Methods of using immune organoids to predict drug efficacy

Abstract

The present disclosure relates to methods of using immune organs to evaluate the efficacy of a therapeutic agent. The present disclosure also provides compositions useful in the methods.

Description

Cross-reference of related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 621,935, filed January 17, 2024, the disclosure of which (including drawings) is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to a method for predicting the efficacy of therapeutic agents using immune organoids. This disclosure also provides compositions of immune organoids and therapeutic agents for use in said method. Background Technology

[0003] A key challenge in evaluating the efficacy of immunotherapeutic drugs is determining humoral and cellular responses and / or immunostimulatory or immunosuppressive phenotypes. Therefore, it is crucial to have suitable models for testing potential drugs and their effects on the immune system. Drugs entering clinical development have a high consumption rate (approximately 95%), but the cost of developing new drugs is rising (approximately $800 million). This high consumption rate is attributed to the current methods used for drug discovery, efficacy testing, and drug development in two-dimensional (2D) cell-culture analysis and in vivo animal models. Currently, mice and non-human primates (NHPs) are used, but there is considerable evidence that drugs safe in mice are dangerous in humans, and vice versa. This is due to the significant differences in physiology, tissue structure, and proteins between mice and NHPs and humans. Not to mention, mice are also inbred, single-sex, and kept under sterile conditions. Therefore, there is an urgent need for improved tools and techniques for testing the efficacy of immunotherapies, which remain unmet. Summary of the Invention

[0004] This disclosure shows the development of a new method for evaluating the efficacy of therapeutic agents using 3D immune organoids.

[0005] This article provides a method for evaluating the efficacy of a therapeutic agent. The method comprises (a) contacting a three-dimensional immune organoid comprising a plurality of self-assembled primary immune cells and a plurality of stem cells obtained from one or more secondary lymphoid organs with a therapeutic agent, and (b) measuring the immune response of the three-dimensional immune organoid to the therapeutic agent after contact.

[0006] In some embodiments, the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+.

[0007] In some embodiments, one or more secondary lymphoid organs are derived from the spleen, lymph nodes, Peyer's patch, and / or MALT.

[0008] In some embodiments, immune organoids are human immune organoids.

[0009] In some embodiments, immune organoids further include peripheral blood mononuclear cells.

[0010] In some embodiments, secondary lymphoid organs are obtained from living patients, surgical excisions, fine needle aspirates, biopsies, and deceased patients.

[0011] In some embodiments, a plurality of primary immune cells include B cells, T cells, plasmablasts, plasma cells, ILCs, granulocytes, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof. In some embodiments, B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or combinations thereof. In some embodiments, T cells include naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, CD4 effector memory T cells, CD8 effector memory T cells, or combinations thereof. In some embodiments, dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, bone marrow-like dendritic cells, or combinations thereof.

[0012] In some embodiments, the plurality of primary cells further include one or more stromal cells, follicular dendritic cells, and fibroblastic reticular cells.

[0013] In some embodiments, the diameter of the immune organoids is 8000 µm or less.

[0014] In some embodiments, immune organoids include germinal centers and / or B / T cell regions.

[0015] In some embodiments, immune organoids produce antibodies. In some embodiments, the antibodies are IgG, IgM, or IgA antibodies. In some embodiments, the antibodies have complete humoral function. In some embodiments, the antibodies bind to human and non-human targets. In some embodiments, human targets include proteins, sugars, and nucleic acids. In some embodiments, non-human targets include infectious disease antigens, venoms, poisons, and small molecules.

[0016] In some embodiments, the therapeutic agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from the group consisting of vaccines, antibodies, gene therapy, cell therapy, small molecules, and nanobodies.

[0017] In some embodiments, measuring an immune response includes measuring the number of immune cells, immune cell proliferation, immune cell phenotype, immune cell polarization, antibody production, cytokine production, chemokine production, B cell receptor changes, T cell receptor changes, and / or immune memory. In some embodiments, measuring B cell receptor changes includes measuring antibody isotype switching, clonal expansion, somatic hypermutation, and / or memory B cells. In some embodiments, measuring T cell receptor changes includes measuring T cell clonal expansion.

[0018] This article also provides a composition comprising a three-dimensional immune organoid containing multiple self-assembled primary immune cells obtained from one or more secondary lymphoid organs and multiple stem cells, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+; and a therapeutic agent.

[0019] In some embodiments, one or more secondary lymphoid organs are derived from the spleen, lymph nodes, Pyayne lymph plexus, and / or MALT.

[0020] In some embodiments, the plurality of immune cells are human immune cells.

[0021] In some embodiments, the plurality of immune cells includes 2 × 10 6 One or fewer cells.

[0022] In some embodiments, the composition further comprises peripheral blood mononuclear cells.

[0023] In some embodiments, the multiple immune cells are obtained from living patients, surgical excisions, fine needle aspirates, biopsies, and deceased patients.

[0024] In some embodiments, the plurality of immune cells include B cells, T cells, plasmablasts, plasma cells, ILCs, granulocytes, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof. In some embodiments, B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or combinations thereof. In some embodiments, T cells include naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, CD4 effector memory T cells, CD8 effector memory T cells, or combinations thereof. In some embodiments, dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, bone marrow-like dendritic cells, or combinations thereof.

[0025] In some embodiments, the plurality of primary cells further include one or more stromal cells, follicular dendritic cells, and fibroblastic reticular cells.

[0026] In some embodiments, the diameter of the immune organoids is 8000 µm or less.

[0027] In some embodiments, immune organoids include germinal centers and / or B / T cell regions.

[0028] In some embodiments, immune organoids produce antibodies. In some embodiments, the antibodies are IgG, IgM, or IgA antibodies. In some embodiments, the antibodies have complete humoral function. In some embodiments, the antibodies bind to human and non-human targets. In some embodiments, human targets include proteins, sugars, and nucleic acids. In some embodiments, non-human targets include infectious disease antigens, venoms, poisons, and small molecules.

[0029] In some embodiments, the therapeutic agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from the group consisting of vaccines, antibodies, gene therapy, cell therapy, small molecules, and nanobodies.

[0030] The headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter.

[0031] While various features of this disclosure may be described in the context of a single embodiment, features may also be provided individually or in any suitable combination. Conversely, although this disclosure may be described herein in the context of a single embodiment for clarity, embodiments may also be implemented in a single embodiment. Attached Figure Description

[0032] The features of this disclosure are specifically set forth in the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of this disclosure, and in the drawings:

[0033] Figure 1 This study demonstrates the morphological changes in immune organoids after treatment with therapeutic agents.

[0034] Figure 2A-2C This demonstrates the stem cell population present in immune organoids after treatment with an experimental influenza subunit vaccine. Figure 2A and 2B This presentation quantifies the stem cell types in representative immune organoids from total viable cell cultures on day 7. Cell frequencies were determined by flow cytometry. The plotted values ​​are from two different donors. Figure 2C This presentation showcases a representative flow cytometry analysis of stem cell populations contained in representative immune organoids from day 7 cultures. The cells shown are pre-selected onto live, single CD45+CD34+CD45RA- cells. The data presented are from two donors.

[0035] Figures 3A-3F This demonstrates that the diversity of immune cell populations in human immune organoids is maintained after immunotherapy treatment. Figure 3A This presentation shows a representative flow cytometry analysis of B (CD19+) and T (CD3+) cells contained in representative immune organoids from cultures dated to day 7 following stimulation with an experimental influenza subunit vaccine on day 0. The cells shown were pre-selected onto live, single CD45+ cells. Data presented were from two donors. Figure 3B This presentation shows a representative flow cytometry analysis of plasmablasts (CD27+CD38++) stimulated with donor-unvaccinated Imovax rabies vaccine. The cells shown are pre-selected onto live, single CD45+ and CD19+ cells. Figure 3C This study presents the quantification of CD138+ plasma cell populations in immune organoids after exposure to five developmental influenza mRNA vaccine conditions and one unstimulated control. Analysis was performed using samples from three independent donors, and data are presented as mean ± standard deviation (n = 4 technical replicates / conditions). Figure 3D This image shows representative flow cytometry analyses of NK cells in unstimulated control immune organoids (left) and immune organoids (right) 7 days after treatment with an experimental antibody therapeutic agent designed to activate the immune system. The cells shown are pre-selected onto live, single CD45+ cells. NK cells are characterized by those that are CD56+CD16+. Figure 3E This study presents a representative flow cytometry analysis of granulocyte populations in immune organoids following subunit influenza vaccination. The analysis is based on high side-scattering (SSC) data. 高 ) and CD15 expression gated cells and plotted to show SSCs 高 The presence of a CD15+ granulocyte population. Figure 3F This presentation showcases a representative flow cytometry analysis of immune organoid myelomorphic populations after treatment with a subunit influenza vaccine. Flow cytometry plots illustrate dendritic cells (CD11b+CD45+), monocytes (CD14+CD11b+), and macrophages (which can be considered SSCs against CD14+). A sequential gating strategy was used to identify distinct populations for each myelomorphic group.

[0036] Figures 4A-4C This demonstrates that immune organoids undergo B cell differentiation after treatment. Figure 4ARepresentative images from flow cytometry analysis are presented, showing representative immune organoid B cell differentiation at days 0, 7, 14, and 21 post-treatment. All cells shown were previously gated onto total B cells (CD3-CD19+CD45+). Phenotypes shown: CD38-CD27-naïve B cells, CD38-CD27+ memory B cells, CD38+CD27-pre-GC B cells, and CD38+CD27+ GC B cells. A drawing from a representative donor is also presented. Figure 4B This study quantifies the presence of B cells in immune organoids on days 0, 7, 14, and 21 following immunization with hemagglutinin. Cell frequencies were determined by flow cytometry. Cells were previously gated onto total B cells (CD3-CD19+CD45+). Figure 4C This study presents the quantification of CD138+ plasma cell populations in immune organoids after exposure to five developmental mRNA influenza vaccine conditions and one unstimulated control. Analysis was performed using samples from three independent donors, and data are presented as mean ± standard deviation (n = 4 technical replicates / conditions).

[0037] Figures 5A-5C The immune organoids after immunotherapy treatment are shown to be composed of T cell subtypes. Figure 5A This presentation shows a representative flow cytometry analysis of T cell populations in immune organoids on day 7 after treatment with an influenza vaccine using experimental adjuvants. Analysis was performed on selected pass T cells (CD19-CD3+), and the data are presented as total T cells, CD4+ and CD8+ T cells, CD4+ naive T cells (CD3+CD4+CD45RA+), CD4+ memory T cells (CD3+CD4+CD45RO+), and CD4+ effector memory cells (CD3+CD4+CD45RO+CCR7-). Data from two representative donors are presented, along with relevant markers. Figures 5B-5C This presentation shows a representative flow cytometry analysis of T cell subgroups in immune organoids treated with influenza vaccine using experimental adjuvants on day 0. Analysis was performed on selected gated T cells (CD19-CD3+), followed by subgroup-specific gating. The presented population includes naive CD8+ T cells (CD3+CD8+CD45RA+), memory CD8+ T cells (CD3+CD8+CD45RO+), CD8+ effector memory cells (CD3+CD8+CD45RO+CCR7-), regulatory T cells (CD3+CD4+CD25+), γδ T cells (CD3+CD27+), and T follicular helper cells (CD3+CD25+CXCR5+). Data from two representative donors are presented, along with indications of relevant markers.

[0038] Figures 6A-6C This study demonstrates that immune organoids are composed of other immune cell types, including bone marrow-like dendritic cells, plasmacytoid dendritic cells, and dendritic cells. It also presents a representative flow cytometry analysis of dendritic cell populations in immune organoids after treatment with an experimental subunit of influenza vaccine. Figure 6A The flow cytometry plot shows CD14+CD11c+ myeloid dendritic cells from two independent donors. Figure 6B Analysis of plasmacytoid dendritic cells (CD123+) pre-gated on the CD45RA+CD11c+ population. Figure 6C Demonstrates the identification of CD11b+CD45+ dendritic cells. The data presented are representative plots from independent experiments.

[0039] Figures 7A-7C This demonstrates that, after treatment with the experimental subunit influenza vaccine, immune organoids consist of other immune cell types, including stromal cells, fibroblasts, reticular cells, and follicular dendritic cells. A representative flow cytometry analysis of the stromal population in the treated immune organoids is also presented. Figure 7A The image shows a flow cytometry plot of a CD45-stromal cell population. Figure 7B Demonstrates the identification of fibroblast reticulocytes (PDPN+CD31+). Figure 7C This presentation shows an analysis of follicular dendritic cells (CD45+PDPN+CD35+). The data presented are representative plots from independent donors.

[0040] Figure 8 This demonstrates that the size and morphology of immune organoids can be key indicators of therapeutic efficacy. Representative bright-field images of immune organoids are shown on days 5 and 21, following administration of the developmental mRNA influenza vaccine on day 0. The blue line indicates the area of ​​a representative organoid subgroup, and the diameter of each organoid is calculated from this area. Rows A to E represent different vaccine formulations, and columns 1-5 represent decreases in mRNA concentration.

[0041] Figures 9A-9C This demonstrates how immune organoids respond to specific treatments designed to drive the differentiation of plasmablasts and plasma cells, mirroring the function of human lymph nodes, to form germinal centers. Figure 9A This image shows a representative bright-field image of organoids immunized on day 14 after receiving an inactivated Hep A vaccine. The shallower structures in the organoids, outlined by dashed circles, are consistent with the morphology of the germinal centers. Figure 9B Representative images stained by flow cytometry show that immune organoids consist of B (CD19+) and T cell (CD3+) regions. All cells were previously gated onto live, single CD45+ cells. The drawing is from representative day 7 immune organoids treated with an experimental influenza subunit vaccine. Figure 9CRepresentative images from flow cytometry analysis show that immune organoids are composed of germinal center B cells. The cells shown are gated onto live, single CD45+, CD3-, and CD19+ cells, with the germinal center B cells being CD27+CD38+. The plots are from four representative day 14 immune organoids treated with hemagglutinin.

[0042] Figures 10A-10E This demonstrates how immune organoids respond to vaccination by forming antigen-specific antibodies. Figure 10A This presentation showcases antigen-specific IgG antibodies derived from immune organoids on days 4, 8, 11, and 15 of 12 different doses and formulations of an experimental influenza subunit vaccine. The data presented are from a representative donor. Figure 10B Representative images from flow cytometry analysis are presented, showing plasmablast differentiation of immune organoids following vaccination with an adjuvanted influenza subunit vaccine. The plots are from representative D0 and D14 vaccinated immune organoids. The cells shown are previously gated onto live single and total B cells (CD19+ CD3- CD45+). Phenotypes shown include: CD38-CD27-naïve B cells, CD38-CD27+ memory B cells, CD38+CD27- GC pre-B cells, CD38+CD27+ GC B cells, and CD38++CD27+ plasmablasts. A plot from a representative donor is also presented. Figure 10C This study demonstrates the characteristics of antigen-specific immunoglobulin production following vaccination in two independent donors. Concentrations of IgG1-4 subtypes, IgM, and IgA were measured in response to treatments A and B, which were two different doses of rabies vaccine. Analyses were performed on four technical replicates / conditions, and concentrations are reported in pg / mL. Data are presented as mean ± standard deviation (n=4). Figure 10D The presentation shows antigen-specific IgM antibodies in immune organoids stimulated twice with influenza subunit vaccines using six different formulations of experimental adjuvants on days 4, 8, 11, and 14. A drawing from a representative donor is also included. Figure 10E The presentation shows antigen-specific IgG antibodies in immune organoids stimulated twice with influenza subunit vaccines using six different formulations of experimental adjuvants on days 4, 8, 11, and 14. A drawing from a representative donor is also included.

[0043] Figure 11A-11D This demonstrates that immune organoids can respond to foreign antigens and can also be stimulated to break tolerance, thereby generating antibodies against human targets. Figure 11AShowing influenza-specific IgM and IgG antibodies from day 11 immune organoids derived from unstimulated control organoids and 12 different stimulation conditions consisting of influenza hemagglutinin protein and various experimental adjuvants. A drawing from a representative donor is also shown. Figure 11B Showing SARS-CoV-2 specific IgM and IgG antibodies from day 11 immune organoids derived from unstimulated control organoids and 12 different stimulation conditions consisting of SARS-CoV-2 spike protein and various experimental adjuvants. A drawing from a representative donor is also shown. Figure 11C Displaying myelin-specific IgM and IgG antibodies from day 11 immune organoids derived from unstimulated control organoids and eight different stimulation conditions consisting of myelin proteins and various experimental adjuvants. A drawing from a representative donor is also shown. Figure 11D Showing NK cell-specific IgM and IgG antibodies from day 11 immune organoids derived from unstimulated control organoids and 12 different stimulation conditions consisting of NK cell proteins and various experimental adjuvants. A drawing from a representative donor is also shown.

[0044] Figure 12A-12G This demonstrates that immune organoids can comprehensively evaluate the efficacy of immunotherapy through multi-parameter analysis of cellular responses. Figure 12A This study presents a time-dependent analysis of lymphocyte populations in immune organoids after treatment with a combination of experimental therapeutic antibodies and a set of different experimental adjuvants. CD3+ T cells and CD19+ B cells were quantified by flow cytometry at days 0, 7, and 14 post-inoculation. Data points represent individual replicates, and different symbols indicate individual treatment conditions. Figure 12B This study presents a representative flow cytometry analysis of CD4+ memory T cell immune organoid populations based on CD45RO and CD45RA expression. The analysis was previously gated onto live single CD45+, CD3+ cells. A plot illustrates the distribution of CD45RO+ CD4+ memory T cells and CD45RA+ CD4+ naive T cells from two independent donors in response to the experimental subunit of influenza vaccine. Data are presented as a percentage of total CD4+ T cells, and in both samples, the CD45RO+ population was dominant (89.6% and 83.2%) compared to the CD45RA+ population (4.59% and 9.99%). Figure 12C This study demonstrates the immunoglobulin production characteristics following vaccination in two independent donors. Concentrations of IgG1-4 subtypes, IgM, and IgA were measured in response to treatments A and B, where treatments A and B represent two different doses of rabies vaccine. Analyses were performed on four technical replicates / conditions, and concentrations are reported in pg / mL. Data are presented as mean ± standard deviation (n=4). Figure 12D This study presents an analysis of IL-10 and IFN-γ production in immune organoids treated with and untreated experimentally designed therapeutic antibodies to stimulate the immune system in three independent donors. Intercytokine levels were measured under untreated and after treatment A. Data are presented as mean ± standard deviation. Figure 12E This study demonstrates CXCL10 production in immune organoids measured in five independent donors (AEs) under untreated and treatment A, where treatment A consisted of an experimentally designed therapeutic antibody designed to stimulate the immune system. Data are presented as mean ± standard deviation. Figure 12F This study demonstrates the selective expansion of B and T cell populations in immune organoids after treatment with experimental immunomodulators, tested alone and in various combinations, on day 0. The data were quantified from flow cytometry data of organoids from day 14. Twenty-four different conditions were included, comprising single treatments and combinations. Data are presented as mean ± standard deviation (n=3). Figure 12G This presentation shows representative flow cytometry analyses of different B cell phenotypes on day 0 after untreated, rabies vaccine-only, or rabies vaccine-plus-investigative therapeutic antibody treatment. The analyses were previously gated onto live, single CD45+ and CD19+ cells. Plots show the distribution of naive B cells (CD27-CD38-), memory B cells (CD27+CD38-), pre-GC B cells (CD27-CD38+), germinal center (GC) B cells (CD27+CD38+), and plasmablasts (CD27+CD38++).

