenotype produced by the method of any of embodiments 82 to 101, and providing said immune cells having a specific phenotype in a pharmaceutically acceptable carrier. 123. The use according to embodiment 123, wherein the pharmaceutically acceptable carrier is a liquid medium suitable for injection. 124. The use according to embodiment 123, wherein the pharmaceutically acceptable carrier is a biocompatible hydrogel or tissue scaffold. 125. A three-dimensional cell-based assay system comprising: a first planar or essentially planar layer of extracellular matrix or hydrogel within a culture vessel; a first cell below or mixed within the first layer; a second layer comprising second cells, said second layer being essentially planar; A system including: 126. The system of embodiment 125, wherein the first cell is selected from the group consisting of an immune cell, a target cell, and a combination of an immune cell and a target cell. 127. The system of embodiment 125 or 126, wherein the second cells are selected from the group consisting of immune cells, target cells, and a combination of immune cells and target cells. 128. A system described in any of embodiments 125 to 127, wherein the system further comprises a cell type selected from the group consisting of stromal cells, fibroblasts, endothelial cells, immune cells, normal tissue cells, diseased cells, pathogens, bacteria, and viruses. 129. The system of any of embodiments 125-128, wherein the second layer comprises an extracellular matrix or a hydrogel. 130. The system described in any of embodiments 125 to 129, wherein the second cell is selected from the group consisting of a tumor cell, a non-tumor diseased mammalian cell, a pathogen, a bacterium, a virus, a source of a chemokine, a source of a chemoattractant, and a source of a stimulus that enables an immune response. 131. The system described in any of embodiments 125 to 130, wherein the system comprises immune cells selected from the group consisting of T cells, B cells, dendritic cells, macrophages, NK cells, peripheral blood mononuclear cells (PBMCs), and derivatives thereof. 132. The system of any of embodiments 125-131, wherein the second cell is of human, animal, or non-animal origin. 133. The system according to any of embodiments 125 to 132, wherein the system comprises a biomaterial selected from the group consisting of polymers of natural or synthetic origin; decellularized tissue, proteins, dextran, alginate, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, combinations thereof, and chemically modified derivatives thereof formulated for chemical or physical crosslinking. 133. The system of any of embodiments 125-132, comprising at least one layer comprising a polymer polymerized using electromagnetic radiation, temperature, or time. 134. The system described in embodiment 133, wherein the at least one layer is selected for biocompatibility, elastic modulus, porosity, and degradability to allow nutrient exchange and cellular interaction within or between layers. 135. A system described in any of embodiments 125 to 134, wherein the first layer is the product of a standard or customized molding, photolithography, or bioprinting step. 136. The system of any of embodiments 125-135, wherein the first layer has a specified thickness between 10 micrometers and 2 millimeters. 137. The system of any of embodiments 125-135, wherein the first layer has a specified thickness between 100 micrometers and 1 millimeter.

Claims

1. 1. A method for reducing the time it takes to expand an immune cell population, comprising: Obtaining a three-dimensional construct comprising a planar or essentially planar first layer of extracellular matrix or hydrogel in a culture vessel, said three-dimensional construct having tumor cells provided beneath or mixed therein; Culturing immune cells in contact with the three-dimensional construct; providing an incubation period sufficient for the immune cells to proliferate and generate an expanded immune cell population; and recovering the expanded immune cell population from the construct. A method comprising:

2. 10. The method of claim 1, wherein the expanded immune cell population is modified after harvesting, wherein the expanded immune cell population is modified by genetic modification, further expansion, or a combination thereof.

3. 10. The method of claim 1, wherein the three-dimensional construct further comprises a second layer comprising second cells, the second layer being above the first layer.

4. The method of claim 3 , wherein the second cell is a stromal cell.

5. 1. A method for expanding a specific immune cell phenotype, comprising: generating a three-dimensional culture comprising a first layer comprising tumor cells and a second layer comprising stromal cells; contacting the three-dimensional culture with a plurality of immune cells to generate a co-culture, wherein the plurality of immune cells is in contact with the second layer; incubating the co-culture for a period of time sufficient for clonal expansion of an expanded immune cell population comprising the immune cells; and harvesting the expanded immune cell population. A method comprising:

6. The method of claim 5 , wherein the immune cells are peripheral blood mononuclear cells.

7. 6. The method of claim 5, wherein the three-dimensional culture is patterned into distinct regions or directions.

8. 6. The method of claim 5, wherein the immune cells having a selected phenotype are memory T cells.

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