[0045] Figures 13A-13B This study demonstrates that immune organoids can achieve antibody type switching and memory B cell induction in both initial and memory responses after vaccination. Figure 13A This study presents a time-dependent analysis of the anti-rabies antibody response, showing the changes in IgM and IgG concentrations over time on day 0 post-rabies vaccination. Data are presented as mean ± standard deviation (n=3, N=3). The donors were previously confirmed to have no rabies antigen exposure. Figure 13B Representative flow cytometry analyses are presented, showing B cell differentiation in immune organoids before and after experimental influenza subunit vaccination. Analysis was performed on CD19+ gated cells, demonstrating CD27-to-CD38 expression. Data presented are from two independent organoid cultures. Detailed Implementation

[0046] To address the lack of sufficient models to evaluate the efficacy of immunotherapeutic agents, the disclosures provided herein offer 3D in vitro immune organoids cultured in the presence of an immunotherapeutic agent, which fully summarize the cellular complexity and key functions of in vivo secondary lymphoid organs in individuals treated with this immunotherapeutic agent.

[0047] This disclosure provides a method for evaluating the efficacy of therapeutic agents (e.g., immunotherapeutic agents) to ensure that a drug is effective when tested in patients. No other system can capture true patient heterogeneity to ensure that a drug is effective regardless of age, sex, race, genetic background, and exposure history.

[0048] definition

[0049] Unless the context clearly indicates otherwise, the singular forms “a” and “the” include the plural. For example, the term “a cell” includes one or more cells, including mixtures thereof. As used herein, “A and / or B” is intended to include all the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0050] It should be understood that the aspects and embodiments of this disclosure described herein include "comprising aspects and embodiments," "consisting of," and "substantially consisting of." As used herein, "comprising" is synonymous with "including," "containing," or "characterized in," and is inclusive or open-ended, not excluding other unlisted elements or method steps. As used herein, "consisting of" excludes any element, step, or component not specified in the claimed composition or method. As used herein, "substantially consisting of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any statement of the term "comprising" herein (especially in the description of compositional components or method steps) should be understood to cover those compositions and methods that are substantially composed of and consist of the cited components or steps.

[0051] If a range of values ​​is provided, it should be understood that this disclosure covers every intermediate value between the upper and lower limits of the range and any other value or intermediate value in the range shown, accurate to one-tenth of the lower limit unit unless otherwise expressly indicated in the context. The upper and lower limits of these smaller ranges may be included independently within smaller ranges, which are also covered in this disclosure except for any limit specifically excluded from the range. If the range includes one or both of the limit values, this disclosure also includes ranges that exclude any or both of the included limit values.

[0052] All scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any scope listed can be readily identified as sufficiently descriptive and capable of being divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all language such as “highest,” “at least,” “greater than,” “less than,” and the like includes the listed numbers and refers to a scope, which can subsequently be divided into subscopes as discussed above. Finally, those skilled in the art will understand that a scope includes each individual member. Thus, for example, a group having 1-3 objects means a group having 1, 2, or 3 objects. Similarly, a group having 1-5 objects means a group having 1, 2, 3, 4, or 5 objects, etc.

[0053] It should be understood that certain features of this disclosure described in the context of a single embodiment for clarity may also be provided in combination in a single embodiment. Conversely, various features of this disclosure described in the context of a single embodiment for simplicity may also be provided individually or in any suitable sub-combination. This disclosure specifically covers and discloses all combinations of embodiments relating to this disclosure, as if each and every combination were disclosed individually and explicitly. In addition, this disclosure also specifically covers and discloses all sub-combinations of various embodiments and their elements, as if each and every such sub-combination were disclosed individually and explicitly.

[0054] Although features of this disclosure may be described in the context of a single embodiment, features may also be provided individually or in any suitable combination. Conversely, while this disclosure may be described in the context of a single embodiment for clarity reasons, it may also be practiced in a single embodiment. Any disclosed patent applications referenced herein, as well as any other disclosed references, documents, drafts, and scientific literature, are incorporated herein by reference for any purpose. In the event of any conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0055] The method disclosed herein

[0056] This disclosure particularly provides a method for evaluating the efficacy of a therapeutic agent. The method comprises (a) contacting a three-dimensional immune organoid comprising a plurality of self-assembled primary immune cells and a plurality of stem cells obtained from one or more secondary lymphoid organs with a therapeutic agent, and (b) measuring the immune response of the three-dimensional immune organoid to the therapeutic agent after contact.

[0057] Immune organoids

[0058] As described above, one aspect of this disclosure relates to a method for evaluating the efficacy of a therapeutic agent. The method comprises contacting a three-dimensional immune organoid, comprising a plurality of self-assembled primary immune cells and a plurality of stem cells derived from one or more secondary lymphoid organs, with the therapeutic agent.

[0059] As used in this article, "three-dimensional immune organoids" can be a composition of live immune cells arranged in a three-dimensional or multi-layer configuration (rather than a single layer).

[0060] In some embodiments, immune organoids are generated in vitro. Those skilled in the art will readily recognize that the immune organoids described herein are also non-natural.

[0061] Generally, organoids are artificial constructs generated in vitro to mimic or resemble the function and / or tissue structure of an organ, tissue, or part thereof. As used herein, organoids may be cellular structures obtained through expansion of immune cells and stem cells and composed of self-organized tissue-specific cell types. In this disclosure, the term "organoid" may be used to refer to normal (e.g., non-tumor) organoids. Depending on the specific tissue and / or organ being modeled or simulated, organoids may include one or more (e.g., 1, 2, 3, 4, or more) differentiated cell types.

[0062] immune cells

[0063] The immune cells comprising the three-dimensional immune organoids of this disclosure are derived from one or more secondary lymphoid organs. As used herein, “derived from” generally refers to the source of the primary cells that form the organoid. In some embodiments, “derived from one or more secondary lymphoid organs” may mean that the organoid is formed without any passages of primary cells from secondary lymphoid organs. In some embodiments, “derived from one or more secondary lymphoid organs” may mean that the organoid is formed after one passage of primary cells. In some embodiments, “derived from one or more secondary lymphoid organs” may mean that the organoid is formed after more than one passage of primary cells. Secondary lymphoid organs are sites that elicit adaptive immune responses and maintain lymphocytes. Example secondary lymphoid organs include lymph nodes (LNs), spleen, Peyer's lymph plexus (PP) and mucosa-associated lymphoid tissue (MALT), popliteal tonsils, and tonsils. In some embodiments, primary immune cells are obtained from the spleen, lymph nodes, Peyer's lymph plexus, and / or MALT.

[0064] Secondary lymphoid tissue can be obtained from mammals (i.e., donors or patients) (e.g., humans, dogs, cats, rabbits, monkeys, chimpanzees, cattle, pigs, or goats). Secondary lymphoid tissue can be obtained directly from living patients, surgical excisions, fine-needle aspirations, biopsies, or deceased patients. In some embodiments, secondary lymphoid tissue is obtained from humans, thereby generating human immune organoids.

[0065] In some embodiments, secondary lymphoid tissue is obtained and dissociated mechanically, enzymatically, or both. In some embodiments, secondary lymphoid tissue is dissociated using proteolytic enzymes and / or collagenases. In some embodiments, secondary lymphoid tissue is dissociated enzymatically using Accutase (StemCell Technologies), Accuxax (StemCell Technologies), trypsin, trypsin / EDTA, collagenase, dispersase, TrypLE Express (Thermo Fisher Scientific), TrypLE Select (Thermo Fisher Scientific), or any combination thereof. In some embodiments, secondary lymphoid tissue is mechanically dissociated by grinding (e.g., using a pipette). In some embodiments, the resulting single-cell suspension of cells is filtered to remove any non-dissociated cellular material.

[0066] Primary immune cells derived from secondary lymphoid tissue can be any hematopoietic cell functionally involved in the initiation and / or execution of innate and / or adaptive immune responses, such as typically CD3 or CD4 positive cells. Indigenous types of primary immune cells include (but are not limited to) dendritic cells, mast cells, NK cells, plasmablasts, macrophages, B cells, T cells, plasma cells, innate lymphoid cells (ILCs), and granulocytes. In some embodiments, the plurality of immune cells includes B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, plasma cells, ILCs, granulocytes, and combinations thereof.

[0067] In some embodiments, B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or combinations thereof. In some embodiments, B cells primarily include naive B cells. In some embodiments, B cells differentiate upon stimulation to produce pre-GC B cells, GC B cells, memory B cells, or combinations thereof. In some embodiments, naive B cells are CD38-CD27-. In some embodiments, memory B cells are CD38-CD27+. In some embodiments, pre-GC B cells are CD38+CD27-. In some embodiments, GC B cells are CD38+CD27+.

[0068] In some embodiments, T cells include naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, CD4 effector memory cells, CD8 effector memory cells, or combinations thereof. In some embodiments, naive CD4 T cells are CD4+CCR7+CD45RA+. In some embodiments, naive CD8 T cells are CD8+CD45RA-CD27+. In some embodiments, memory CD4 T cells are CD4+CCR7+CD45RA+ or CD4+CD45RA-CD45RO+. In some embodiments, memory CD8 T cells are CD8+CD45RA-CD45RO+. In some embodiments, T regulatory cells are CD3+CD4+CD25+. In some embodiments, T follicular helper cells are CD3+CXCR5+CD25+. In some embodiments, γδ T cells are gdTCR+CD3+CD27+.

[0069] In some embodiments, dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, bone marrow-like dendritic cells, or combinations thereof. In some embodiments, the dendritic cells are CD45+CD11b+ dendritic cells. In some embodiments, the dendritic cells are CD14+CD11c+ bone marrow-like dendritic cells. In some embodiments, the dendritic cells are CD123+ plasmacytoid dendritic cells.

[0070] In some embodiments, the monocytes are CD14+CD11b+ monocytes.

[0071] In some embodiments, the macrophages are CD68+ macrophages. In some embodiments, the macrophages are CD14+ macrophages.

[0072] In some embodiments, the plasmablasts are CD38++CD27++ plasmablasts.

[0073] In some embodiments, the NK cells are CD56+ NK cells. In some embodiments, the NK cells are CD56+CD16+ NK cells.

[0074] In some embodiments, granulocytes are SSCs 高 CD15+ granulocytes.

[0075] In some embodiments, immune organoids as described herein generate and / or modify T cells. For example, novel T cell phenotypic changes may occur in immune organoids, such as an increase in T helper cells. In some embodiments, novel cytotoxic T cells and / or memory T cells may be generated in the immune organoids described herein. Analyses including (but not limited to) flow cytometry may be used to identify said phenotypic changes to detect markers on T cells. Cytotoxic T cells may be further identified, for example, using T cell cytotoxicity assays.

[0076] In some embodiments, the amount of multiple immune cells present in the organoids of this disclosure may be from about 1%, 5%, 10%, 25%, 50% to about 55%, 60%, 75%, 80%, 90%, or 95% of the total number of cells present in the organoids. In some embodiments, the organoids of this disclosure include primary immune cells in an amount of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the total number of cells present in the organoids. If the organoids of this disclosure include primary immune cells in an amount less than 100% of the total number of cells present in the organoids, then any suitable amount of stem cells, stromal cells, fibroblasts, reticular cells, and follicular dendritic cells may constitute the remaining cell number / percentage.

[0077] In some embodiments, B cells are present in the immune organoids of this disclosure at an amount of about 1%, 5%, 10%, 15%, or 20%. In some embodiments, T cells are present in the immune organoids of this disclosure at an amount of about 1%, 5%, 10%, or 15%. In some embodiments, NK cells are present in the immune organoids of this disclosure at an amount of about 1%, 5%, 10%, or 15%. In some embodiments, macrophages are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, monocytes are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, dendritic cells are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, plasmablasts are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, plasma cells are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, ILCs are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, granulocytes are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, or 5%.

[0078] stem cells

[0079] As described above, immune organoids also include various stem cells. Stem cells are characterized by the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers identified by the lack of specific antibody binding. Stem cells can also be identified through in vitro and in vivo functional analyses, particularly analyses related to their ability to produce multiple differentiated progeny. The immune organoid stem cells described in this article are identified as CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+. Therefore, stem cells can include hematopoietic stem cells. Hematopoietic stem cells refer to a subgroup of pluripotent stem cells that produce all types of blood or immune cells, including bone marrow-like (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NKT cells, NK cells). "Stem cells" can refer to cells that maintain their self-renewal capacity through mitotic division and can differentiate into various specialized cell types. Stem cells may also include mesenchymal stem cells. Mesenchymal stem cells comprise stem cells that can be obtained, for example, from primary cultures of bone marrow, peripheral blood, skin, hair roots, muscle tissue, endometrium, blood, umbilical cord blood, and various tissues. Mesenchymal stem cells can differentiate into all or several types of bone cells, chondrocytes, and adipocytes.

[0080] Flow cytometry and immunofluorescence, as described in the examples below, can be used to identify stem cells. These methods are known in the industry and involve the use of antibodies to detect the presence or absence of various protein markers on the surface of cells (e.g., CD34, CD45RA, ITGA3, EPCR, CD90, CD73, and CD105).

[0081] Other cell types

[0082] In some embodiments, immune organoids further comprise peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from peripheral blood and identified as any blood cell with a round nucleus (i.e., lymphocytes, monocytes, natural killer cells (NK cells), or dendritic cells). The addition of PBMCs increases the size of the organoid immune repertoire. For example, in some embodiments, PBMCs are isolated from the same donor as secondary lymphoid tissue. In some embodiments, PBMCs are isolated from different donors.

[0083] In other embodiments, the immune organoids further comprise one or more stromal cells, fibroblastic reticular cells, and follicular dendritic cells. Stromal cells are a class of cells that form specific types in connective tissue in vivo. Fibroblastic reticular cells are stromal cells found in secondary lymphoid organs. Follicular dendritic cells are non-hematopoietic cells derived from the stromal matrix. They are integrated into a stromal network within a continuous lymphoid organ. The presence of these cell types in the immune organoids described herein contributes to the deposition of extracellular matrix and the overall architecture of the three-dimensional immune organoids. For example, stromal cells can be identified by CD105+, CD29+, CD44+, CD90+, and CD45-, and fibroblastic reticular cells can be identified by PDPN+ and CD31+ using the methods described in the examples herein.

[0084] In some embodiments, stromal cells are present in the immune organoids of this disclosure at an amount of about 0.5%, 1%, 1.5%, or 2%. In some embodiments, fibroblastic reticular cells are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, follicular dendritic cells are present in the immune organoids of this disclosure at an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0085] structure

[0086] In some embodiments, the diameter and / or maximum size of the immune organoids disclosed herein is about 8000 μm, 7500 μm, 7000 μm, 7000 μm, 6500 μm, 6000 μm, 5500 μm, 5000 μm, 4500 μm, 4000 μm, 3500 μm, 3000 μm, 2500 μm, 2000 μm, 1500 μm, 1000 μm, 500 μm, 250 μm, 100 μm, 50 μm or smaller. In some embodiments, organoids may comprise a total of about 1,500, 2,000, or 5,000 to about 10,000, 25,000, or 50,000 cells, or a total of about 1,000, 5,000, 10,000, or 50,000 to about 75,000, 100,000, 150,000, 250,000, 500,000, 750,000, 1,000,000, 50,000,000, or 100,000,000 cells. In some embodiments, the immune organoids of this disclosure may comprise about 1 million, 2 million, or 5 million to about 10 million, 25 million, 50 million, or 100 million cells / mL. In some embodiments, the organoids of this disclosure may comprise about 10 million cells / mL or 20 million cells / mL. In some embodiments, the organoids of this disclosure may comprise from about 5 million or 10 million cells / mL to about 15 million or 20 million cells / mL. The organoids of this disclosure may be of any suitable three-dimensional or multi-layered shape. In some embodiments, the organoids of this disclosure are in the form of spherical bodies. In some embodiments, the organoids of this disclosure may self-organize in a suspension or culture medium.

[0087] In some embodiments, the immune organoid contains a germinal center and / or a B / T cell region. The germinal center (GC) is a subanatomical structure that controls the transformation of B cells into antibody-producing cells. In the GC, somatic mutations occur in the genes encoding their B cell receptors, which, upon successful selection, can lead to the emergence of B cell clones that bind antigens with high affinity. As described by Stebegg et al., “Regulation of the Germinal Center Response,” Front. Immunol. 9 (2018), the GC is divided into two distinct chambers: a light zone and a dark zone. In some embodiments, the immune organoid of this disclosure contains both a light zone and a dark zone. The dark zone (DZ) contains a reticular cell network that produces CXCL12 and is a site for GC B cell proliferation and somatic hypermutation (SHM). The central blast then follows the CXCL13 gradient to enter the light zone (LZ) as a central cell via its CXCR5 expression. In the germinal center (LZ), the central cell captures antigens presented on its internalized follicular dendritic cells, processes them, and subsequently presents them to T follicular helper cells for selection. This process is regulated by T follicular regulatory cells also present in the LZ. After receiving a survival signal from Tfh cells, the central cell re-enters the DZ for further proliferation and SHM, after which it leaves the GC as a memory B cell or a plasma cell secreting high-affinity antibodies. Therefore, the presence of dark and light regions in the immune organoids of this disclosure can be identified using techniques including (but not limited to) immunofluorescence as described in the examples. In some embodiments, the germinal center in the immune organoid is characterized as CXCR4. + CD83 + Ki67 + and IgD + In some embodiments, the B and T cell regions in the germinal centers of immune organoids are characterized as CD3+. + and CD20 + .

[0088] Antibody production

[0089] In some embodiments, the immune organoids of this disclosure may be capable of producing antibodies. Antibodies are proteins used by the immune system to identify and neutralize foreign substances (e.g., pathogenic bacteria and viruses). Antibodies recognize unique molecules of pathogens (called antigens). Antibodies can exist in different types (called isotypes or species). In placental mammals, there are five types of antibodies, called IgA, IgD, IgE, IgG, and IgM, which are further subdivided into subclasses (e.g., IgA1, IgA2). Therefore, the immune organoids of this disclosure may be capable of producing IgA, IgD, IgE, IgG, IgM, and combinations thereof. The prefix "Ig" represents immunoglobulin, while the suffix indicates the type of heavy chain contained in the antibody: heavy chain types α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ (mU) produce IgA, IgG, IgD, IgE, and IgM, respectively. In some embodiments, the immune organoids of this disclosure produce IgG antibodies. In some embodiments, the immune organoids of this disclosure produce IgA antibodies. In some embodiments, the immune organoids of this disclosure produce IgM antibodies.

[0090] B cell antibody isotypes change during cell development and activation. Immature B cells that have never been exposed to antigens express only IgM and IgD isotypes in a cell-surface bound form. B lymphocytes in this reactive form are called "naïve B lymphocytes." Naïve B lymphocytes express both surface IgM and IgD. The co-expression of these two immunoglobulin isotypes enables B cells to respond to antigens. B cell activation occurs after cell-bound antibody molecules are linked to antigens, leading to cell division and differentiation into antibody-producing cells (called plasma cells). In this activated form, B cells initially produce antibodies in a secretory form rather than a membrane-bound form. Some daughter cells of activated B cells undergo isotype conversion, which is the mechanism by which antibody production changes from IgM or IgD to other antibody isotypes that play a defined role in the immune system, such as IgE, IgA, or IgG. Therefore, the immune cells of the immune organoids disclosed herein may also include those that have undergone or will undergo isotype conversion.

[0091] Antibodies are crucial for the development of humoral immune responses, in which they are produced by B cells and secreted into the blood and / or lymph in response to antigenic stimuli. In a properly functioning immune response, antibodies specifically bind to antigens (e.g., pathogens) on the cell surface, thereby marking cell destruction by phagocytic and / or complement-mediated mechanisms. In short, after binding to target cells, antibodies participate in several important functions, including antibody-dependent cytotoxicity (ADCC), phagocytosis (opsonization), and complement-dependent cytotoxicity (CDC).

[0092] Therefore, in some embodiments, the immune organoids have full humoral functionality. Specifically, in some embodiments, antibodies generated by the immune organoids of this disclosure can function in ADCC. ADCC is an in vitro or in vivo process in which an antibody binds to an antigen on the cell surface, then connects to an immune effector cell via a sequence within the antibody's Fc domain, thereby causing the cell to release a toxin that can kill the bound cell. ADCC activity can be measured using methods known in the art, including (but not limited to) in vitro methods known in the art.

[0093] In some embodiments, antibodies generated by the immune organoids of this disclosure may function in CDC. CDC refers to an in vitro or in vivo process in which an antibody binds to an antigen on the surface of a eukaryotic or prokaryotic cell, then links to a C1q protein via a sequence within the antibody's Fc domain, thereby initiating a classical complement cascade capable of killing the bound cell. CDC activity can be measured using methods known in the art, including (but not limited to) in vitro methods known in the art.

[0094] In some embodiments, antibodies produced by the immune organoids of this disclosure may play a role in opsonization. Opsonization is a process in which antibodies bind to antigens on the cell surface, then bind to immune cells via sequences within their Fc domains, leading to phagocytosis, consumption, and ultimately killing of antibody-bound cells by the immune cells. Opsonization activity may be measured using methods known in the art, including (but not limited to) in vitro methods known in the art.

[0095] In some embodiments, immune organoids as described herein exhibit at least one of ADCC, CDC, and opsonization activities. In some embodiments, immune organoids as described herein exhibit at least two of ADCC, CDC, and opsonization activities. In some embodiments, immune organoids as described herein exhibit all three of the ADCC, CDC, and opsonization activities.

[0096] In some embodiments, antibodies produced by the immune organoids of this disclosure may also undergo somatic hypermutation. Somatic hypermutation refers to a process of enhanced mutations in genes that are thought to require activation-induced cytidine deaminase (AID) (a mistaken DNA repair enzyme). SHM was initially described based on the observed increase in mutations in immunoglobulin gene regions encoding the light and heavy chain variable regions in B lymphocytes after antigen stimulation. For example, AID is discussed in Smith et al., Trends Genet. 20:224-227 (2004). The presence of somatic hypermutation can be identified using methods that include (but are not limited to) measuring AID upregulation by, for example, quantitative PCR. B cell receptor sequencing can also be used to identify mutation sets.

[0097] In some embodiments, the immune organoids described herein produce plasmablasts and antigen-specific antibodies against targets exposed in the patient donor (recall response) and targets not exposed in the patient.

[0098] In some embodiments, the immune organoids described herein generate an autoimmune response against an autoantigen.

[0099] In some embodiments, antibodies produced by the immune organoids described herein can bind to human and non-human targets. Examples of human targets include (but are not limited to) proteins, sugars, and nucleic acids. Examples of non-human targets may include (but are not limited to) infectious disease antigens, venoms, poisons, and small molecules.

[0100] Therapeutic agents

[0101] As described above, the method includes the step of contacting the three-dimensional immune organoids described above with a therapeutic agent.

[0102] As used herein, a therapeutic agent can refer to a molecule or composition that has an effect on cells. The therapeutic agents used in the methods of this disclosure may be selected from one or more of the following classes of therapeutic agents: immunotherapeutic agents, autoantigens, tumor-specific peptides, checkpoint inhibitors, alkylating agents, antimetabolites, metabolic agonists, metabolic antagonists, plant alkaloids, mitotic inhibitors, antitumor agents, antibiotics, topoisomerase inhibitors, radiotherapy agents, chemotherapy agents, antibodies, nanobodies, photosensitizers, stem cell transplantation, vaccines, cytotoxic agents, cell growth inhibitors, tyrosine kinase inhibitors, proteasome inhibitors, interferons, interleukins, intercalating agents, targeted therapy agents, gene therapy, small molecule drugs, hormones, steroids, cell therapy agents, viral vectors, and nucleic acid therapeutic agents.

[0103] In some embodiments, the therapeutic agent is an autoantigen. In some embodiments, the autoantigen is associated with autoimmune diseases. Examples of autoimmune diseases include (but are not limited to) multiple sclerosis (MS), peripheral neuropathy, Sjogren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behcet's disease, bullous pemphigoid, celiac disease, Devic's disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, mixed vasculitis, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, systemic lupus erythematosus, Graves' disease, myasthenia gravis (MG), pemphigus vulgaris, antiglomerular basement membrane disease (Goodpasture syndrome), Hashimoto's thyroiditis, autoimmune hepatitis, or combinations thereof. In some aspects, autoantigens include β-cell proteins, insulin, islet antigen 2 (IA-2), glutamate decarboxylase (GAD65), zinc transporter 8 (ZNT8), myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), protein lipid protein (PLP), myelin-associated glycoprotein (MAG), citrullinated antigen, synovial proteins, aquaporin-4 (AQP4), nicotinic acetylcholine receptor (nAChR), desmosome core protein-1 (DSG1), desmosome core protein-2 (DSG2), thyroid-stimulating hormone receptor, type IV collagen, thyroglobulin, thyroid peroxidase, thyroid-stimulating hormone receptor (TSHR), or combinations thereof. In some embodiments, the autoantigen is MOG and the autoimmune disease is multiple sclerosis (MS).

[0104] In some embodiments, the autoantigen is an inducible autoantigen. In some embodiments, the inducible autoantigen is an NK cell receptor protein. In some embodiments, the NK cell receptor protein is NKG2D.

[0105] Autoantigens can be contacted with immune organoids as described herein at any dose to seek testing the efficacy of the autoantigen. In some embodiments, autoantigens are contacted with immune organoids at doses of about 1.0 µg / mL, 2.0 µg / mL, 3.0 µg / mL, 4.0 µg / mL, 5.0 µg / mL, 6.0 µg / mL, 7.0 µg / mL, 8.0 µg / mL, 9.0 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, 35 µg / mL, 40 µg / mL, 45 µg / mL, or 50 µg / mL.

[0106] In some embodiments, the therapeutic agent is an immunotherapeutic agent. "Immunotherapeutic agent" can refer to any medical intervention that induces, suppresses, or enhances a patient's immune system to treat a disease. In some embodiments, immunotherapy can activate a patient's innate and / or adaptive immune responses (e.g., T cells) to more effectively target and eliminate pathogens or cure a disease (e.g., cancer or an immune disorder).

[0107] In some embodiments, immunotherapeutic agents are selected from the group consisting of vaccines, antibodies, gene therapies, cell therapies, small molecules, and nanobodies.

[0108] Any of the therapeutic agents used in the methods disclosed herein may also be combined with one or more adjuvants that can enhance the immune response (humoral and / or cellular). Adjuvants may include (but are not limited to) Pam3CSK4 (0.1-1 µg / mL), MALP-2 (50-500 ng / mL), Poly I:C (1-50 µg / mL), Poly ICLC (1-25 µg / mL), MPL (0.1-10 µg / mL), GLA (0.5-5 µg / mL), flagellin (0.1-2 µg / mL), imiquimod (1-10 µM), resiquimod (0.1-5 µM), gardiquimod (0.5-5 µM), CpG ODN (0.1-10 µM), MDP (1-20 µg / mL), M-Tri-DAP (5-50 µg / mL), cGAMP (1-20 µg / mL), and ADU-S100 (0.5-10 µg / mL). µM), aluminum hydroxide (50-250 µg / mL), aluminum phosphate (50-250 µg / mL), calcium phosphate (100-500 µg / mL), MF59 (2-20% v / v), AS03 (2-20% v / v), AS04 (5-50 µg / mL), IL-2 (10-100 ng / mL), IL-12 (1-50 ng / mL), IL-15 (1-100 ng / mL), IL-21 (10-100 ng / mL), GM-CSF (10-100 ng / mL), Flt3L (50-200 ng / mL), type I IFN (100-1000 U / mL), TSLP (5-50 ng / mL), QS-21 (1-20 µg / mL), ISCOM (1-25 µg / mL), matrix-M (1-25 µg / mL). (µg / mL), AS01 (1-20 µg / mL), AS02 (1-20 µg / mL), cyclic dinucleotides (0.1-10 µg / mL), R848 (0.1-5 µM), glucopyranoside A (0.1-10 µg / mL), α-GalCer (50-500 ng / mL), muramyl dipeptide (0.5-10 µg / mL), β-glucan (10-100 µg / mL), inulin (50-500 µg / mL), chitosan (1-50 µg / mL), dextran (10-100 µg / mL), mannose derivatives (1-50 µg / mL), montanide (5-20% v / v), Complete Freund's adjuvant (1:1 ratio), Incomplete Freund's adjuvant (1:1 ratio), squalene adjuvant (2-20% v / v), PLGA (10-200 µg / mL), PLA (10-200 µg / mL), PCL (10-200 µg / mL), gold nanoparticles (1-50 µg / mL), liposomes (10-200 µg / mL), CD40 agonists (0.1-10 µg / mL). (µg / mL), OX40 agonist (0.1-10 µg / mL), 4-1BB agonist (0.1-10 µg / mL), GITR agonist (0.1-10 µg / mL), Quillajasaponins (1-50 µg / mL), essential oils (0.01-0.1% v / v), plant extracts (1-100 µg / mL), modified bacterial toxins (0.1-1 µg / mL), AS01 (MPL 1-10 µg / mL + QS-21 1-20 µg / mL), AS04 (MPL 1-10 µg / mL + Alum 50-250 µg / mL), CAF01 (DDA 250-500 µM + TDB 50-100 µM) and IC31 (KLK 100-500 µM + ODN1a 0.1-10 µg / mL). µM).

[0109] vaccine

[0110] In some embodiments, the immunotherapeutic agent is a vaccine. As used herein, a vaccine may comprise any substance administered to elicit a humoral and / or cell-mediated immune response, such as live or attenuated viral and bacterial immunogens and inactivated viruses, tumor-derived, protozoan, organism-derived, fungal, and bacterial immunogens, toxoids, toxins, polysaccharides, proteins, glycoproteins, peptides, cellular vaccines (e.g., using dendritic cells), DNA vaccines, recombinant proteins, glycoproteins, and peptides. Consider using any vaccine in the methods described herein. Example vaccines include (but are not limited to) vaccines for the following diseases: cancer, BCG, cholera, plague, typhoid fever, hepatitis A, B and C, influenza A and B, parainfluenza, polio, rabies, measles, mumps, rubella, yellow fever, tetanus, diphtheria, Haemophilus influenzae type b, tuberculosis, meningococcal and pneumococcal vaccines, adenovirus, HIV, fowlpox, cytomegalovirus, dengue fever, feline leukemia, fowl cholera, HSV-1 and HSV-2, swine cholera, Japanese encephalitis, respiratory syncytial virus, rotavirus, papillomavirus, severe acute respiratory syndrome (SARS), SARS-CoV-2, anthrax, and yellow fever.

[0111] Antigens or immunogens used to prepare vaccines can be derived from a wide range of sources. For example, suitable antigens or immunogens may include infectious agents (e.g., bacteria, fungi, protozoa, parasites, or viruses), infectious agent products (e.g., proteins, peptides, nucleic acids, polysaccharides, glycoproteins, glycolipids, antigens, or antigenic preparations), degenerative disease antigens, atopic disease antigens, autoimmune disease antigens, allogeneic antigens, heteroantigens, metabolic disease enzymes or enzymatic products, recombinant proteins or peptides, chimeric fusion proteins, and / or small molecules.

[0112] Suitable antigens or immunogens may be in the form of whole-cell, purified, or partially purified antigens or antigenic preparations. Suitable antigens or immunogens may be used unmodified, in galenal form, or in combination with a carrier or delivery system (e.g., microspheres, liposomes, nanospheres, and other antigen delivery systems familiar to those skilled in the art).

[0113] Vaccines can be based on antigens prepared or derived from natural sources or produced through recombinant technology.

[0114] In some embodiments, the vaccine may be a vaccine for an infectious disease. In some embodiments, a vaccine for an infectious disease includes antigens or immunogens selected from: microbial structures (cell walls, vesicles, flagella, pili, viral capsids, mantle-associated glycoproteins); microbial toxins (allergens: dust, pollen, hair, food, dander, bee venom, drugs, and other substances that cause allergic reactions); foreign tissues and cells (from transplants and blood transfusions); and the body's own cells that the body cannot recognize as the "normal self" (cancer cells, infected cells, cells involved in autoimmune diseases).

[0115] In one embodiment, the antigen or immunogen may be an infectious agent or a product of an infectious agent. In one embodiment, the antigen or immunogen includes, for example, an inactivated infectious agent that has been killed or otherwise attenuated. In another embodiment, the antigen or immunogen includes a live infectious agent.

[0116] In one embodiment, the infectious agent (or infectious agent product) is a virus, such as (but not limited to) poxvirus (e.g., vaccinia virus), smallpox virus, Marburg virus, flavivirus (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), influenza virus (or antigen, such as F and G proteins or derivatives thereof) (e.g., influenza A); or purified or recombinant proteins thereof (e.g., HA, NP, NA or M proteins or combinations thereof), parainfluenza virus (e.g., Sendai virus). Respiratory syncytial virus (RSV), measles virus, human immunodeficiency virus (or antigens, such as TAT, NEF, GP120, or GP160), human papillomavirus (or antigens, such as HPV6, 11, 16, 18), varicella-zoster virus (or antigens, such as GPL, II, and IE63), herpes simplex virus (e.g., herpes simplex virus I, herpes simplex virus II; or antigens, such as GD or its derivatives, or early proteins, such as ICP27 from HSV1 or HSV2), cytomegalovirus (or antigens, such as GB or its derivatives), Epstein-Barr virus. (or antigens, such as gp350 or its derivatives), JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picoronavir virus, poliovirus, mumps virus, rabies virus, reovirus, rubella virus, cloacal virus, orthomyxovirus, retrovirus, hepatotropic DNA virus, hantavirus, junin virion, filovirus (e.g., Ebola virus), Coxsackie virus, equine encephalitis virus, Rift Valley fever virus, alpha virus (e.g., Chikungunya virus, sindbis virus), hepatitis A virus, hepatitis B virus (or its antigens, such as hepatitis B surface antigen or its derivatives), hepatitis C virus, hepatitis D virus, or hepatitis E virus.

[0117] In one embodiment, the infectious agent is bacteria. Non-limiting examples of suitable bacteria (or bacterial products) used in the vaccines and / or methods of this disclosure include *Neisseria* spp., including *Neisseria gonorrhea* and *Neisseria meningitidis* (or antigens, such as capsular polysaccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, Pi1C, adhesins); *Haemophilus* spp., such as *Haemophilus influenzae*; *Streptococcus pyogenes* (or antigens, such as M protein or fragments thereof, C5A protease, lipoteichoic acid), *Streptococcus agalactiae*, *Streptococcus mutans*; *Haemophilus ducreyi*; and *Moraxella* spp., including *Moraxella catarrhalis*, also known as *Branhamella catarrhalis*. (or antigens, such as high and low molecular weight adhesins and invaginins); *Bordetella* spp., including *B. pertussis* (or antigens, such as *B. pertussis* adhesin, pertussis protein toxin or its derivatives, filamentous hemagglutinin, adenylate cyclase, fimbriae), *B. parapertussis*, and *B. bronchiseptica*; *Mycobacterium* species, including *Mycobacterium tuberculosis* (or antigens, such as ESAT6, antigen 85A, -B or -C), *Mycobacterium bovis*, *Mycobacterium leprae*, *Mycobacterium avium*, *Mycobacterium paratuberculosis*, and *Mycobacterium smegmatis*; *Legionella* spp. The genera *L. pneumophila* (spp.) include *L. pneumophila*; *Escherichia spp.* include enterotoxic *E. coli* (or antigens, such as colonization factors, heat-labile toxins or their derivatives, heat-stable toxins or their derivatives), enterohemorrhagic *E. coli*, and enteropathogenic *E. coli* (or antigens, such as Shiga toxin-like toxins or their derivatives); and *Vibrio spp.* includes *Vibrio cholerae* (V. cholerae).Cholera (or antigens, such as cholera toxin or its derivatives); Shigella spp., including Shigella sonnei, Shigella dysenteriae, and Shigella flexnerii; Yersinia spp., including Y enterocolitica (or antigens, such as Yop protein), Y pestis, and Y pseudotuberculosis; Campylobacter spp., including Campylobacter jejuni (or antigens, such as toxins, adhesins, and invaginins) and Campylobacter coli; Salmonella spp., including Salmonella typhi, Salmonella paratyphi, and Salmonella choleraesuis (S. typhi). *Salmonella choleraesuis*, *Salmonella enteritidis*, *Salmonella typhimurium*, and *Salmonella dysenteriae*; *Listeria* genus, including *Listeria monocytogenes*; *Helicobacter pylori* genus, including *H. pylori* (e.g., urease, catalase, vacuolating toxin); *Pseudomonas* genus, including *P. aeruginosa*; *Staphylococcus* genus, including *Staphylococcus aureus* and *Staphylococcus epidermidis*; *Proteus* genus, for example, *Proteus mirabilis*; *Enterococcus* genus, including *Enterococcus faecalis* and *Enterococcus faecium*. Clostridium species, including *Clostridium tetani* (or antigens, such as tetanus toxin and its derivatives), *Clostridium botulinum* (or antigens, such as botulinum toxin and its derivatives), *Clostridium difficile* (or antigens, such as clostridial toxin A or B and its derivatives), and *Clostridium perfringens*; and Bacillus species, including *Bacillus anthracis* (B.).*Bacillus anthracis* (or antigens, such as botulinum toxin and its derivatives), *Bacillus cereus*, *Bacillus circulans*, and *Bacillus megaterium*; *Corynebacterium* species, including *Corynebacterium diphtheriae* (or antigens, such as diphtheria toxin and its derivatives); *Borrelia* species, including *Borrelia burgdorferi* (e.g., OspA, OspC, DbpA, DbpB), *Borrelia garinii* (or antigens, such as OspA, OspC, DbpA, DbpB), *Borrelia afzelii* (e.g., OspA, OspC, DbpA, DbpB), and *Borrelia andersonii*. (or antigens, such as OspA, OspC, DbpA, DbpB), *B. hermsii*; *Ehrlichia* species, including *E. equi* and the infectious agent of human granulocytic Ehrlichiosis; *Rickettsiaspp*, including *R. rickettsii*; *Chlamydia* species, including *C. trachomatis* (or antigens, such as MOMP, heparin-binding protein), *C. pneumoniae* (e.g., MOMP, heparin-binding protein), *C. psittaci*; *Leptospiraspecies*, including *L. interrogans*; *Streptococcus* species, such as *Streptococcus pyogenes*. *Streptococcus pyogenes*, *Streptococcus agalactiae*, and *Streptococcus pneumoniae*; the genus *Treponema*, including *Treponema pallidum* (or antigens, such as rare outer membrane proteins), *Treponema denticola*, and *Treponema hyodysenteriae*.

[0118] In one embodiment, the infectious agent is a parasite or a parasite-derived product. Non-limiting examples of suitable parasites (or parasite-derived products) used in the vaccines and / or methods of the present invention include *Plasmodium* species, including *P. falciparum*; *Toxoplasma* species, including *T. gondii* (or antigens, such as SAG2, SAGS, Tg34); *Entamoeba* species, including *E. histolytica*; *Babesia* species, including *Babesia microti*; *Trypanosoma* species, including *T. cruzi*; *Giardia* species, including *G. lamblia*; *Leshmania* species, including *L. major*; and *Pneumocystis*. The genera *Trichomonas* include *Pneumocystis carinii*; *Trichomonas* species include *T. vaginalis*; and *Schisostoma* species include *Schistosoma mansoni*.

[0119] In another embodiment, the infectious agent is a fungus or a fungal product. Suitable fungi (or fungal products) used in the invention of vaccines and / or methods include (but are not limited to) the genus *Candida*, including *C. albicans* and *C. parapsilosis*; the genus *Cryptococcus*, including *C. neoformans*; *Aspergillus fumigates* and *Aspergillus niger*; the genus *Fusarium*; the genus *Trychophyton*; the genus *Absidia*, such as *Absidia corymbifera*; the genus *Ajellomyces*, such as *Ajellomyces capsulatus*; and the genus *Arthroderma*, such as *Arthroderma benzii*. * *Benhamiae*; *Blastomyces* species, such as *Blastomyces dermatitidis*; *Cladophialophora* species, such as *Cladophialophora carrionii*; *Coccidioides* spp., such as *Coccidioides immitis*; *Cryptococcus* spp., such as *Cryptococcus neoformans*; *Cunninghamella* species; *Epidermophyton* species, such as *Epidermophyton floccosum*; *Exophiala* spp., such as *Exophialadermatitidis*; *Filobasidiella* spp., such as *Filobasidiella neoformans*; *Fonsecaea spp.*, such as *Fonsecaea pedrosoi*; *Fusarium*, such as *Fusarium solani*; *Geotrichum* spp.*, such as *Geotrichum candidum*.Histoplasma spp., for example, *Histoplasma capsulatum*; Hortaea spp., for example, *Hortaea werneckii*; Issatschenkia spp., for example, *Issatschenkia orientalis*; Madurella spp., for example, *Madurella grisae*; Malassezia spp., for example, *Malassezia furfur*; Microsporum spp., for example, *Microsporum canis*; Mucor spp., for example, *Mucor circinelloides*; Nectria spp. spp., for example, *Nectria haematococca*; *Paecilomyces* spp., for example, *Paecilomyces variotii*; *Paracoccidioides* spp., for example, *Paracoccidioides brasiliensis*; *Penicillium* spp., for example, *Penicillium marneffei*; *Pichia* spp., for example, *Pichia guilliermondii*; *Pneumocystis* spp., for example, *Pneumocystis carinii*; *Pseudallescheria* spp., for example, *Pseudallescheria boydii*; *Rhizopus* spp., for example, *Rhizopus oryzae*. oryzae); Rhodotorula spp, for example Rhodotorula rubra; Scedosporium spp, for example Scedosporium apiospermum; Schizophyllum spp, for example Schizophyllum commune; Sporothrix spp, for example Sporothrix schenckii;Trichophyton spp., such as Trichophyton violaceum; and Trichosporon spp., such as Trichosporon mucoides.

[0120] In another embodiment, the infectious agent is a protozoan or a protozoan-derived product. Suitable protozoa (or protozoan-derived products) used in the vaccines and / or methods of the present invention include (but are not limited to) protozoa (single-celled or multi-celled), such as Plasmodium falciparum; and worms, such as tapeworms, nematodes, and flukes.

[0121] In one embodiment, suitable antigens or immunogens used in the vaccines and methods of the present invention are allogeneic antigens (autoantigens) (e.g., proteins or peptides), lipoproteins, lipids, carbohydrates, nucleic acids, enzymes, structural proteins, secretory proteins, cell surface receptors, and cytokines (e.g., TNF, IFN-γ, IL-1, or IL-6).

[0122] In some embodiments, the vaccine is an influenza vaccine. The influenza vaccine may comprise (but is not limited to) an influenza subunit vaccine, an influenza mRNA vaccine, and an adjuvant. In some embodiments, the influenza vaccine is an influenza subunit vaccine. In some embodiments, the influenza subunit includes hemagglutinin protein (HA). In some embodiments, the influenza vaccine is an influenza mRNA vaccine. In some embodiments, the influenza vaccine is an adjuvanted influenza vaccine.

[0123] In some embodiments, the vaccine is a rabies vaccine.

[0124] In some embodiments, the vaccine is a hepatitis A vaccine.

[0125] In some embodiments, the vaccine is a SARS-CoV-2 vaccine.

[0126] Antibody

[0127] In some embodiments, the immunotherapeutic agent is an antibody. An “antibody” is an immunoglobulin molecule capable of specifically binding to a target (e.g., carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in a variable region of an immunoglobulin molecule. As used herein, the term “antibody” encompasses not only complete polyclonal or monoclonal antibodies, but also any antigen-binding fragment (i.e., “antigen-binding moiety”) or its single chain, fusion proteins including antibodies, and any other modified configurations of immunoglobulin molecules including antigen recognition sites, including (e.g., but not limited to) scFv, single-domain antibodies (e.g., shark and camel antibodies), maxibodies, microantibodies, intracellular antibodies, bivalent antibodies, trivalent antibodies, tetravalent antibodies, v-NAR, and bi-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1 126-1 136). Antibodies can include any type of antibody, such as IgG, IgA, or IgM (or their subspecies), and need not be of any particular type. Immunoglobulins can be classified into different types based on the antibody amino acid sequence in the heavy chain constant region.

[0128] There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different types of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.

[0129] The antibodies used in this disclosure may be monoclonal antibodies. The term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone) and not to a method of its production. Preferably, monoclonal antibodies exist in the form of a homogeneous or substantially homogeneous population. In immunotherapy, monoclonal antibodies (mAbs) are manufactured in vitro to recognize specific target antigens. They are used to treat, for example, solid and hematopoietic tumors, inflammatory conditions, and infections. Most mAbs used clinically target a single antigen, but a few are modified to be bispecific. The monoclonal antibodies contemplated herein may encompass murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.

[0130] "Humanized" antibodies refer to non-human (e.g., murine) antibody forms that chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody), containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) from the recipient are replaced by residues of the CDR from a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and capability. The term "chimeric antibody" can also refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0131] The antibodies disclosed herein may be produced using techniques well known in the art (e.g., recombinant techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques readily known in the art) (e.g., see Jayasena SD, Clin. Chem., 45: 1628-50 (1999) and Fellouse FA et al., J. Mol. Biol., 373(4):924-40 (2007)).

[0132] Immunotherapy antibodies are well-known in the industry and include (but are not limited to) bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ustekinumab, etanercept, gemtuzumab, alemtuzumab, rituximab, and trastuzumab. Monoclonal antibodies (trastuzumab), nimotuzumab, palivizumab, daratumumab, denosumab, dinutuximab, elotuzumab, isatuximab, margetuximab, mogamulizumab, naxitamab, necitumab, obintuzumab, and abeiximab.

[0133] Antibodies can be contacted with immune organoids as described herein at any dose to seek testing of antibody efficacy. In some embodiments, antibodies are contacted with immune organoids at doses of about 0.1 µg / mL, 0.2 µg / mL, 0.3 µg / mL, 0.4 µg / mL, 0.5 µg / mL, 0.6 µg / mL, 0.7 µg / mL, 0.8 µg / mL, 0.9 µg / mL, 1.0 µg / mL, 2.0 µg / mL, 3.0 µg / mL, 4.0 µg / mL, 5.0 µg / mL, 6.0 µg / mL, 7.0 µg / mL, 8.0 µg / mL, 9.0 µg / mL, 10 µg / mL, 15 µg / mL, or 20 µg / mL.

[0134] In some embodiments, the immunotherapeutic antibody is a nanobody. Nanobodies in immunotherapy are well known in the field and described, for example, by Maali et al., “Nanobodies in Cell-Mediated Immunotherapy: On the Road to Fight Cancer,” Frontiers in Immunology 2023; 14: 1012841. Nanobodies, or VHHs (heavy chain variable domains of heavy chain antibodies (HCAbs) only), are derived from camel heavy chain antibodies. Nanobodies, as the smallest natural antigen-binding domains, can have sizes ranging from approximately 2.5 nm in diameter to approximately 4 nm in height and approximately 15 kD in molecular weight. High-affinity nanobodies targeting different targets (including tumor markers) can be selected from phage display libraries using biopanning processes.

[0135] Nanobodies can be readily produced in microorganisms, mammalian cells, or plants. Nanobodies are expressed in high yields, whether in the periplasm of Escherichia coli or the cytoplasm of eukaryotic cells.

[0136] Nanobodies have been used in various applications, including biosensing, protein affinity capture, and protein crystallization. It targets tumor cell surface receptors, particularly for cancer therapeutics, such as HER2 (Hussack G, Raphael S, Lowden MJ, Henry KA, Isolation and characterization of camelid single-domain antibodies against HER2, BMC Res notes (2018) 11(1):866.doi: 10.1186 / s13104-018-3955-8), CAIX (Araste F, Ebrahimizadeh W, Rasooli I, Rajabibazl M, Mousavi Gargari SL). SL), A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoformIX and its usefulness for cancer diagnosis, Biotechnol Letters (2014) 36(1):21-8. doi: 10.1007 / s10529-013-1340-1), TAG-72 (Sharifzadeh Z, Rahbarizadeh F, Shokrgozar MA, Ahmadvand D, Mahboudi F, Rahimi Jamnani F, et al., Development of oligoclonal nanobodies for targeting the tumor-associated glycoprotein 72 antigen, Molecular Biotechnol (2013) 54(2):590-601.doi: 10.1007 / s12033-012-9601-0), DR5 (Huet HA, Grauney JD) (Growney JD, Johnson JA, Li J, Bilic S, Ostrom L, et al., Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction, Monoclonal Antibodies (mAbs) (2014) 6(6):1560-70. doi: 10.4161 / 19420862.2014.975099), c-Met (Slödl TS (Sl. rdahl TS), Denayer T), Moen SH), Standal T), Borset M), rsetM), Ververken C et al., Anti-c-MET nanobody - a new potential drug in multiple myeloma treatment, European Journal of Hematology (2013) 91(5):399-410. doi: 10.1111 / ejh.12185; Chen T, Liu X, Hong H, Wei H, Novel single-domain antibodies against the EGFR domain III epitope exhibit the anti-tumore effect, Journal of Translational Medicine (2020) 18(1):376. doi: 10.1186 / s12967-020-02538-y), mesothelin (Tang Z, Feng M, Gao W, Feng Y, Chen W, Chaudhary A, et al., A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cellsurface), Molecular Cancer Ther (Mol Cancer Ther) (2013) 12(4):416-26. doi: 10.1158 / 1535-7163.MCT-12-0731), AgSK1 (Rashidi SK, Mousavi Gargari SL, Ebrahimizadeh W, Targeting colorectal cancer cell lines using nanobodies; AgSK1 as a potential target, Iranian J Biotechnol (2017) 15(2):78-86. doi: 10.15171 / ijb.1472) and CD33 (Romão E, Krasniqi A, Maes L, Vandenbrande C, Sterckx YG, Stijlemans B) B) et al., Identification of nanobodies against the acute myeloid leukemia marker CD33, International Journal of Molecular Sciences (Int J Mol Sci) (2020) 21(1):310. doi:10.3390 / ijms21010310.

[0137] Currently, approximately 16 therapeutic nanobodies have entered clinical trials for various disease types (Arbabi-Ghahroudi M, Camelidsingle-domain antibodies: Promises and challenges as lifesaving treatments, International Journal of Molecular Sciences (2022) 23(9):5009. doi: 10.3390 / ijms23095009).

[0138] Gene therapy

[0139] In some embodiments, the immunotherapeutic agent is a gene therapy. The term "gene therapy" is used in the industry in its general sense. Simply put, "gene therapy" refers to the transfer of genetic material of interest (e.g., DNA or RNA polynucleotides) into host cells and / or tissues to treat or prevent disease symptoms. The target genetic material encodes a product (e.g., a protein, peptide, or functional RNA) that is desired to be produced in vivo. For example, the target genetic material may encode an enzyme, hormone, receptor, or peptide with therapeutic value.

[0140] According to this disclosure, any method available in the industry for gene therapy may be used. For example, gene therapy technology is described in the following literature: Alnasser, Gene 769:145246 (2021); Kohn et al., Gene Therapy 30:738-746 (2023); Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932. (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); and May, TIBTECH 11(5):155-215 (1993). Commonly known recombinant DNA techniques for gene therapy are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993) and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0141] The main immune targets of gene therapy include (but are not limited to) intercytokine / chemokine genes, tumor-associated antigens, fusion proteins containing tumor antigens, and genetically modified tumor cells or immune cells. These immune targets are described in detail in the industry, for example, Akbulut, “Immune Gene Therapy of Cancer”, Turk J Med Sci 202 50(7): 1679-1690.

[0142] Cell therapy

[0143] In some embodiments, the immunotherapeutic agent is a cell therapy. Cell therapy refers to a treatment method that expands normal cells or biotechnologically modified cells in vitro and then transplants or infuses them into a patient. The newly introduced cells may replace damaged cells to rebuild tissue structure and function (stem cell therapy technology) or have a strong immune-killing function (immunocellular therapy technology) to achieve the purpose of treating diseases.

[0144] For the purposes of the methods disclosed herein, any type of cell therapy may be considered. For example, cell therapy may include pluripotent stem cell therapy, adult stem cell therapy, cancer stem cell therapy, fibroblast therapy, chondrocyte therapy, keratinocyte therapy, hepatocyte therapy, pancreatic islet cell therapy, T-cell therapy, dendritic cell therapy, natural killer cell therapy, and macrophage cell therapy. These types are described in more detail in, for example, El-Kadiry et al., Frontiers in Medicine 8 (2021).

[0145] In some embodiments, cell therapy is receptive cell therapy. "Receptive cell therapy" refers to immunotherapy in which an individual is given immune cells to help them fight a disease (such as cancer) or viral infection. For example, in cancer therapy, T cells are harvested from an individual's own blood (or from a donor's blood) or tumor tissue, grown in large numbers, and then returned to the individual to help them fight cancer. Types of receptive cell therapy include tumor-infiltrating lymphocyte ("TIL") therapy, T cell receptor ("TCR") therapy, and chimeric antigen receptor T cell (CAR-T cell) therapy.

[0146] "Tumor-infiltrating lymphocyte ("TIL") therapy" or "TIL therapy" refers to an immunotherapy that uses receptive cell therapy that utilizes lymphocytes within or near a tumor and possesses the ability to recognize the tumor. In TIL therapy, lymphocytes (such as T cells) within or near the tumor are isolated and then treated with a substance that causes them to grow rapidly in large numbers. These lymphocytes are then returned to the individual.

[0147] "T-cell receptor therapy" or "TCR therapy" refers to a class of receptive cell therapies that involve modifying an individual's or donor's T or immune cells to express specific or particular T-cell receptors or TCRs.

[0148] "Chimeric antigen receptor T-cell therapy" or "CAR-T therapy" refers to a receptive cell therapy that transforms one or more portions of the T-cell receptor into extracellular binding portions (such as antibodies or antibody fragments). Tumor-associated antigens (TAAs) or tumor-specific antigens can be used to target the extracellular binding portions (such as antibodies or antibody fragments).

[0149] Small molecules

[0150] In some embodiments, the immunotherapeutic agent is a small molecule. "Small molecule" refers to a composition with a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about 0.5 kD. Small molecules may include nucleic acids, peptides, polypeptides, peptide-like substances, peptides, carbohydrates, lipids, or components of these or other organic or inorganic molecules. In some embodiments, the small molecule is a small molecule-based immunomodulator. The small molecule includes those that target the innate immune system, the adaptive immune system, and the tumor microenvironment. Small molecule-based immunomodulators refer to non-steroidal agents that reduce the production or secretion of pro-inflammatory cytokines, resulting in a reduction of pro-inflammatory responses, or otherwise modulate the immune system. The small molecules mentioned are known in the industry and described in more detail in, for example, Wu et al., Acta Pharm Sin B 12:4287-4308 (2022); Zong et al., Signal Transduction and Targeted Therapy 6 (2021); and Dhanak et al., Cell Chemical Biology 24 (2017). Examples of small molecule immunomodulators include p38 kinase inhibitors such as VX 702 (Vertex Pharmaceuticals), SCIO 469 (Scios), doramapimod (Boeringer Ingelheim), RO 30201195 (Roche), and SCIO 323 (Scios); TACE inhibitors such as DPC 333 (Bristol Myers Squibb); ICE inhibitors such as pranalkasan (Vertex Pharmaceuticals); and IMPDH inhibitors such as mycophenolate (Roche) and meremepodib (Vertex Pharmaceuticals).

[0151] touch

[0152] This allows the therapeutic agents described herein to be contacted with immune organoids in a manner involving exposure of the organoids to therapeutic levels of known or unknown therapeutic agents. Typically, the agent is dissolved in solution to a (predicted) therapeutically effective concentration and added to a culture in a vessel for maintaining the culture. The therapeutic agent dosage range will vary depending on the specific composition and the therapeutic agent being contacted with the organoids.

[0153] In some embodiments, the therapeutic agent is exposed to organoids for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the therapeutic agent is exposed to organoids for about 5 days. In some embodiments, the therapeutic agent is exposed to organoids for about 7 days. In some embodiments, the therapeutic agent is exposed to organoids for about 11 days. In some embodiments, the therapeutic agent is exposed to organoids for about 14 days. In some embodiments, the therapeutic agent is exposed to organoids for about 21 days. In some embodiments, the therapeutic agent is exposed to organoids for 25 days. In some embodiments, the therapeutic agent is exposed to organoids for about 28 days.

[0154] The therapeutic agent described herein may also be contacted with the immune organoid at various time points during immune organoid formation. In some embodiments, the therapeutic agent is contacted with the immune organoid on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 0. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 1. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 2. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 3. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 3. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 4. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 5. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 6. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 7. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 8. In some embodiments, the therapeutic agent is contacted with the immune organoid on day 9. In some embodiments, the therapeutic agent is exposed to the immune organoids on day 10. In some embodiments, the therapeutic agent is exposed to the immune organoids on day 11. In some embodiments, the therapeutic agent is exposed to the immune organoids on day 12. In some embodiments, the therapeutic agent is exposed in a primary-boost regimen. For example, in some embodiments, the therapeutic agent is exposed to the immune organoids on days 0, 1, 2, 3, 4, 5, and 6, and then exposed again to the immune organoids on days 14, 15, 16, 17, 18, 19, and 20.

[0155] The vaccine can be contacted with immune organoids as described herein at any dose to test the vaccine's efficacy. In some embodiments, the vaccine is contacted with immune organoids at doses of about 0.1 µg / mL, 0.2 µg / mL, 0.3 µg / mL, 0.4 µg / mL, 0.5 µg / mL, 0.6 µg / mL, 0.7 µg / mL, 0.8 µg / mL, 0.9 µg / mL, 1.0 µg / mL, 2.0 µg / mL, 3.0 µg / mL, 4.0 µg / mL, 5.0 µg / mL, 6.0 µg / mL, 7.0 µg / mL, 8.0 µg / mL, 9.0 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, 35 µg / mL, 40 µg / mL, 45 µg / mL, or 50 µg / mL.

[0156] In some embodiments, the vaccine is applied to the immune organoids at doses of approximately 0.1 IU / mL, 0.5 IU / mL, 1 IU / mL, 1.5 IU / mL, 2 IU / mL, 2.5 IU / mL, 5 IU / mL, 10 IU / mL, 15 IU / mL, 20 IU / mL, 25 IU / mL, 30 IU / mL, 35 IU / mL, 40 IU / mL, 45 IU / mL, 50 IU / mL, 55 IU / mL, 60 IU / mL, 65 IU / mL, 70 IU / mL, 75 IU / mL, 80 IU / mL, 85 IU / mL, 90 IU / mL, 95 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, or 160 IU / mL.

[0157] In some embodiments, the vaccine is an influenza subunit vaccine that is in contact with immune organoids at doses of about 0.5 µg / mL, 0.6 µg / mL, 0.7 µg / mL, 0.8 µg / mL, 0.9 µg / mL, 1.0 µg / mL, 2.0 µg / mL, 3.0 µg / mL, 4.0 µg / mL, 5.0 µg / mL, 6.0 µg / mL, 7.0 µg / mL, 8.0 µg / mL, 9.0 µg / mL, or 10 µg / mL. In some embodiments, the vaccine is an influenza mRNA vaccine that is exposed to immune organoids at doses of approximately 0.1 µg / mL, 0.2 µg / mL, 0.3 µg / mL, 0.4 µg / mL, 0.5 µg / mL, 0.6 µg / mL, 0.7 µg / mL, 0.8 µg / mL, 0.9 µg / mL, 1.0 µg / mL, 2.0 µg / mL, 3.0 µg / mL, 4.0 µg / mL, 5.0 µg / mL, 6.0 µg / mL, 7.0 µg / mL, 8.0 µg / mL, 9.0 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, or 35 µg / mL.

[0158] In some embodiments, the vaccine is a rabies vaccine that is in contact with an immune organoid at a dose of about 2.5 IU / mL, 5 IU / mL, 10 IU / mL, 15 IU / mL, 20 IU / mL or 25 IU / mL.

[0159] In some embodiments, the vaccine is a hepatitis A vaccine that is in contact with an immune organoid at a dose of approximately 20 IU / mL, 25 IU / mL, 30 IU / mL, 35 IU / mL, 40 IU / mL, 45 IU / mL, 50 IU / mL, 55 IU / mL, 60 IU / mL, 65 IU / mL, 70 IU / mL, 75 IU / mL, 80 IU / mL, 85 IU / mL, 90 IU / mL, 95 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, or 160 IU / mL.

[0160] Measuring immune response

[0161] The methods described herein further involve measuring the immune response of three-dimensional immune organoids to a therapeutic agent after a contact step. An important indicator of drug efficacy is how the drug acts on the immune system. Measuring the immune response may include measuring any aspect of the immune response, such as the induction of an innate or adaptive immune response. In some embodiments, the methods described herein involve measuring an innate immune response. In some embodiments, the methods described herein involve measuring an adaptive immune response.

[0162] Innate immune responses refer to non-antigen-specific reactions, such as the release of soluble effector compounds and non-specific phagocytosis performed by dendritic cells, macrophages, etc. They also refer to cellular changes that enhance the ability of cells to present antigens and / or regulate antigen-specific adaptive immune responses. Monocyte lineage cells mediate the initiation and progression of inflammation and other early immune responses through direct cytotoxicity, secretion of soluble factors, and / or regulation of adaptive immune responses. These include the expression of adhesion molecules on monocyte-derived cells and potential vascular endothelial cells, as well as the release of cytokines, chemokines, tissue-destructive metalloproteinases, and reactive oxygen species.

[0163] Adaptive immune responses may involve antibody production or activation of specific immune competent cells, or both. Adaptive immune responses encompass both cellular and humoral immune responses.

[0164] In some embodiments, the methods described herein involve measuring cellular immune responses. Cellular immunity is generally associated with the activation of macrophages, natural killer (NK) cells, antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to stimuli. More generally, cellular immunity is not antibody-dependent but is associated with the activation of immune system cells. Cellular immune responses are characterized, for example, by the activation of antigen-specific cytotoxic T lymphocytes capable of inducing apoptosis in somatic cells (e.g., virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens) on their surface; activation of macrophages and natural killer cells, thereby enabling them to destroy pathogens; and stimulation of cells to secrete various cytokines that can influence the function of other cells involved in adaptive and innate immune responses. Specifically, cellular immune responses involve the presentation of peptide epitopes in conjunction with class II or class I MHC molecules to respectively activate antigen-specific CD4. + T helper cells and / or CD8 + Cytotoxic T cells. This response may also involve the activation or recruitment of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils, or other components of the innate immune system. This can be (for example) detected by proliferation assays (CD4+). -T cell (T cell) or CTL (cytotoxic T lymphocyte) analysis is used to determine the presence of a cell-mediated immune response.

[0165] In some embodiments, the methods described herein involve measuring humoral immune responses. Humoral immunity is generally associated with antibody production and its possible accompanying accessory processes. Humoral immune responses can generally be characterized as, for example, Th2 activation and intercytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell generation. Humoral immunity can also generally be associated with the effector functions of antibodies, including pathogen and toxin neutralization, classical complement activation, and opsonin-promoted phagocytosis and pathogen elimination. In some embodiments, humoral immune responses are measured by detecting germinal center formation. In some embodiments, humoral immune responses are measured by detecting antibody type switching. In some embodiments, humoral immune responses are measured by detecting memory B cell induction.

[0166] In some embodiments, the immune response is measured by measuring both innate and adaptive immune responses.

[0167] In some embodiments, immune responses are measured by measuring the number of immune cells, immune cell proliferation, immune cell phenotype, immune cell polarization, antibody production, cytokine production, chemokine production, changes in B cell receptors, changes in T cell receptors, and / or immune memory.

[0168] Immune cell proliferation / number

[0169] In some embodiments, immune cell proliferation is measured. Any suitable method known in the art can be used to measure immune cell proliferation. For example, lymphocyte proliferation can be measured using carboxyfluorescein diacetate succinimide diester (CFSE) dilution analysis or by incorporation of [31-l]-thymidine. Additionally, techniques that can identify immune cell types based on markers (such as flow cytometry as described in the examples provided herein) can be used to measure the number of immune cells, such as CD3+ T cells, CD19+ B cells, CD56+ and / or CD16+ NK cells, CD27+CD8++ plasmablasts, CD138+ plasma cells, CD15+ granulocytes, CD11b+CD45+ dendritic cells, CD14+CD11c+ myeloid dendritic cells, CD123+ plasmacytoid dendritic cells, CD45- stromal cells, PDPN+CD31+ fibroblasts and reticular cells, CD45+PDPN+CD35+ follicular dendritic cells, CD27+CD38+ germinal center B cells, CD14+CD11b+ monocytes, and CD68+ or CD14+ macrophages.

[0170] In one embodiment, an increase in immune cell proliferation and / or the number of immune cells in organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. In one embodiment, a decrease in immune cell proliferation and / or the number of immune cells in organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. In one embodiment, no change in immune cell proliferation and / or the number of immune cells in organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0171] In some embodiments, compared with a control, the increase in the proliferation of immune cells and / or the number of immune cells in immune organoids cultured with the therapeutic agent is an increase of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0172] In some embodiments, compared with a control, the reduction in the proliferation of immune cells and / or the number of immune cells in immune organoids cultured with the therapeutic agent is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0173] "Control" refers to a sample or standard used for comparison with the test sample. In some embodiments, the control is a three-dimensional organoid obtained from a healthy individual who has never been exposed to any therapeutic agent. In some embodiments, the control is a historical control or a standard reference value or range of values ​​(e.g., a previously tested control sample).

[0174] Immune cell phenotype

[0175] In some embodiments, immune cell phenotypes are measured. Phenotypic characterization of cell populations via surface markers can be performed by individual staining of cells (such as flow cytometry as described in the examples provided herein) or by preparing tissue sections of the population in situ according to standard methods. Determining the expression characteristics of surface markers (i.e., immunophenotypic characterization) via antibodies can be direct (using labeled antibodies) or indirect (using a second labeled antibody with primary specificity against the cell marker), thereby amplifying the signal. Alternatively, the presence of antibody binding can be determined by various methods, including (but not limited to) immunofluorescence microscopy and radiography. Similarly, antibody binding levels can be monitored by flow cytometry, a technique for correlating fluorescent dye levels with the amount of antigen present on the cell surface that specifically binds labeled antibodies. Differential expression of a series of surface markers on a cell population provides a method for identifying and separating said populations.

[0176] In some embodiments, changes in the immune cell phenotype in organoids cultured with the therapeutic agent, compared to a control, indicate that the therapeutic agent of interest is effective. In some embodiments, no change in the immune cell phenotype in organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0177] For example, in some embodiments, therapeutically treated immune organoids can exhibit time-dependent changes in B cell phenotype. As demonstrated in the examples herein, different populations can emerge at different time points. For instance, a significant population can progress from major naive B cells (CD38-CD27-) on day 0 to germinal center B cells (CD38+CD27+) on days 7-14, and finally to plasmablasts (CD38+++CD27+) on day 21, demonstrating successful activation and maturation of B cells within the organoid system.

[0178] Similarly, in some embodiments, immune organoids treated with therapeutic agents can exhibit time-dependent plasmablast differentiation. As demonstrated in the examples herein, organoids can exhibit an increase in CD38++CD27+ plasmablasts in response to vaccination.

[0179] Immune cell polarization

[0180] In some embodiments, immune cell polarization is measured. Immune cell polarization is the process by which immune cells respond to specific signals, adopt different procedures, and perform specific functions. Various methods known in the art can be used to measure immune cell polarization, including (but not limited to) intracellular cytokine staining, flow cytometry, ELISA, ELISpot, MSD, and / or Luminex methods.

[0181] In some embodiments, immune cell polarization is measured by measuring the production of intercytokines (including, for example, IL10, CXCL10, IFN-γ, IL-17, TNF, IL-4, and IL-2). In some embodiments, IL10 production is measured. In some embodiments, CXCL10 production is measured. In some embodiments, IFN-γ production is measured.

[0182] Techniques for measuring intercytokines and chemokines are known to those skilled in the art. For example, enzyme-linked immunosorbent assay (ELISA) can be used to measure the levels of intercytokines produced by immune organoids. In one embodiment, the EMDmillipore LUMINEX® xMAP® multiplexer can be used to measure intercytokines levels. In some embodiments, bead-based multiplexers are used to measure the production of intercytokines and chemokines. In some embodiments, the bead-based multiplexer is LEGENDplex™.

[0183] In some embodiments, changes in immune cell polarization in organoids cultured with the therapeutic agent, compared to a control, indicate that the therapeutic agent of interest is effective. In some embodiments, no change in immune cell polarization in organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0184] Antibody production

[0185] In some embodiments, antibody production is measured. Antibody production can indicate the immune system's response to a particular therapeutic agent and thus serve as an indicator of efficacy, or in some cases, an indicator of insufficient efficacy. This is particularly relevant to vaccines for infectious diseases and cancer vaccines, where the production of antibodies against the antigen of interest is the desired outcome. For other therapies, antibodies may be produced against the treatment itself, leading to potential resistance to the therapy.

[0186] Antibody production can be measured using methods known in the industry. In some embodiments, antibody production is measured using an ELISA assay. In some embodiments, antibody production is measured using a bead-based multiplex assay. In some embodiments, the bead-based multiplex assay is LEGENDplex™. In some embodiments, the production of specific IgG, IgM, and IgA antibodies against immune organoids is measured using an ELISA as described in the examples provided herein. In some embodiments, IgG antibody production is measured. In some embodiments, IgG1 antibody production is measured. In some embodiments, IgG2 antibody production is measured. In some embodiments, IgG3 antibody production is measured. In some embodiments, IgG4 antibody production is measured. In some embodiments, IgM antibody production is measured. In some embodiments, IgA antibody production is measured.

[0187] In some embodiments, an increase in antibody production in immune organoids cultured with a therapeutic agent compared to a control indicates that the therapeutic agent of interest is effective. Alternatively, in some embodiments, an increase in antibody production in immune organoids cultured with a therapeutic agent compared to a control indicates that the therapeutic agent of interest is ineffective. In some embodiments, a decrease or no change in antibody production in immune organoids cultured with a therapeutic agent compared to a control indicates that the therapeutic agent of interest is ineffective.

[0188] Cytokine / Chemokine Production

[0189] In some embodiments, in the methods disclosed herein, the immune response is measured by measuring the production of intercytokines and / or chemokines. Evaluating the intercytokine / chemokine response in response to immunotherapy is highly relevant because these signaling molecules play a crucial role in activating and regulating the immune response, a primary goal of immunotherapy. Immunotherapy that induces an appropriate intercytokine response is a good indicator of drug efficacy.

[0190] Intercytokines are 8-30 kDa proteins and glycoproteins produced by many cell types and used as signaling molecules in cell-cell communication. They play a central role in the immune system and are involved in a variety of immune, inflammatory, and infectious diseases. Measurable examples of intercytokines include (but are not limited to) IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17F, IL18, IL19, IL20, IL21, IL22, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RA, and IL36B. 7. IL38, IL1A, IL1B, IL1RN, IFNA, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16, IFNA17, IFNA21, IFNB, IFNg, IL-1a, IL-1b, IL-6, IL-13, IL-17a, TNFA, TNFB, TRAIL, TL1, BAFF, APRIL, RANKL, CD40LG, EDA, FASLG, CD70. In some embodiments, IL10 production is measured. In some embodiments, IFNγ production is measured.

[0191] Chemokines are small, secreted chemokines that regulate cell localization and recruitment to tissues, playing crucial roles in embryogenesis, tissue development, and immune responses. To date, approximately 50 chemokines and 20 chemokine receptors have been identified. Chemokines and their receptors have been reported to play important roles in immune cell migration and inflammation, as well as in tumor initiation, promotion, and progression. (Marcuzzi E et al., Chemokines and Chemokine Receptors: Orchestrating Tumor Metastasization, International Journal of Molecular Sciences, 2018 Dec 27;20(1):96). Based on their prominent functions, chemokines can be broadly classified into two categories: inflammatory and homeostatic chemokines. Among inflammation-induced inflammatory chemokines, some non-limiting examples measurable in the methods of this disclosure include CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, and CXCL14. On the other hand, homeostatic chemokines (e.g., but not limited to) CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, and CXCL13 are constitutively expressed and involved in homeostatic leukocyte transport. In some embodiments, the chemokines include CXC chemokines or isoforms or derivatives capable of binding thereto. In some non-limiting exemplary embodiments, the chemokines may be CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, U83, and vCXC1. In some embodiments, chemokines include CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28.In some embodiments, the chemokines include CXCL12, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, and XCL2. In some embodiments, the chemokines include vMIPII, U83, or vCXC1. In some embodiments, the chemokine is CXCL10.

[0192] Techniques for measuring intercytokines and chemokines are known to those skilled in the art. For example, enzyme-linked immunosorbent assay (ELISA) can be used to measure the levels of intercytokines produced by immune organoids. In one embodiment, the EMDmillipore LUMINEX® xMAP® multiplexer can be used to measure intercytokines levels. In some embodiments, bead-based multiplexers are used to measure the production of intercytokines and chemokines. In some embodiments, the bead-based multiplexer is LEGENDplex™.

[0193] In some embodiments, an increase in the production of cytokines and / or chemokines in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. Alternatively, in some embodiments, an increase in the production of cytokines and / or chemokines in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective. In some embodiments, a decrease or no change in the production of cytokines and / or chemokines in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0194] BCR / TCR changes

[0195] In other embodiments, the immune response is measured by analyzing changes in the BCR and / or TCR. In one embodiment, changes in the BCR are measured. In another embodiment, changes in the TCR are measured. In yet another embodiment, changes in both the BCR and TCR are measured.

[0196] Both BCR and TCR changes can be measured using sequencing techniques, such as using a 10X genomics platform and a 5' kit to obtain BCR and TCR libraries, and then using NGS technology to sequence and analyze changes in the BCR and TCR libraries.

[0197] Changes in BCR that can be measured by sequencing in the methods disclosed herein include (but are not limited to) antibody isotype conversion, clonal expansion, and somatic hypermutation. Isotype conversion and somatic hypermutation are described in more detail above. Clonal expansion is the process by which daughter cells are generated from parental cells. During B cell clonal expansion (and T cell clonal expansion), numerous copies of B cells with affinity and specificity for the same antigen are produced. This can be identified by sequencing as described above.

[0198] In some embodiments, changes in BCR will result in measurable memory B cells. For example, in some embodiments, immune organoids treated with a therapeutic agent exhibit an increase in the number of memory B cells in response to the therapeutic agent. Memory B cells can be measured using methods known in the art, including (but not limited to) flow cytometry for detecting CD27+CD19+ cells. In some embodiments, memory B cells are measured using flow cytometry for detecting CD19+CD27+CD38- cells.

[0199] In some embodiments, an increase in B and / or T cell clonal expansion in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. Alternatively, in some embodiments, an increase in B and / or T cell clonal expansion in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective. In some embodiments, a decrease or no change in B and / or T cell clonal expansion in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0200] In some embodiments, an increase in memory B cells in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. In some embodiments, a decrease or no change in memory B cells in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0201] In some embodiments, an increase in isotype conversion of immune organoids cultured with the therapeutic agent compared to a control indicates that the therapeutic agent of interest is effective. In some embodiments, a decrease or no change in isotype conversion of immune organoids cultured with the therapeutic agent compared to a control indicates that the therapeutic agent of interest is ineffective.

[0202] In some embodiments, an increase in somatic hypermutation in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. In some embodiments, a decrease or no change in somatic hypermutation in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0203] Immune Memory

[0204] In other embodiments, the immune response is measured by measuring the formation of immune memory. Immune memory represents an important aspect of the mammalian immune response. Memory responses form the basis of the effectiveness of many therapeutic agents (e.g., vaccines in particular) due to increased response to subsequent exposures and long-term protection derived from immunotherapy, and strong induction of immune memory indicates preferred drugs. Therefore, the formation of immune memory is a key indicator of therapeutic efficacy. As used herein, “immune memory” refers to the state in which long-lived antigen-specific lymphocytes are available and capable of rapidly generating a response upon repeated exposure to a specific antigen. Memory B cells are long-lived B cells that express BCR on their surface but do not secrete large amounts of antibodies. Memory B cells circulate throughout the body and rapidly respond to trigger a new immune response upon re-encounter of the antigen. Memory B cells have undergone clonal expansion and differentiation, as well as affinity maturation, thus enabling them to divide more frequently and produce antibodies with higher affinity. Memory T cells can be either CD4+ or CD8+. These memory T cells have a lower threshold for MHC activation and therefore require less antigen to activate compared to naive T cells.

[0205] In some embodiments, immune memory is measured by identifying and quantifying memory T cell and memory B cell subgroups using techniques known in the art, such as flow cytometry as described in the examples herein. In some embodiments, identifying the memory T cell subgroup includes identifying CD45RO, CCR7, CD62L, and CD127 on T cells. In some embodiments, identifying the memory B cell subgroup includes identifying CD27 on B cells and CD138 on plasma cells.

[0206] In some embodiments, the formation of immune memory in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is effective. In some embodiments, a decrease or no change in immune memory in immune organoids cultured with the therapeutic agent, compared to a control, indicates that the therapeutic agent of interest is ineffective.

[0207] Other methods

[0208] This article also provides a method for predicting the range of drug efficacy outcomes across various patient groups. This method involves generating three-dimensional immune organoids, which include multiple self-assembling primary immune cells derived from one or more secondary lymphoid organoids and multiple stem cells from various donor pools. Instances of genetically diverse donors include various populations, such as intersex and multi-ethnic donors.

[0209] Analyze the various immune responses of immune organoids from a large number of donors to therapeutic agents and analyze the differential responses among the tested organoids to provide a variety of useful data, including (but not limited to) the total percentage of a representative human population in which the drug may be effective, or key phenotypic characteristics (e.g., sex or race) of individuals in a human population who are most likely to render the drug ineffective.

[0210] The immune organoids and therapeutic agents described above can be used in the methods and compositions of this disclosure.

[0211] Composition

[0212] As described in more detail below, this disclosure relates, in one aspect, to compositions of three-dimensional immune organoids comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+; and a therapeutic agent.

[0213] The immune organoids and therapeutic agents described above can be used in the methods and compositions of this disclosure.

[0214] Example

[0215] While certain alternatives have been disclosed herein, it should be understood that various modifications and combinations are possible and covered within the true spirit and scope of the appended claims. Therefore, it is not intended to be limited to the exact abstract and disclosure presented herein.

[0216] Example 1: Method

[0217] Tissue collection and freezing. Whole spleens and lymph nodes from healthy donors (e.g., deceased donors (all ages) or living donors (all ages)) were collected by a certified clinician, segmented, and placed in hypothermosol + 2x Pen / Strep, 1x Normocin, and stored at 4°C for transport until further processing. Hypothermosol culture medium was prepared and the workspace was ready. Cryopreservation tubes were labeled with the date, tissue type, batch ID, and name [attached to the cryopreservation tube label template]. Upon arrival at the laboratory, the tissues were completely immersed in fresh hypothermosol + 2x Pen / Strep, 1x Normocin, and the untreated segments were placed on ice (4°C). Using a disposable scalpel, the tissues were carefully dissected into 5 mm x 5 mm x 5 mm sections and placed in 24-well plates for dissection. Wash the tissue with 1 ml of 0.5 mM EDTA and then incubate at 37°C in 1 ml of 0.5 mM EDTA for 1 hour (adjust and record changes as needed). Incubate the tissue with 1 ml of Acumax at 37°C for 1 hour, and terminate the reaction with 1 ml of complete culture medium. Count the cells using an NC-200 and record the viable cell count and survival rate. Centrifuge the cells at 250 x g for 10 minutes. Aspirate the cells and centrifuge at 10 x 10⁻⁶ cm⁻¹. 6 Cells / mL / vial are resuspended in a 1 mL cryopreservation bag. Cells are then transferred to pre-barcoded cryovials. The vials are transferred to a gradient cooling box (Mr. Frosty) and stored overnight at -80°C, then transferred to LN2 the following day. Barcodes are recorded in the LN2 inventory (LN2 number, hotel number, case number, and case placement).

[0218] Organoid formation. After thawing, a ROCK inhibitor (e.g., Y-27632) was added to the complete culture medium. The cryovials were partially submerged in a 37°C bath and thawed until only a small amount of ice remained. Cells were transferred from the cryovials to 50 ml conical tubes. 1 ml of complete culture medium was added dropwise, rotating the tube between each drop until 9 ml of complete culture medium had been added. The tubes were rotated at 200 x g for 5 minutes. The supernatant was discarded and the cells were resuspended in 2 ml of complete culture medium. Cells were counted using an NC-200 micrometer while rotating the tubes again (200 x g, for 5 minutes) during counting. The resuspended volume was calculated to achieve 100 µl / well in the upper chamber (total 1 x 10⁻⁶ cells / well). 6 Up to 2×10 6(cells). Complete medium supplemented with 1 µg / ml BAFF, and 200 µl of supplemented medium is added to each well of (e.g.) transfer plate, ULA plate, flat plate, V-shaped plate, or base. The stimulant is added directly to the medium, and then incubated overnight at 37°C. Organoids are supplemented with BAFF-supplemented complete medium every 3 days.

[0219] Add PBMCs. Once formed, the organoid is supplemented with adaptive cells from PBMCs.

[0220] Flow cytometry. Prepare staining buffer (1x DPBS + 0.5% BSA or 1x DPBS + 1% FBS) and design a flow cytometry panel using a Biolegend spectrometer (https: / / www.biolegend.com / en-us / spectra-analyzer). Remove the organoids containing the panel from the incubator and place them in a pre-cleaned biosafety cabinet. Lyse the organoids to produce a single-cell suspension. Count the cells and place them in flow cytometry tubes. Add 2 mL of staining buffer to each tube. Centrifuge the cells at 500g for 5 minutes and resuspend them in approximately 50 µL of staining buffer. Add the appropriate amount of primary antibody according to the manufacturer's instructions and incubate at 4°C in the dark for 30 minutes. The antibody group included CD3, CD4, CD8, CD19, CD38, CD27, CD127, CD138, PDPN, CD11c, HLA-DR, CD123, live / dead, CD45, CD45RO, CD14, CD56, CD68, CD25, CXCR5, PD-1, CD35, CD20, CD11b, and CFSE. The samples were washed twice with 2 mL of flow staining buffer, the supernatant was aspirated, and the cells were resuspended in the residual buffer. Secondary antibodies were added according to the manufacturer's instructions, and the cells were incubated at 4°C in the dark for 30 minutes. The samples were washed twice with 2 mL of flow staining buffer, the supernatant was aspirated, and the test samples were resuspended in 250 µL of staining buffer, while the unstained control was resuspended in 350 µL. If necessary, the cells were fixed at this point using 1% paraformaldehyde. A monochromatic compensation control was prepared, and the samples were analyzed.

[0221] ELISA. First, prepare the analytical diluent (1% BSA in 1x PBS, free of +Mg and +Ca) and wash buffer (1x PBS + 0.05% Tween -20). Coat the ELISA plate with coating buffer diluted from 5x ELISA coating buffer to 1x working solution in deionized water. Coat columns “1” and “2” with coating buffer diluted 1:300 with IgG capture antibody (100 µL / well). Coat the remaining wells with coating buffer containing influenza A recombinant protein at a concentration of 0.1 µg / well (100 µL per well). Cover the plate with a membrane and incubate at 20–25°C on a shaker for 1 hour or cover with a membrane and incubate overnight at 4°C. Block the ELISA plate by washing each well with 300 µL of the prepared wash buffer. Repeat this process until a total of 4 times are complete. Add 200 µL of analytical diluent to each well of the plate and incubate on a shaker at room temperature for a total of 1 hour. Prepare all standards during plate sealing. Dilute the standards to a concentration of 1000 ng / mL using analytical diluent. Add 500 µL of analytical diluent to each tube. Add 500 µL of AD from the initially prepared standard (200 ng / mL). This is now tube 1 with a final concentration of 100 ng / mL. Continue serial dilutions twice to the final tube. Each tube contains only analytical diluent.

[0222] Remove the plate from the shaker and wash it a total of 4 times. Add the prepared standard in duplicate to the wells at 100 µL and determine the sample dilution. Based on the sample dilution, add the analytical diluent to all wells of the plate, for a total volume of 100 µL / well. Place the plate on the shaker at room temperature for 2 hours.

[0223] To add the detection antibody-HRP, prepare a detection antibody dilution at a ratio of 1:100,000. Remove the plate from the shaker and wash it a total of 4 times. Add 100 µL of the prepared detection antibody solution to all wells. Cover the plate with a membrane and place it on a shaker for 1 hour.

[0224] The next step is 2TMB substrate incubation and reaction termination. Prepare the TMB substrate and protect it from light. Add 5.5 mL of the solution to each well. Remove the plate from the shaker and wash a total of 5 wells with 300 µL of the prepared wash buffer per well. Add 100 µL of the prepared TMB solution to each well. Place the plate in the dark and monitor for color change. When the fourth standard has developed color, add 100 µL of ELISA stop solution or (2N H2SO4) to each well.

[0225] Acquire data using a plate reader. For data analysis, the repeatability of the absorbance value should be within 10%. Plot a standard curve for the IgG standards (known concentrations) and the sample, and plot the average absorbance value for each standard concentration minus the blank value for each standard on the vertical (Y) axis. Plot the corresponding human IgG concentrations on the horizontal (X) axis in relation to the absorbance values. This is used to extrapolate the concentration of unknown samples.

[0226] Automatic image segmentation for area and diameter. Open the brightfield image and equalize its histogram. Set the saturated pixel portion to 0%. Perform Canny edge detection using a Gaussian kernel radius pixel resolution set according to the settings. Run a maximum filter with a radius set according to the settings. This filter produces a running window that uses the maximum value of neighboring pixels instead of the center pixel. Perform a morphological closing operation with an iteration number set to 10 and a count set to 3. This fills in any remaining small holes in the image. Run a morphological opening operation with an iteration number set to 10 and a count set to 3. This eliminates small structures such as debris located on the outer side of organoids. Overlay the outline of the segmented region onto the original brightfield image and check the segmentation accuracy. Adjust the program parameters if necessary, focusing particularly on the high threshold setting for Canny edge detection. Calculate the area using Analyze > Measure. Calculate the diameter using the formula d = 2√(A / π).

[0227] Sandwich antigen-specific IgG ELISA. Thaw the plate containing the supernatant on ice for several hours on the day of sample transfer. Prepare the analytical diluent, which also serves as a blocking buffer (1% BSA in 1x PBS, excluding +Mg and +Ca). Add 4 ml of 7.5% BSA to 26 ml of DPBS (total = 30 ml). Store the analytical diluent at 4°C for short-term use and at -20°C for long-term use. Prepare the wash buffer (1X PBS + 0.05% Tween 20). Prepare the 10X wash buffer (10x PBS + 0.5% Tween 20) by adding 2.5 ml of Tween 20 to 500 ml of 10x PBS. Add 900 ml of MiliQ water to 100 ml of 10X buffer to prepare 1 l of 1X wash buffer. Store any remaining 10X wash buffer at 4°C for later use.

[0228] Coat the ELISA plate. Prepare the coating solution as follows (1 µg / ml antibody, 1x coating buffer), ensuring a total volume of 100 µl / well. Dilute 5x ELISA coating buffer to 1x working solution in deionized water. Add the capture antibody to a final concentration of 1 µg / ml. Add 100 µl / well. Cover the plate with the membrane and incubate on a shaker with gentle vortexing at 20–25°C for 1 hour or overnight at 4°C.

[0229] Close the ELISA plate. Wash the plate as follows. Load wash buffer into the plate washer. Wash the plate four times with 250 µl / well of wash buffer. Close the plate with 200 µl / well of analytical diluent. Cover the plate with the membrane and incubate on a shaker at room temperature for 1 hour or overnight at 4°C.

[0230] Capture antigens / antibodies. Wash the plate four times with 250 µl / well of wash buffer. Prepare the capture antigen / antibody solution as follows. Prepare sufficient solution to ensure a total volume of 100 µl / well. Add the capture antigen / antibody to the analytical diluent to achieve a final concentration of 1 µg / ml. Coat the plate with 100 µl / well of the prepared antigen / antibody solution. Cover the plate and incubate on a shaker at room temperature for 1 hour.

[0231] Sandwich antigen-specific IgM / IgA ELISA. On the day of sample transfer, thaw the plate containing the supernatant on ice for several hours. Prepare the analytical diluent, which also serves as a blocking buffer (1% BSA in 1x PBS, excluding +Mg and +Ca). Add 4 ml of 7.5% BSA to 26 ml of DPBS (total = 30 ml). Store the analytical diluent at 4°C for short-term use and at -20°C for long-term use. Then prepare the wash buffer (1X PBS + 0.05% Tween 20). Prepare the 10X wash buffer (10x PBS + 0.5% Tween 20) by adding 2.5 ml of Tween 20 to 500 ml of 10x PBS. Add 900 ml of MiliQ water to 100 ml of 10X buffer to prepare 1 l of 1X wash buffer. Store any remaining 10X wash buffer at 4°C for later use.

[0232] Coat the ELISA plate. Prepare the coating solution as follows (1 µg / ml antibody, 1x coating buffer), ensuring a total volume of 100 µl / well. Dilute 5x ELISA coating buffer to 1x working solution in deionized water. Add the capture antibody to a final concentration of 1 µg / ml and add 100 µl / well. Cover the plate with the membrane and incubate on a shaker with gentle vortexing at 20–25°C for 1 hour or overnight at 4°C.

[0233] Block the ELISA plate. Load washing buffer into the plate washer and wash the plate four times with 250 µl / well of washing buffer. Block the plate with 200 µl / well of analytical diluent. Cover the plate with the membrane and incubate on a shaker at room temperature for 1 hour or overnight at 4°C.

[0234] The plate was then washed four times with 250 µl / well of wash buffer. The capture antigen / antibody solution was prepared as follows: Prepare sufficient solution to ensure a total volume of 100 µl / well. The capture antigen / antibody was added to the analytical diluent to achieve a final concentration of 1 µg / ml. The plate was coated with 100 µl / well of the prepared antigen / antibody solution. The plate was covered and incubated on a shaker at room temperature for 1 hour.

[0235] Luminex. Harvest the cell culture supernatant at the time point of interest. Ensure that cells are not collected when removing the supernatant from the organoid culture. Store aliquots at -80°C. Coat carboxylated Luminex beads with the monoclonal antibody required for the study using the two-step carbodiimide procedure described by the manufacturer. Wash 5 x 10⁵ cells / ml in 100 μL distilled water. 6 The microspheres were prepared by centrifugation at 8000 xg for 2 minutes, followed by removal of the supernatant. The beads were resuspended in 80 µl of 100 mM sodium dihydrogen phosphate at pH 6.2. 10 µl of 50 mg / mL sulfo-NHS (diluted in dH2O) and 10 µl of 50 mg / mL EDC (diluted in dH2O) were added to the microspheres and gently vortexed. The beads were incubated in a hot mixer at room temperature, gently mixed, and at 1000 rpm for 20 minutes. The microspheres were washed three times with 250 µl of 50 mM MES at pH 5.0. The beads were resuspended in 100 µl of 50 mM MES at pH 5.0 and mixed with 25 µg of monoclonal antibody in the same buffer to a final volume of 500 µl. The coupling reaction was incubated at 1000 rpm and room temperature for 2 hours. The beads were washed and resuspended in PBS containing 1% bovine serum albumin and 0.05% sodium azide. Coupling efficiency was determined by incubating 2000 coated beads with 50 µl of 1 µg / ml R-phycoerythrin (PE) goat anti-mouse IgG (H+L) antibody. The beads were washed twice with 500 µl PBS-0.05% Tween 20 (PBS-T) and resuspended in 125 µl PBS-T for analysis on a Luminex instrument.

[0236] Intraocular BCR and TCR sequencing. Organoids are cultured and stimulated with and without selected immunotherapies. Organoids are harvested at the time point of interest (between day 0 and day 25). Tubes are labeled with well locations to maintain single organoid / single-well resolution. Organoids can be rapidly frozen and stored at -80°C. o RNA isolation was performed at -80°C or immediately after harvest. A single-cell suspension of organoids was generated in each well. RNA was isolated from each well using the Qiagen RNA Isolation Kit according to the manufacturer's instructions. RNA quantity and quality were measured using a Nanodrop spectrophotometer and stored at -80°C. o Continue to the next step under C. Send the RNA to iRepertoire for library preparation and sequence the BCR and TCR in each well. Analyze the data using software provided by iRepertoire to evaluate BCR and TCR library characteristics, including monoclonal variation and BCR CDR3 mutation rate.

[0237] Single-cell BCR and TCR sequencing. Culture organoids and stimulate them with and without the selected immunotherapy. Harvest organoids at the time point of interest (between day 0 and day 25). Generate a single-cell suspension of the organoid in each well. Count the cells in each well and resuspend them to the appropriate concentration. Load the single cells onto a 10X Genomics microarray. Use the 10X Genomics 5' Immunophenotyping Kit to generate single-cell BCR and TCR libraries. Check library volume and purity. Send the libraries to Novogene for sequencing and analysis. Evaluate BCR and TCR library characteristics, including monoclonal variation and BCR CDR3 mutation rate.

[0238] Somatic hypermutation. Cells were harvested from day 0 and the control endpoint. Cells were washed with FACS buffer and cultured with 4 µg / mL of the biotinylated recombinant protein of interest in Fc blocking. Cells were also cultured with a fluorescently labeled antibody group to determine B cell lineages (CD38+CD27+). B cells with GC or plasmablast phenotypes were sorted into 96-well plates. RNA was isolated from individual B cells, and cDNA was synthesized, tagged with unique DNA barcodes, and collected by plate. Gene-specific PCR was used to amplify immunoglobulin remutation and mild remutation regions. Libraries were sent for sequencing and sequence analysis. Fastq files were generated and quality filtered using demultiplexing with MiSeq Reporter. Paired reads were assembled and separated according to well number and common sequence. Reads from each well were clustered into operable taxonomic units. Operable taxonomic units were analyzed using IMGTHigh V-QUEST. Clonal families are defined by the use of the same V and J genes and at least 70% amino acid consistency in the heavy and light chain CDR3 loci.

[0239] LegendPlex. All reagents were brought to room temperature before use. The multiplexed standards were reconstituted using analysis buffer. Next, the standards were serially diluted in analysis buffer according to the specific kit protocol. The supernatant was then appropriately added to the analysis buffer. The trap beads were then vortexed for 1 minute. 25 µL of analysis buffer, 25 µL of diluted sample or standard, and 25 µL of mixed beads were then added to each well of a 96-well V-plate. The plate was incubated at room temperature with shaking at ~800 rpm for 2 hours. Next, the beads were rotated and the supernatant was removed without disturbing the bead clusters. The wells were washed once with 200 µL of 1X wash buffer / well. Next, 25 µL of detection antibody was added and the plate was incubated at room temperature with shaking at ~800 rpm for 1 hour. Next, without washing, 25 µL of SA-PE was added. The plate was incubated at room temperature with shaking at ~800 rpm for 30 minutes. Next, the beads were rotated and the supernatant was removed again. The wells were washed once and the beads were resuspended in 150 µL of 1X wash buffer. The sample was read using a flow cytometer.

[0240] Example Stem cell populations exist in immune organoids treated with immunotherapy.

[0241] This example demonstrates that, similar to control immune organoids, the administered immune organoids contain a population of stem cells found only in secondary lymphoid organs, not in the blood. These stem cells proliferate and play a role in both lymph node transport and function, as well as in the transport and function of the immune organoids.

[0242] In simple terms, immune organoids were generated using the method described in Example 1. Some organoids were left untreated while others were cultured for 25 days with various doses of different immunotherapies. Immunotherapy was added only on day 1. The day 25 cultures were stained against stem cell markers, and flow cytometry was performed using antibodies specific to CD45, CD34, CD45RA, CD201 (EPCR), CD49c (ITGA3), CD105, and CD73.

[0243] Example Stem cell populations exist in immune organoids.

[0244] This example demonstrates that immune organoids contain a population of stem cells found only in secondary lymphoid organs, not in the blood. These stem cells proliferate and play a role in both lymph node transport and function, as well as in the transport and function of immune organoids.

[0245] In simple terms, immune organoids were generated using the method described in Example 1. Cultures from day 7 of vaccination were stained with stem cell markers, and flow cytometry was performed using antibodies specific to CD45, CD34, CD45RA, CD201 (EPCR), CD49c (ITGA3), CD105, and CD73. Figure 2A-2C The results demonstrate the regulation of stem cell populations characterizing after therapeutic treatment. These cells are crucial during immunotherapy treatment because they play a vital role in maintaining the structure of lymph nodes and lymphoid tissues, as well as in the formation and maintenance of carrier memory cells. They can also serve as an indicator of successful immune activation and the potential for long-term treatment durability.

[0246] Example Size of immune organoids treated with immunotherapy

[0247] This example demonstrates that organoids bred using immunotherapy (vaccines, antibodies, gene therapy, cell therapy, small molecules) did not exhibit differences in organoid integrity. Therefore, immunotherapy did not alter organoids to the point of rendering them useless or nonfunctional, and organoids can be used to screen for immunotherapy.

[0248] In simple terms, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were cultured for 25 days with various doses of different immunotherapies. Immunotherapy was added only on day 1. The day 25 cultures were imaged using bright-field microscopy, and their diameter, shape, integrity, and opacity were measured.

[0249] Example Size of immune organoids treated with immunotherapy

[0250] This example demonstrates that immunotherapy treatments can have significant effects on the size, structure, and morphology of immune organoids, depending on successful activation of the immune system and successful drug delivery. While immunotherapy does not alter organoids to the point of rendering them useless or nonfunctional and thus usable for screening immunotherapies, suboptimal formulations or concentrations can lead to cell inactivation and proliferation, or toxicity and cell death. However, the size and morphology of immune organoids are highly dependent on the type of treatment and how it is designed to influence the immune system. Immune cell activation and inhibition can have different effects on organoid size, and significant growth can be observed after successful stimulation due to cell proliferation.

[0251] In simple terms, immune organoids are generated using the method described in Example 1. Figure 8 The AE row represents different influenza mRNA vaccine formulations, and columns 1-5 represent reductions in mRNA concentration. Immunotherapy is added only on day 0. Figure 8 As shown, day 5 and day 21 cultures were imaged and their diameters measured using bright-field microscopy. All organoids exhibited morphological changes in response to vaccination; however, higher concentrations of mRNA showed greater morphological changes and the formation of larger structures around the organoids compared to lower doses. In this example, the effect of different mRNA concentrations on organoid morphology was greater than that of the vaccine formulation. These morphological differences disappeared by day 21, and the diameters were similar under different conditions. These early morphological differences led to accelerated antibody and cellular responses at earlier time points, but by day 21, all organoids showed the same response at all concentrations and in all vaccine formulations (data not shown).

[0252] Example Immunotherapy-treated organoids Formed within hours and still alive at least sky

[0253] This example demonstrates the ability of immunotherapy-treated organoids to form their basic structure and begin functioning after one day. These organoids are currently still viable in the culture medium. Days or more.

[0254] In simple terms, immune organoids are generated using the method described in Example 1. Some organoids are left untreated, while others are cultured for 25 days with various doses of different immunotherapies. The organoids in... Visualization was performed using bright-field microscopy after 1 hour and continued until the 1st hour. We also used flow cytometry to evaluate cell viability, quantified the data, and compared them under different conditions.

[0255] Example Immunotherapy-treated organoids Formed within hours and still alive at least sky

[0256] This example demonstrates the ability of immune organoids treated with immunotherapy to form their basic structure and begin to function after one day.

[0257] In simple terms, immune organoids were generated using the method described in Example 1. Some organoids were left untreated while others were cultured with the experimental subunit vaccine on day 0, and the organoids were visualized using bright-field microscopy after 24 hours and then up to day 28.

[0258] like Figure 1 The images shown depict representative longitudinal bright-field images of immune organoids after therapeutic treatment, revealing morphological changes over time. Immune organoids mature within 24 hours. Initial transcriptional responses and cell expansion occur within the first 24 hours. Heaven to the First Day. The initial germinal center reaction and antibody reaction occur on day. Heaven to the Di The peak antibody reaction occurred on the 1st day. sky.

[0259] Example Immunotherapy-treated organoids contain various populations of immune cells. It includes both innate and adaptive cells.

[0260] This example demonstrates that, similar to untreated immune organoids, immunotherapy-treated immune organoids are composed of the same various types of immune cells found in human lymph nodes. These cells work together to govern the structure and function of lymph nodes.

[0261] In short, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were cultured for 25 days with various doses of different immunotherapies. Cultures were stained with markers for B cells, T cells, NK cells, macrophages, monocytes, dendritic cells, plasma cells, ILCs, granulocytes, and plasmablasts, such as CD3, CD4, CD8, CD19, CD38, CD27, CD127, CD138, CD11c, HLA-DR, CD123, live / dead, CD45, CD45RO, CD14, CD56, CD68, CD25, CXCR5, PD-1, CD35, CD20, CD11b, and CFSE. Cells were analyzed by flow cytometry. Data were quantified and analyzed using FlowJO, with comparisons made under all conditions.

[0262] Example 9: Immunotherapy-treated immune organoids contain various immune cell populations, including both innate and adaptive cells.

[0263] This example demonstrates that immune organoids are composed of the same diverse range of immune cell types found in human lymph nodes, and that this diversity is maintained after treatment. Regulation of specific immune cells is a crucial indicator of treatment efficacy, depending on the characteristics of the target product. For instance, plasmablasts or plasma cell differentiation are important cellular considerations for vaccine efficacy. Alternatively, in evaluating the efficacy of many antibody-based therapeutic strategies, the immune response may be disrupted, for example, by destroying germinal center B cells or depleting B, T, and other immune cells.

[0264] In short, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were cultured for 7 days with various doses of different immunotherapies. The cultures were stained with markers targeting B cells, T cells, NK cells, macrophages, monocytes, dendritic cells, plasma cells, granulocytes, and plasmablasts. Cells were analyzed by flow cytometry. Data were quantified and analyzed using FlowJO, with comparisons made under all conditions.

[0265] like Figure 3A The study demonstrated the use of an influenza subunit vaccine to treat organoids, in which the ratio of CD19+ B cells to CD3+ T cells was evaluated. Figure 3B In this study, organoids were treated with rabies vaccine, and the induction of CD27+CD38++ plasmablasts after rabies vaccination was evaluated.

[0266] Next, experimental influenza mRNA vaccines with different formulations were used to treat immune organoids from three independent donors. For example... Figure 3C As shown in the study, all five vaccine conditions resulted in the presence of CD138+ plasma cells in immune organoids, suggesting that the vaccines may be effective.

[0267] Then, the presence of NK cells was examined. Immune organoids from different donors were administered for 7 days without stimulation or with experimental antibody therapeutic agents. Figure 3D As demonstrated, antibody stimulation resulted in a significant increase (61% vs. 1.18%) in the percentage of CD56+CD16+ NK cells within a representative immune organoid from one of the donors. This substantial increase confirms successful immune activation and successful pathway targeting. The antibody effectively promotes NK cell expansion and potentially enhances innate immune responses, highlighting the ability of immune organoids to provide valuable insights into the mechanism of antibody action.

[0268] Next, the granulocyte population was studied. Immune organoids derived from a single donor were administered an influenza subunit vaccine 7 days prior. Figure 3E As shown in the paper, vaccination leads to the production of large SSCs in immune organoids. 高 The CD15+ granulocyte population is consistent with the hypothesis that successful vaccination triggers an early granulocyte response following the initial inflammatory response.

[0269] Finally, the immune organoids from a single donor were treated with an influenza subunit vaccine for 7 days. Figure 3F As demonstrated, a sequential gating strategy targeting each bone marrow look group was used to identify different populations of dendritic cells (CD11b+CD45+), monocytes (CD14+CD11b+), and macrophages (which can be considered as SSCs and CD14+) to evaluate how these immune cells are affected after vaccination.

[0270] Example 10: Immunotherapy-treated immune organoids are composed of other immune cell types.

[0271] This example shows that, similar to untreated immune organoids, immunotherapy-treated immune organoids are composed of rarer immune cell types that are essential for human lymph nodes.

[0272] In simple terms, immune organoids were generated using the method described in Example 1. Some organoids were left untreated while others were cultured for 25 days with various doses of different immunotherapies. The cultures were stained against bone marrow-like DCs (CD11c+), plasma cell-like DCs (CD123+), conventional DCs (CD11b+CD45+), and CD14+ DCs (CD14+CD11c+), stromal cells (CD45-), and fibroblast reticulum cells (CD31+PDPN+).

[0273] Example 11: Immunotherapy-treated immune organoids are composed of other immune cell types.

[0274] This example demonstrates that immune organoids, composed of rarer immune cell types that are crucial to human lymph nodes, can be used to evaluate the population effect after immunotherapy treatment.

[0275] In short, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were treated with an experimental influenza subunit vaccine on day 7. Cultures were stained and flow cytometry was performed on bone marrow-like DCs (CD11c+), plasma cell-like DCs (CD123+), conventional DCs (CD11b+CD45+), and CD14+ DCs (CD14+CD11c+), stromal cells (CD45-), and fibroblast reticulum cells (CD31+PDPN+).

[0276] like Figures 6A-6C and Figures 7A-7C The study showed that vaccine-treated immune organoids contained CD14+CD11c+ myeloid dendritic cells (CD14+CD11c+). Figure 6A ), plasmacytoid dendritic cells (CD123+) Figure 6B ), CD11b+CD45+ dendritic cells ( Figure 6C ), CD45-stromal cells ( Figure 7A ), fibroblasts and reticular cells (PDPN+CD31+) Figure 7B ) and follicular dendritic cells (CD45+PDPN+CD35+) Figure 7C ).

[0277] Example Immunotherapy-treated immune organoid germinal centers

[0278] This example demonstrates the need for germinal centers with both light and dark zones to encapsulate lymph node biology, which are currently unavailable in in vitro techniques. The organoid platform described in this paper uniquely generates germinal centers and thereby replicates key features of human biology.

[0279] In short, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were cultured for 25 days with various doses of different immunotherapies. The day 14 cultures were stimulated with a hepatitis B vaccine, and the organoids were visualized under bright-field microscopy.

[0280] Then confocal microscopy was performed to image the germinal centers of day 14 immune organoids containing B (CD20) and T (CD3) cells, plasmablasts (CD138), BCL6+ cells, and PD1+ cells.

[0281] Example 13: Immunotherapy-treated immune organoids contain cells that function identically to germinal centers.

[0282] This example demonstrates the necessity and functionality of germinal centers. These data indicate that these centers are fully functional and replicate all the essential aspects of lymph node function, representing the first type of organ technology capable of doing so.

[0283] Flow cytometry staining revealed that immune organoids consist of B and T cell regions. Flow cytometry was also performed to identify germinal center B cells.

[0284] Example Immunotherapy-treated immune organoids form germinal centers and contain cells that function identically to germinal centers.

[0285] This example shows that immune organoids treated with immunotherapies designed to stimulate the immune system (in this case, vaccines) form germinal centers containing B and T cell regions.

[0286] In short, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were vaccinated against hepatitis A on day 0. The organoids were visualized under bright-field microscopy on day 14.

[0287] like Figure 9A As shown in the image, immune organoids form germinal centers in response to hepatitis A vaccination. The lighter structures in the organoids, outlined in red, correspond to the morphology of the germinal centers.

[0288] Next, the presence of B and T cell regions in the immune organoids was examined after vaccination with the experimental influenza vaccine. Flow cytometry was performed to analyze CD19+ B cells and CD3+ T cells in the organoids on day 14. Figure 9B As shown in the study, immune organoids contain CD19+ B cells and CD3+ T cells, consistent with the presence of B and T cell regions.

[0289] Finally, stimulation with hemagglutinin protein confirmed the presence of germinal center B cells within the organoid. Flow cytometry was performed to detect... Figure 9C The CD27+CD38+ germinal center B cells shown in the figure.

[0290] Therefore, immune organoids replicate all the basic aspects of lymph node function, making them the first organoid technology capable of doing so.

[0291] Example 15: Immune organoids undergo changes after treatment Cell differentiation

[0292] This example demonstrates that the disclosed immune organoid platform uniquely generates germinal centers and thereby replicates key features of human biology.

[0293] First, we demonstrate the diverse responses of immune organoids to stimulation, including B cell differentiation. During this process, immune organoids stimulated on day 0 are primarily naive B cells, which then differentiate into pre-GC B cells, GC B cells, memory B cells, plasmablasts, and plasma cells. These B cell phenotypes are consistent with the responses of human B cells to lymph node infection, vaccination, or other stimuli.

[0294] In simple terms, immune organoids are generated using the method described in Example 1. For example... Figure 4A As shown in the study, B cell differentiation was monitored by flow cytometry over a 21-day period following treatment with hemagglutinin protein, with different populations emerging at different time points. Progression from the primary naïve B cells (CD38-CD27-) on day 0 to a significant population of germinal center B cells (CD38+CD27+) on days 7-14, and finally to plasmablasts (CD38+++CD27+) on day 21, demonstrates successful activation and maturation of B cells within the organoid system. Figure 4B As shown in the study, the amount of pre-GC and GC B cells in organoids treated with hemagglutinin protein increased over time, indicating successful B cell differentiation.

[0295] Finally, the plasma cell population was examined in the immune organoids after vaccination. In short, immune organoids were generated using the method described in Example 1, and immune organoids from three independent donors were treated with a set of five different formulations of experimental mRNA influenza vaccines. Figure 4C As demonstrated, all five vaccine conditions induced CD138+ plasma cells in comparative immune organoids compared to the unstimulated control, suggesting potential vaccine efficacy. This timing analysis of B cell differentiation validates the ability of immune organoids to summarize key aspects of germinal center responses and subsequent B cell differentiation. Furthermore, organoids provide further insights into the temporal nature of these responses in evaluating vaccine efficacy.

[0296] Example 16: Immune organoids after immunotherapy treatment Cell subtype composition

[0297] This example demonstrates that immune organoids are composed of T cells, including T cell subtypes consistent with those found in human lymph nodes.

[0298] In simple terms, immune organoids were generated using the method described in Example 1. Some organoids were left untreated, while others were treated with an influenza vaccine containing experimental adjuvants on day 0. Flow cytometry was performed on day 7 using markers of various T cell populations. Figures 5A-5BThe vaccine-treated immune organoids exhibited CD4+ naive T cells (CD3+CD4+CD45RA+), CD4+ memory T cells (CD3+CD4+CD45RO+), CD4+ effector memory cells (CD3+CD4+CD45RO+CCR7-), naive CD8+ T cells (CD3+CD8+CD45RA+), memory CD8+ T cells (CD3+CD8+CD45RO+), CD8+ effector memory cells (CD3+CD8+CD45RO+CCR7-), regulatory T cells (CD3+CD4+CD25+), γδ T cells (CD3+CD27+), and T follicular helper cells (CD3+CD25+CXCR5+).

[0299] The results showed that immune organoids can respond to immunotherapy in a manner consistent with human lymph nodes.

[0300] Example 17: Immune organoids are composed of B and T cells that can be regulated in response to various stimuli.

[0301] This example demonstrates that immunotherapy-treated immune organoids consist of a large mixture of different immune cells that are activated and proliferating upon stimulation. B and T cells represent an example of significant differences in cell abundance after stimulation of immune organoids using six different stimulation conditions, some of which preferentially amplified the B cell population while others amplified the T cell population.

[0302] Example Immunotherapy-treated organoids exhibit a fully adaptive immune response.

[0303] This example demonstrates that organoids produce plasmablasts and antigen-specific antibodies against targets exposed in the patient donor (recall response) and targets not exposed in the patient. Antibodies produced by organoids have the ability to switch species in accordance with adaptive immune responses, such as IgM to IgG antibodies.

[0304] Example Immunological organoids can detect antibody production after immunotherapy treatment.

[0305] This example demonstrates that organoids treated with immunotherapy can produce humoral responses specific to the immunotherapy used. For some immunotherapies, evaluating specific antibody responses is particularly important because it indicates the immune system's response to the drug. This is especially relevant to vaccines for infectious diseases and cancer vaccines, where the production of antibodies against the antigen of interest is the desired outcome. For other therapies, antibodies against the treatment itself may be produced, leading to potential resistance to the therapy. Therefore, antibody responses produced by immune organoids can be a key indicator of drug efficacy or, in some cases, lack of efficacy.

[0306] In summary, immune organoids were generated using the method described in Example 1. Organoids were established in three different donors. Some organoids were left untreated, while others were cultured for 25 days with various doses of different immunotherapies. The vaccine was added to the organoids on day 0. Supernatants were collected at different time points from day 0 to day 21, and ELISA was performed to evaluate the production of specific IgG, IgM, and IgA antibodies. Untreated organoids showed similar low or absent specific antibody responses, while drug-treated organoids exhibited neutralizing antibody responses against the antigen of interest (ELISA).

[0307] Example Immune organoids respond to vaccination by forming antigen-specific antibodies

[0308] This example demonstrates that, upon stimulation, immune organoids undergo a complete immune response encompassing a fully adaptive immune response. This includes complete B cell differentiation, from naïve B cells to germinal center B cells, as well as mature cell-like memory B cells, plasmablasts, and plasma cells. Extensive antibody production occurs throughout the process, but antigen-specific and higher-affinity antibodies are produced after germinal center formation and differentiation into more mature B cells. IgM production occurs initially temporally and then species-specifically—converting to IgG and IgA. IgG isoforms are also visible in immune organoids.

[0309] First, antigen-specific IgG antibodies were measured over time under different stimulation conditions. In short, immune organoids were generated using the method described in Example 1. Organoids were stimulated with 12 different formulations of experimental influenza subunit vaccines. The presence of antigen-specific IgG antibodies in the organoids was analyzed by ELISA on days 4, 8, 11, and 15.

[0310] like Figure 10AThe study measured antigen-specific IgG responses in 12 different experimental influenza vaccine formulations in immune organoids over a 15-day period. ELISA data revealed significant variations in anti-HA IgG production across different stimulation conditions, with several formulations exhibiting different temporal patterns. Notably, stimuli 7 and 11 showed sustained antibody production reaching higher concentrations (>400 ng / mL) at later time points (days 11–15), while others (such as stimuli 1–4) showed minimal IgG production (<100 ng / mL) throughout the observation period. Stimulation 6 showed a gradual increase in antibody concentration over time, indicating a more delayed but gradual immune response.

[0311] Next, the ability of immune organoids to differentiate into plasmablasts after vaccination was analyzed. In short, immune organoids were generated using the method described in Example 1. The organoids were inoculated with an influenza vaccine containing experimental adjuvants, and flow cytometry was performed on day 0 or day 14 to evaluate CD38 and CD27 expression on individual cells. Figure 10B As shown in the study, the organoids exhibited an increase in CD38++CD27+ plasmablasts in response to vaccination.

[0312] Then, antigen-specific immunoglobulin characteristics were examined in two different donors. In short, immune organoids were generated using the method described in Example 1, and treated with either Treatment A or Treatment B (two different doses of rabies vaccine). On day 7, the supernatant was collected and the LEGENDplex Human Immunoglobulin Isotyping Panel was performed to evaluate the concentrations of IgG1-4 subtypes, IgM, and IgA corresponding to Treatment A and Treatment B. Figure 10C As shown in the study, both donors exhibited similar patterns of immunoglobulin production, and the levels of IgG1, IgM, and IgA were significantly elevated under both treatment conditions.

[0313] Finally, as Figure 10D and 10EAs shown in this study, immune organoids were used to evaluate anti-HA antibody responses following vaccination with different formulations and doses of experimental influenza vaccines. Data showed successful immunoglobulin species conversion, progressing from an early IgM response to later IgG production. All formulations were tested at two doses (1 > 2), with stimuli 3 and 4 being the most immunogenic, demonstrating sustained IgM responses (OD 0.8–1.0) and robust IgG production (70–80 ng / mL) by day 15. Although most formulations showed early IgM responses between days 4 and 8, there were considerable differences in the magnitude and kinetics of antibody production under different conditions. Higher doses generally induced stronger responses, but this dose dependence varied across formulations. Notably, stimuli 5 and 6 showed relatively weak IgG responses, even with initial IgM production, suggesting that these formulations may be less effective in promoting species conversion or maintaining antibody responses.

[0314] Example Immune organoids can break tolerance to generate antibodies against human targets.

[0315] This example demonstrates how immune organoids produce IgM and IgG antibodies against foreign antigens, including influenza and SARS-CoV-2. Immune tolerance is a key property of systemic immunity, a characteristic replicated by immune organoids through their lack of response to stimuli. Another key property of systemic immunity is the ability to break tolerance (i.e., autoimmune diseases and allergic diseases). Under certain stimuli, immune organoids can be induced to model tolerance breaking, a key feature of immune organoids producing antibodies against human targets for therapeutic purposes (i.e., cancer and autoimmune diseases). The organoids' ability to generate autoimmune responses against autoantigens (myelin) is crucial for the first-ever modeling of autoimmune and allergic diseases.

[0316] In short, immune organoids were generated using the method described in Example 1. Some organoids were untreated, while others were stimulated with influenza hemagglutinin, SARS-CoV-2 spike protein, myelin, or NK cell proteins in combination with different experimental adjuvants. On day 11, ELISA was performed to detect IgM and IgG antibodies.

[0317] like Figure 11A-11DAs demonstrated, organoids exhibit versatility in modeling conventional vaccine responses and autoimmune phenomena. While stimulation with influenza HA and SARS-CoV-2 spike proteins elicited expected antigen-specific antibody responses, notably, when combined with specific adjuvant formulations, the organoids also successfully generated antibodies against autoantigens (myelin and NK cell receptor proteins). Specifically, Stimulation 2 consistently produced the highest IgG response (uptake rate ~0.3–0.5) across all tested antigens, while IgM responses showed more variable patterns under different conditions. The ability to break immune tolerance in this control system is particularly significant for immunotherapy development, as it demonstrates the potential to overcome autoantigen mechanisms that often limit antitumor immune responses. This feature could be especially valuable for cancer immunotherapy development, where generating an effective immune response against autoantigens (tumor-associated antigens) is crucial but challenging due to innate tolerance mechanisms. Therefore, immune organoids provide a unique platform for screening and optimizing stimuli formulations that can effectively break tolerance.

[0318] Example 22: Immunotherapy-treated immune organoids exhibit changes in cell phenotype, number, and polarization.

[0319] This example demonstrates that untreated immune organoids, compared to each other, possess similar cell phenotypes, numbers, and polarization, but differ from those treated with immunotherapy. Immunotherapy has a significant impact on immune cell phenotype, immune cell numbers (immunotherapy can cause some immune cells to proliferate more than others), and immune cell polarization. These characteristics of the immune system influence drug efficacy, and this is not something that can be read in the human system, which is used to predict patient outcomes. Therefore, organoids can be used to read how drugs act on the immune system, which indicates drug efficacy.

[0320] In simple terms, immune organoids are generated using the method described in Example 1. Some organoids are left untreated while others are cultured for 25 days with various doses of different immunotherapies.

[0321] Organoid plates were placed under a bright-field microscope and images were captured. Software was used to visually observe shape and integrity, quantify opacity, and compare results under different conditions.

[0322] Organoids were also harvested and depolymerized into single-cell suspensions. The suspensions were stained using live / dead cells and read using a cell counter. Data were quantified and analyzed using FlowJO, with comparisons made under different conditions. Groups included: CD3, CD4, CD8, CD19, CD38, CD127, CD27, CD138, PDPN, CD11c, HLA-DR, CD123, live / dead cells, CD45, CD45RO, CD14, CD56, CD68, CD25, CXCR5, PD-1, CD35, CD20, CD11b, and CFSE.

[0323] Example Immunological organoids can detect the production of cytokines and chemokines after immunotherapy.

[0324] This example demonstrates that organoids can detect the production of intercytokines / chemokines after immunotherapy treatment, a key indicator of drug efficacy, or in some cases, its absence. Evaluating the intercytokine / chemokine response to immunotherapy treatment is highly relevant because these signaling molecules play a crucial role in activating and regulating the immune response, a primary goal of immunotherapy. Immunotherapy that induces an appropriate intercytokine response is a good indicator of drug efficacy.

[0325] In summary, immune organoids were generated using the method described in Example 1. Organoids were established in three different donors. Some organoids were left untreated, while others were cultured with various doses of different immunotherapies. The organoids survived for 25 days. Supernatants were collected at different time points from day 0 to day 21, and Luminex was performed to evaluate intercytokine and chemokine production. The untreated organoids exhibited distinct intercytokine and chemokine response characteristics compared to the treated organoid group.

[0326] Example Immunotherapy-treated organoids exhibit immune memory

[0327] This example demonstrates that organoids possess the ability to evaluate the formation of immune memory, a key indicator of immunotherapy efficacy. The formation of immune memory is crucial to the efficacy of certain immunotherapies (especially vaccination) (due to increased response to subsequent exposure and long-term protection derived from immunotherapy), and strong induction of immune memory indicates preferred drugs.

[0328] In summary, immune organoids were generated using the method described in Example 1. Organoids were established from three different donors. Some organoids were left untreated while others were cultured with various doses of different immunotherapies. The organoids survived for 25 days. Organoids were harvested at various time points from day 0 to day 21 and dissociated after collection. Cells were stained against a wide range of phenotypic / other surface markers (groups: CD3, CD4, CD8, CD19, CD38, CD27, CD138, live / dead, CD45RO, CD25, CXCR5, CD127, CD20, CFSE), read, quantified, and plotted using a cell counter.

[0329] Organoids in the vaccinated cohort exhibited above-baseline immune memory formation, as indicated by the identification and quantification of memory T cell subgroups (CD45RO, CCR7, CD62L, and CD127) as well as memory B cells (CD27) and plasma cells (CD138), features not observed in the control cohort (flow cytometry).

[0330] Example Immune organoids allow for a comprehensive evaluation of the efficacy of immunotherapy through multi-parameter analysis of cellular responses.

[0331] This example demonstrates that immune organoids can comprehensively assess the efficacy of immunotherapeutic agents through multiple complementary readouts within a physiologically relevant human immune microenvironment. The system captures key parameters, including cytotoxic responses, proliferative capacity, and detailed immunophenotypic analysis, revealing therapy-induced changes in cellular composition and activation state. The systemic generalization of the ability of immune cells to polarize and differentiate provides crucial insights into the mechanistic basis of therapeutic responses. Furthermore, the quantification of antibody production and isotype conversion, along with the profiling of coupled cytokines and chemokines, provides a detailed understanding of humoral and cellular immune responses. Current drug development workflows lack predictive preclinical models that effectively reflect human immune responses. Traditional in vitro systems fail to capture the complex cell interactions and organizational structures required for adequate immune function, and animal models often struggle to predict human immune responses due to substance-specific differences. The immune organoid platform bridges this critical gap by providing a physiologically relevant human immune microenvironment capable of generating functional readouts across multiple immune parameters. The ability to evaluate multiple aspects of immune responses within a single platform using donor-matched human cells makes immune organoids a valuable tool for therapeutic screening and optimization. This system makes drug development decisions more informed by providing comprehensive immune profiling before advancing candidates to clinical trials, potentially increasing success rates and reducing development costs in immunotherapy development.

[0332] First, a temporal analysis of lymphocyte populations in immune organoids was performed after treatment with experimental therapeutic antibodies in combination with a set of different experimental adjuvants. In short, immune organoids were generated using the method described in Example 1. Organoids were treated with the antibody and adjuvant combination on day 0, and flow cytometry was performed on CD3+ T cells and CD19+ B cells on days 0, 7, and 14. Figure 12A As shown in the study, CD19+ B cells decreased over time, while CD3+ T cells increased over time, consistent with plasma cell induction and indicating that the adjuvant successfully promoted T cell proliferation.

[0333] Next, we examined the ability of immune organoids to generate CD4+ memory T cells in response to immunotherapy treatment. In short, we generated immune organoids from two donors using the method described in Example 1. The organoids were treated with an experimental influenza subunit vaccine on day 0, and flow cytometry was performed on CD45RO+CD4+ memory T cells and CD45RA+CD4+ naive T cells on day 7. Figure 12B As shown, the CD45RO+ population was dominant in both samples (89.6% and 83.2%) compared to the CD45RA+ population (4.59% and 9.99%). This indicates that immune organoids can generate CD4+ memory T cells in response to immunotherapy.

[0334] Then, antigen-specific immunoglobulin characteristics were examined in two different donors after vaccination. In short, immune organoids were generated using the method described in Example 1, and treated with either Treatment A or Treatment B, where Treatment A and B were two different doses of rabies vaccine. On day 7, the supernatant was collected and the LEGENDplex™ human immunoglobulin isotyping panel was performed to evaluate the concentrations of IgG1-4 subtypes, IgM, and IgA corresponding to Treatment A and Treatment B. Figure 12C As shown in the study, both donors exhibited similar patterns of immunoglobulin production, and the levels of IgG1, IgM, and IgA were significantly elevated under both treatment conditions.

[0335] Next, the production of cytokines and chemokines in response to treatment was examined in the immune organoids. In short, immune organoids were generated using the method described in Example 1. Cytokine and chemokine levels were measured in untreated conditions and after treatment A, where treatment A was an experimentally designed therapeutic antibody intended to stimulate the immune system. Figure 12DAs shown in the study, IL-10 production increased uniformly across all donors after treatment, reaching similar concentrations (approximately 600-700 pg / mL). IFN-γ production exhibited donor-specific changes, with treated donors B and C showing substantially higher levels (approximately 200,000 pg / mL) compared to donor A. Similarly, as... Figure 12E As shown in the study, treatment A induced a significant increase in CXCL10 secretion in all donors (p = 0.0013, ratio-paired t-test). Donor-specific changes were observed, with baseline levels ranging from approximately 500 to 11,000 pg / mL and treatment levels ranging from 1,000 to 15,000 pg / mL.

[0336] Next, selective expansion of B and T cell populations was measured in immune organoids following treatment with different immunomodulators. In short, immune organoids were generated using the method described in Example 1. Organoids were treated with 24 different experimental immunomodulators on day 0, and flow cytometry was performed on day 14 to evaluate the number of viable B and T cells. The 24 immunomodulators were tested individually and in various combinations to assess their potential synergistic effects on lymphocyte populations. Figure 12F As shown, treatment-specific effects on lymphocyte populations are evident, with treatments 1-7 primarily expanding T cells (viviple >80%), while treatments 8-12 preferentially supported B cell expansion (viviple >70%), consistent with expectations for different immunomodulatory conditions. Because the combination of different immunomodulators was used to balance effects with varying success rates, the remaining conditions exhibited different effects on the two populations. The control condition maintained a relatively balanced ratio of the two cell types.

[0337] Finally, on day 5, B cell phenotypes in immune organoids after different stimuli were measured by flow cytometry. In short, immune organoids were generated using the method described in Example 1. Organoids were either untreated (control), treated with rabies vaccine alone to promote robust germinal centers, or treated with a combination of rabies vaccine and an investigational antibody therapeutic designed to disrupt germinal center formation. All treatments were performed on day 0. Figure 12GAs shown in the study, in the unstimulated control, most B cells exhibited the naïve phenotype (68.6% CD38-CD27-), with minimal germinal center (GC) formation (0.69% CD38+CD27+). Vaccination alone induced a robust germinal center response, confirmed by an increase in GC B cells (18.8%) and plasmablasts (48.1%), and a corresponding decrease in naïve B cells (6.17%). The addition of a therapeutic antibody appeared to slightly modulate this response, resulting in a decrease in GC B cells (14.1%) but an increase in plasmablast formation (56.7%). Flow cytometry results indicated minimal to no effect on the germinal centers or plasmablast populations themselves, suggesting the need to adjust antibody concentrations or the dosing window. Although the effect on the B cell subpopulations was minimal to no, many effects on the total B cell count require further investigation.

[0338] Example Immunological organoids can achieve antibody type switching and memory after immunotherapy treatment. Cell induction

[0339] This example demonstrates that immune organoids can generate memory B cells in response to immunotherapy.

[0340] In short, immune organoids are generated using the method described in Example 1. The organoids are treated with rabies vaccine, and a time-varying analysis of the anti-rabies antibody response is performed.

[0341] like Figure 13A As shown, IgM levels peaked between days 7 and 9, then gradually declined, while IgG levels remained low until day 16, then substantially increased by day 21, consistent with the antibody response kinetics of the initial antigen. Upon initiation of the initial response, a transient upregulation of IgM is expected initially, followed by a shift to IgG, with the IgG response peaking around day 21.

[0342] Next, we examined B-cell differentiation in response to immunotherapy treatment. In short, we generated immune organoids using the method described in Example 1. The organoids were treated with an experimental influenza subunit vaccine, and flow cytometry was performed on day 7 following treatment on day 0. Figure 13B As shown in the study, prior to treatment, the organoids primarily contained naive B cells (93.3%). After treatment, both different organoid populations exhibited a transformation towards a memory B cell phenotype, with 71.9% and 67.0% of the organs, respectively, being memory B cells. This data suggests that immune organoids can generate memory B cells in response to immunotherapy.

[0343] Example 27: Immunotherapy-treated organoids exhibit changes in the BCR / TCR repertoire.

[0344] This example demonstrates that organoids can be used to evaluate changes in BCR and TCR in response to immunotherapy, thereby providing insights into the efficacy, durability, and safety of immunotherapies to guide treatment modalities and improve clinical outcomes. Evaluating the efficacy of immunotherapy includes assessing changes in the BCR and TCR repertoire. BCR changes associated with immunotherapy efficacy may include antibody isotype conversion; high prevalence of certain BCR sequences, indicating clonal expansion of B cells specific to the antigen of interest; somatic hypermutations of BCR sequences, leading to the production of high-affinity antibodies specific to the antigen of interest; and the presence and changes in memory B cells specific to the antigen of interest. TCR changes associated with immunotherapy efficacy include clonal expansion of TCRs specific to the target antigen.

[0345] In summary, immune organoids were generated using the method described in Example 1. Organoids were established in three different donors. Some organoids were left untreated while others were cultured with various doses of different immunotherapies. The organoids survived for 25 days. Organoids were harvested at various time points from day 0 to day 21. Nucleic acids were purified. Organoid cells were treated using a 10X genomics platform and a 5' kit to obtain BCR and TCR libraries, which were then sequenced using NGS technology, and changes in the BCR and TCR libraries were analyzed (ontology sequencing).

[0346] Clonal expansion of B cells specific to the target vaccine was increased in the vaccinated organoids, but absent in the unvaccinated ones. The organoids also exhibited changes in the memory B cell population (increased CD27+CD19+ cells) in response to vaccination. Changes in TCR revealed clonal expansion of antigen-specific T cells in the vaccinated cohort.

[0347] Example Immunotherapy-treated immune organoids can increase the cell population that responds to drugs.

[0348] This example demonstrates that organoids can predict increases in cell populations that change with drug efficacy.

[0349] In simple terms, immune organoids are generated using the method described in Example 1. Organoids are established in three different donors. Some organoids are left untreated while others are cultured with drugs known to cause / not cause changes in cell number. The organoids can survive for 25 days. The organoids are harvested and depolymerized into single-cell suspensions, the cells are stained against a wide range of phenotypic / other surface markers (including CFSEs used to track proliferation), read on a cell counter, quantified, and plotted.

[0350] Untreated organoids have the standard cell-to-cell ratio as previously described (original patent), while treated organoids have an expanded population of immune cells (e.g., more B cells, more T cells, etc.).

[0351] Example 29: Immune organoids can predict the range of drug outcomes for different patient groups.

[0352] This example demonstrates that immune organoids can predict the range of outcomes present in real patient cohorts.

[0353] Organoids were co-cultured with drugs whose efficacy characteristics are known to differ across patient backgrounds (e.g., sex, age, etc.). These donor-dependent effects were reproduced in the organoids.

[0354] All publications and patent applications mentioned in this disclosure are incorporated herein by reference as if each individual publication or patent application were expressly and individually cited.

[0355] No cited reference is acknowledged as prior art. The arguments presented in the references state the claims made by their authors, and the applicant reserves the right to challenge the accuracy and relevance of the cited documents. It should be clearly understood that although this document references numerous sources (including scientific journal articles, patent documents, and textbooks), it does not acknowledge that any of these documents constitutes part of general common knowledge in the industry.

[0356] The general methods described herein are intended for illustrative purposes only. Upon review of this disclosure, those skilled in the art will recognize other alternative methods and solutions, which are included within the spirit and scope of this application.

Claims

1. A method for evaluating the efficacy of a therapeutic agent, the method comprising: (a) Contacting a three-dimensional immune organoid comprising multiple self-assembled primary immune cells and multiple stem cells derived from one or more secondary lymphoid organs with a therapeutic agent, and (b) Measure the immune response of the three-dimensional immune organoids to the therapeutic agent after the contact.

2. The method according to claim 1, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+.

3. The method according to any of the preceding claims, wherein the one or more secondary lymphoid organs are derived from the spleen, lymph nodes, Pierre's lymph plexus, and / or MALT.

4. The method according to any one of the preceding claims, wherein the immune organoid is a human immune organoid.

5. The method according to any of the preceding claims, wherein the immune organoids further comprise peripheral blood mononuclear cells.

6. The method according to any of the preceding claims, wherein the secondary lymphoid organ is obtained from a living patient, surgical excision, fine needle aspirate, biopsy, or a deceased patient.

7. The method according to any one of the preceding claims, wherein the plurality of primary immune cells include B cells, T cells, plasmablasts, plasma cells, ILC, granulocytes, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

8. The method of claim 7, wherein the B cells comprise one or more naïve B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or combinations thereof.

9. The method of claim 7, wherein the T cells comprise naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, CD4 effector memory T cells, CD8 effector memory T cells, or combinations thereof.

10. The method of claim 7, wherein the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, bone marrow-like dendritic cells, or combinations thereof.

11. The method according to any of the preceding claims, wherein the plurality of primary cells further comprises one or more stromal cells, follicular dendritic cells, and fibroblastic reticular cells.

12. The method according to any of the preceding claims, wherein the diameter of the immune organoid is 8000 µm or less.

13. The method according to any of the preceding claims, wherein the immune organoid comprises a germinal center and / or a B / T cell region.

14. The method according to any of the preceding claims, wherein the immune organoid produces antibodies.

15. The method of claim 14, wherein the antibody is an IgG, IgM, or IgA antibody.

16. The method of claim 15, wherein the antibody has complete humoral functionality.

17. The method of claim 16, wherein the antibody binds to human and non-human targets.

18. The method of claim 17, wherein the human target comprises proteins, sugars, and nucleic acids.

19. The method of claim 18, wherein the non-human target includes infectious disease antigens, venom, poisons, and small molecules.

20. The method according to any one of the preceding claims, wherein the therapeutic agent is an immunotherapeutic agent.

21. The method of claim 20, wherein the immunotherapeutic agent is selected from the group consisting of: vaccines, antibodies, gene therapy, cell therapy, small molecules, and nanobodies.

22. The method according to any one of the preceding claims, wherein measuring the immune response includes measuring the number of immune cells, immune cell proliferation, immune cell phenotype, immune cell polarization, antibody production, cytokine production, chemokine production, B cell receptor changes, T cell receptor changes, and / or immune memory.

23. The method of claim 22, wherein measuring the changes in B cell receptors includes measuring antibody isotype switching, clonal expansion, somatic hypermutation, and / or memory B cells.

24. The method of claim 22, wherein measuring the changes in T cell receptors includes measuring T cell clonal expansion.

25. A composition comprising: A three-dimensional immune organoid comprising multiple self-assembled primary immune cells derived from one or more secondary lymphoid organs and multiple stem cells, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+. Therapeutic agents.

26. The composition of claim 25, wherein the one or more secondary lymphoid organs are derived from the spleen, lymph nodes, Pierre's lymph plexus, and / or MALT.

27. The composition according to any one of claims 25 to 26, wherein the plurality of immune cells are human immune cells.

28. The composition according to any one of claims 25 to 27, wherein the plurality of immune cells comprises 2 × 10 6 One or fewer cells.

29. The composition according to any one of claims 25 to 28, further comprising peripheral blood mononuclear cells.

30. The composition according to any one of claims 25 to 29, wherein the plurality of immune cells are obtained from a living patient, surgical excision, fine needle aspirate, biopsy, or a deceased patient.

31. The composition according to any one of claims 25 to 30, wherein the plurality of immune cells comprises B cells, T cells, plasmablasts, plasma cells, ILC, granulocytes, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

32. The composition of claim 31, wherein the B cells comprise one or more naïve B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or combinations thereof.

33. The composition of claim 31, wherein the T cells comprise naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, CD4 effector memory T cells, CD8 effector memory T cells, or combinations thereof.

34. The composition of claim 31, wherein the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, bone marrow-like dendritic cells, or combinations thereof.

35. The composition according to any one of claims 25 to 34, wherein the plurality of primary cells further comprises one or more stromal cells, follicular dendritic cells and fibroblastic reticular cells.

36. The composition according to any one of claims 25 to 35, wherein the diameter of the immune organoid is 8000 µm or less.

37. The composition according to any one of claims 25 to 36, wherein the immune organoid comprises a germinal center and / or a B / T cell region.

38. The composition according to any one of claims 25 to 37, wherein the immune organoid produces antibodies.

39. The composition of claim 38, wherein the antibody is an IgG, IgM, or IgA antibody.

40. The composition of claim 39, wherein the antibody has complete humoral functionality.

41. The composition of claim 40, wherein the antibody binds to human and non-human targets.

42. The composition of claim 41, wherein the human target comprises proteins, sugars, and nucleic acids.

43. The composition of claim 42, wherein the non-human target comprises infectious disease antigens, venom, poisons, or small molecules.

44. The method according to any one of claims 25 to 43, wherein the therapeutic agent is an immunotherapeutic agent.

45. The method of claim 44, wherein the immunotherapeutic agent is selected from the group consisting of: vaccines, antibodies, gene therapy, cell therapy, small molecules, and nanobodies.

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